Ascending projections of simple and complex cells in layer 6 of the cat striate cortex.

Receptive field properties vary systematically across the different layers of the cat striate cortex. Understanding how these functional differences emerge requires a precise description of the interlaminar connections and the quality of information that they transmit. This study examines the contribution of the two physiological types of neuron in layer 6, simple and complex, to the cortical microcircuit. The approach was to make whole-cell recordings with dye-filled electrodes in vivo to correlate visual response property with intracortical projection pattern. The two simple cells we stained projected to layer 4, as previously reported (Gilbert and Wiesel, 1979; Martin and Whitteridge, 1984). Six of the eight complex cells that we labeled projected to the superficial layers, a pathway not previously described in the cat. The remaining two cells targeted the infragranular layers. Layer 4 is dominated by simple cells, whereas layers 5 and 2+3 are mainly composed of complex cells (Hubel and Wiesel, 1962; Gilbert, 1977). Hence, our results indicate that the ascending projections of simple cells in layer 6 target other simple cells. In parallel, the ascending projections of a population of complex cells in layer 6 favor other complex cells. Anatomical experiments in several species (Lund and Boothe,1975; Burkhalter,1989; Usrey and Fitzpatrick, 1996; Wiser and Callaway, 1996) had also demonstrated that layer 6 gives rise to two separate intracortical pathways. Pooling the results of these anatomical studies with our own suggests a common feature of the laminar organization: cells that project to different intracortical targets have distinct functional characteristics.

Identification and quantification of the indole alkaloid ibogaine in biological samples by gas chromatography-mass spectrometry.

A sensitive and highly selective analytical chemical method for measuring the indole alkaloid ibogaine in biological samples has been developed. The method utilizes organic extraction, derivatization with trifluoroacetic anhydride, and detection by combined gas chromatography-mass spectrometry. The deuterated analog of ibogaine, O-[Cd3]-ibogaine, was synthesized and used as an internal standard for the method. Standard curves, constructed from variable amounts of ibogaine (50-400 ng) and a fixed amount of internal standard (250 ng) were linear. The method has an approximate detection limit of at least 20 ng/mL of tissue extract (180 ng/g tissue), with a coefficient of variation of 8 to 12.5%. Chemical stability studies with the method found that aqueous ibogaine solutions (1-10 mg/mL) could be stored at 10 degrees for up to 7 months with no more than 10% loss. The method was also used to measure brain ibogaine levels in rats 1 and 19 hr after a single dose of drug (40 mg/kg, i.p.); the results suggest a rapid disappearance of the drug after i.p. dosing. The method will help reveal the pharmacokinetic properties of this putative anti-addictive agent in animals and humans.

Differential effects of ibogaine pretreatment on brain levels of morphine and (+)-amphetamine.

Previous studies in rats have shown that ibogaine inhibits neurochemical and behavioral effects of morphine yet potentiates similar effects of (+)-amphetamine. To assess whether these different functional interactions have a metabolic basis, brain levels of morphine and (+)-amphetamine were measured by gas chromatography-mass spectrometry after ibogaine pretreatment (19 h before injection of morphine or (+)-amphetamine). Ibogaine pretreatment had no effect on brain morphine levels, either at 30 min or 2 h after morphine injection; however, ibogaine significantly increased brain amphetamine levels at 30 min and, to a greater extent, at 2 h after (+)-amphetamine injection. These and other data suggest that ibogaine irreversibly inhibits an amphetamine-metabolizing enzyme. The functional interactions between ibogaine and (+)-amphetamine, but not those between ibogaine and morphine, may result from a hepatic drug-drug interaction.

Hepatocytes express blood coagulation factor XII (Hageman factor).

The liver synthesizes blood coagulation factor XII (Hageman factor). The specific cell that expresses factor XII, however, has not been previously identified. We used primary rat hepatocytes cultured in serum-free medium to study the transcription, de novo synthesis, and secretion of factor XII. A 32P-labeled human factor XII complementary DNA probe was used for RNA blot hybridization. A single band of hybridization at 2.4 kilobases appeared in blots of polyadenylated RNA derived from 24-hour hepatocyte cultures. This corresponds to the known size of factor XII-processed primary transcript (messenger RNA). Cultured hepatocytes secreted labeled factor XII when tritiated leucine was added to the medium, indicating that the hepatocytes used 3H-leucine to synthesize factor XII de novo. In these hepatocyte cultures immunoreactive factor XII levels progressively increased in 24 hours and factor XII clotting activity increased in parallel. Cycloheximide inhibited the accumulation of both immunoreactive and coagulant factor XII. Secreted factor XII from the rat hepatocytes comigrated with authentic rat plasma factor XII at 80,000 molecular weight in a Western immunoblot. These data indicate that cultured hepatocytes transcribe, synthesize, and secrete authentic factor XII.

Sexual differentiation of play behavior in the ferret.

Three experiments were performed to elucidate the endocrine mechanisms responsible for sex differences in the prepubertal play behavior of ferrets. In Experiment 1, gonadally intact adolescent males exhibited higher levels of "stand-over" behavior than females did in tests between 63 and 123 days of age with gonadally intact female partners of the same age. In Experiment 2, ferrets exposed to androgen or to ovarian steroids over Days 5-20 of postnatal life subsequently exhibited significantly higher levels of stand-over behavior in tests with female partners than did control females gonadectomized on Day 5 and not subsequently given any steroids. However, none of the subjects in Experiment 2 exhibited levels of stand-over behavior comparable to those of the gonadally intact males in Experiment 1. In Experiment 3, males gonadectomized and implanted subcutaneously with testosterone capsules on Day 70 and tested with female partners at 84-96 days of age exhibited levels of stand-over behavior comparable to those observed in Experiment 1 in gonadally intact males of the same age (Weeks 12-14). Males gonadectomized on Day 70 and given no hormone at the time of testing exhibited significantly lower levels of this behavior. Significantly lower levels of this behavior were also exhibited by males gonadectomized on Day 35 and females gonadectomized on Day 70 regardless of whether they were tested with testosterone present after Day 70. Sex differences in the expression of prepubertal play behavior of ferrets apparently result from differential exposure of males and females to androgen over an extended postnatal period.

Plasma and pineal melatonin levels in female ferrets housed under long or short photoperiods.

Ovohysterectomized female ferrets were housed in controlled environment rooms in which the daily lighting schedule was either 15L:9D (long days) or 9L:15D (short days). After 2 weeks some ferrets in each group were given an intrajugular catheter: beginning 1 week later, a blood sample was taken daily at one of eight different clock times over an 8 to 10 day period. One additional blood sample plus the pineal gland were collected from these animals and from uncathetarized animals in each group after decapitation at different clock times. Both plasma melatonin concentrations and pineal melatonin content were elevated in a square-wave pattern during the dark hours, with the duration of elevation being longer in ferrets kept under the short days. These results suggest that differences in the duration of nocturnal increments in melatonin secretion may mediate the stimulatory and inhibitory effects of long and short days, respectively, on ovarian activity in female ferrets.

Normal differentiation of masculine sexual behavior in male ferrets despite neonatal inhibition of brain aromatase or 5-alpha-reductase activity.

Male ferrets born in the laboratory received subcutaneous Silastic capsules containing either the aromatase inhibitor, androst-1,4,6-triene-3, 17-dione (ATD), the 5 alpha-reductase inhibitor, testosterone-17 beta-carboxylic acid (17 beta C), or no hormone, for 15 days beginning on the day of birth; an additional group of females received empty Silastic capsules. All ferrets were gonadectomized when 11 weeks of age and were subsequently tested for masculine sexual behavior after a latin-square sequence of treatments with subcutaneous Silastic capsules containing testosterone (T), estradiol (E), or dihydrotestosterone (DHT). After T, control males displayed significantly more neck gripping, mounting and pelvic thrusting than control females, and males treated neonatally with ATD or 17 beta C were no less responsive than control males. After DHT, little masculine sexual behavior was shown by any group. After E, the duration of mounting was significantly longer in control and ATD males than in control females or 17 beta C males. Subsequently, however, there were no differences between control and 17 beta C males on any parameter of masculine sexual performance, when they were retested sequentially after subcutaneous implantation of E followed by E + DHT. Additional groups of newborn male and female ferrets received subcutaneous capsules containing either ATD, 17 beta C, or no hormone and were killed on postnatal day 7. Administration of ATD, but not 17 beta C, strongly inhibited aromatase activity in the hypothalamus + preoptic area. In all groups, the formation of significantly inhibited cortical 5 alpha-reductase activity. Plasma concentrations of T were equivalent on postnatal day 7 in males given each of the neonatal treatments. These results suggest that behavioral masculinization in the male ferret results primarily from the neonatal action in brain of T itself, and not from its estrogenic or 5 alpha-reduced androgenic metabolites.

Effects of testosterone, dihydrotestosterone, or estradiol administered neonatally on sexual behavior of female ferrets.

Groups of female ferrets born in the laboratory received sc Silastic capsules containing testosterone (T), 5 alpha-dihydrotestosterone (DHT), 17 beta-estradiol (E), or no steroid for 15 days beginning on the day of birth; an additional group of male ferrets received empty sc capsules neonatally. All ferrets were gonadectomized at 11 weeks of age and were subsequently tested for masculine and feminine sexual behaviors while being treated consecutively over an 8-month period with several different gonadal steroids. The ability to display masculine sexual behavior was studied in the absence of replacement hormones and during a counterbalanced sequence of treatments with Silastic capsules containing T, E, or DHT. The maximal amount of neck grip, mount, and pelvic thrusting behavior displayed, regardless of adult endocrine treatment, was significantly greater in control male and neonatally T-treated females than in females that had received no hormone, E, or DHT neonatally. Animals in all five groups displayed equivalent increments in sexual receptivity in response to daily sc injections of increasing dosages of estradiol benzoate. Polyacrylamide gel electrophoresis of plasma collected from newborn female ferrets revealed no binding of either [3H]E or [3H]T, whereas two binding peaks were found for [3H]DHT. After the administration of androgen in adulthood, equivalent clitoral growth and ossification occurred in females given either T or DHT neonatally. These results suggest that in ferrets, behavioral masculinization occurs in response to neonatal exposure to T itself and not to its major neural metabolites, E and DHT. They also show that behavioral defeminization fails to occur in ferrets even after neonatal exposure to pharmacological amounts of E, T, or DHT.

Proliferative colitis in ferrets.

During a 4-month period, 31 of 156 ferrets (Mustela putorius) in a biomedical research program developed protracted diarrhea. Clinical signs were green mucohemorrhagic fecal material, partially prolapsed rectum, anorexia, body weight loss, and dehydration. Nine of the affected animals were necropsied. On gross examination, the descending colon was grossly thick and histologically characterized by marked proliferation of the mucosa, relatively few goblet cells, mixed inflammatory cell infiltrate, and penetration of the mucosal glands through the muscularis mucosa into the submucosa and tunica muscularis. Campylobacter fetus subsp jejuni was isolated from 6 of 9 ferrets with proliferative colitis. Warthin-Starry stained sections of hyperplastic colon revealed large numbers of organisms in the apical portion of epithelial cells, and organisms similar to Campylobacter spp were observed by electron microscopy in hyperplastic colonic epithelium. The proliferative colitis in the ferret is compared with the pathologic and bacterial features of similar intestinal proliferative diseases in swine and hamsters.