Temporal regulation by estrogen of beta-preprotachykinin mRNA expression in the rat ventromedial nucleus of the hypothalamus.

In the rat, reproduction and sexual behavior are controlled by the gonadal steroid regulation of synaptic interactions within the sexually dimorphic limbic-hypothalamic system. The effects of estrogen on the ventromedial nucleus of the hypothalamus, one nucleus within the circuit, are central to the modulation of this behavior. Involvement of the neuropeptide substance P, a member of the tachykinin family of neuropeptides, has been implicated in the regulation of both lordosis behavior and gonadotropin release. However, previous studies have provided conflicting evidence as to whether levels of substance P in the ventromedial nucleus of the hypothalamus are modulated by circulating estrogens. To study this question further, in situ hybridization histochemistry was used to examine levels of beta-preprotachykinin mRNA, which encodes substance P and other tachykinins, in the ventrolateral subdivision of the ventromedial hypothalamus at 10 consecutive timepoints over a 4 day period subsequent to an acute administration of estrogen. Following estrogen treatment, beta-preprotachykinin mRNA expression was increased in cells of the ventrolateral portion of the ventromedial nucleus of the hypothalamus which constitutively express beta-preprotachykinin mRNA; however, there were no statistically significant changes in the number of cells that express detectable levels of beta-preprotachykinin mRNA in the ventrolateral portion of the ventromedial nucleus. Estrogen treatment produced two peaks of beta-preprotachykinin mRNA expression, the first at 2 h and the second at 48 h after the injection of estrogen. These data indicate that estrogen has both rapid and prolonged effects on beta-preprotachykinin mRNA levels, suggesting that estrogen may affect different cellular mechanisms relevant to the induction of beta-preprotachykinin mRNA expression.

Session duration and the VI response function: Within-session prospective and retrospective effects.

Two experiments examined the effects of session duration on responding during simple variable-interval schedules. In Experiment 1, rats were exposed to a series of simple variable-interval schedules differing in both session duration (10 min or 30 min) and scheduled reinforcement rate (7.5 s, 15 s, 30 s, and 480 s). The functions relating response rate to reinforcement rate were predominantly monotonic for the short (10-min) sessions but were predominantly bitonic for the long (30-min) sessions, when data from the entire session were considered. Examination of responding within sessions suggested that differences in the whole-session data were produced by a combination of prospective processes (i.e., processes based on events scheduled to occur later in the session) and retrospective processes (i.e., processes based on events that had already occurred in the session). In Experiment 2, rats were exposed to a modified discrimination procedure in which pellet flavor (standard or banana) predicted session duration (10 min or 30 min). All rats came to respond faster during the short (10-min) sessions than during the first 10 min of the long sessions. As in Experiment 1, the results seemed to reflect the simultaneous operation of both prospective and retrospective processes. The results shed light on the recent controversy over the form of the variable-interval response function by identifying one variable (session duration) and two types of processes (prospective and retrospective) that influence responding on these schedules.

Site-specific effects of intracerebral injections of three neurokinins (neurokinin A, neurokinin K, and neurokinin gamma) on the expression of male rat sexual behavior.

Accumulating evidence indicates that neurokinins play a role in the neural regulation of male rat copulatory behavior. We have previously reported that injections of the neurokinin substance P into the medial preoptic nucleus facilitated male rat copulatory behavior. Recently, a number of other neurokinins, neurokinin K (neuropeptide K), neurokinin A (substance K), and neurokinin gamma (derived from the same gene as substance P), have been identified in the mammalian CNS. Therefore, in a series of experiments we examined the effects on male copulatory behavior following bilateral injections of different doses of neurokinin K (NkK), neurokinin A (NkA), or neurokinin gamma (Nk gamma) into the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BnST), or the caudate/putamen. Bilateral injections of NkK into the MPOA or BnST inhibited the expression of male copulatory behavior. The most marked effect was seen following bilateral injections of 0.25 and 0.52 nmol of NkK into the MPOA and the BnST. These injections produced a dramatic suppression of copulatory behavior in previously sexually vigorous male rats when compared to control injections. In contrast, bilateral injections of three different doses of NkA into the MPOA failed to affect any parameter of male copulatory behavior. Bilateral injections of 0.431 nmol of Nk gamma into the MPOA failed to affect the expression of copulatory behavior, but significantly delayed its initiation when compared to controls. Bilateral injections of 0.251 nmol of NkK into the caudate/putamen had no significant effect on copulatory behavior in sexually vigorous male rats when compared to control injections. The results of the present study provide further support for a role of neurokinins in the regulation of copulatory behavior in male rat. Taken together, these results suggest that the effects of neurokinins upon the expression of male copulatory behavior are site specific for brain regions in the sexually dimorphic vomeronasal pathway which includes the MeA, BnST, and MPOA.

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

Direct spectrophotometry of bilirubin in serum of the newborn, with use of caffeine reagent.

We recently described a direct spectrophotometric method for unconjugated bilirubin, with caffeine reagent (Clin Chem 1986;32:1389-93). Because this method is independent of the protein matrix we used it for the preparation of bilirubin standards (Clin Chem 1987;33:1817-21). Now, in this paper, we utilize the caffeine reagent in setting up a bilirubin method for serum from neonates. This resulted in a two-wavelength (465 and 528 nm) equation, which fully corrects for HbO2 interferences. In combination with a bilirubin standard, this equation may be transformed into a simple relative formula for use with this simple dilution method. We studied this two-wavelength method with 55 neonates' sera, comparing results with those by both the diazo method of Doumas et al. (Clin Chem 1985;31:1779-89) and the borate method of Hertz et al. (Scand J Clin Lab Invest 1974;33:215-30). We found that this new method is independent of hemolysis and of the matrix of the sera. Therefore, it is very suitable for use in neonatology.

Use of the caffeine reagent in direct spectrophotometry of bilirubin.

The molar absorptivity of unconjugated bilirubin in caffeine reagent is independent of the protein matrix. This finding, together with the simplicity of a dilution step for direct spectrophotometry, will improve the calibration methods of bilirubin and make them more nearly accurate. This is encouraging for the development of a new method for bilirubin determination in neonates; moreover, the caffeine reagent has a clearing influence on the turbidity of human sera. These findings should also be important for standardization, especially because the method of Jendrassik and Gróf is also protein-independent. Therefore, the introduction of one reliable, inexpensive, "universal" standard of bilirubin in bovine serum albumin will be of importance for both methods.

Direct potentiometric determination of sodium ion in blood. III. Influence of (bi)carbonate.

We measured the emf of a sodium ion-selective electrode in NaCl-NaHCO3 solutions (160 mmol of Na+, 160-120 mmol of Cl-, and 0-40 mmol HCO3- per liter) with a home-built cell in steady-state after 10 min and with two commercial direct potentiometric analyzers about 20 s after the sample was introduced. Substitution of HCO3- for Cl- resulted in a small decrease in emf. We calculated the effect of chloride ion replacement by bicarbonate ion on the liquid-junction potential and found that this accounted only for one-third of the emf decrease. The exact composition of bicarbonate solutions is closely related to the pH. To control the formation of carbonate, we performed measurements with the home-built cell at two pH values (7.0 and 8.2), controlled by tonometry with carbon dioxide. From the differences in the slope of the emf vs the amount of bicarbonate added at the two pHs we calculated the thermodynamic association constants of the sodium bicarbonate and sodium carbonate complexes (KNaHCO3(0) = 0.53 and KNaCO3- = 24). We conclude that, for the direct potentiometric measurement of sodium ion, only fresh serum should be used, to avoid pH changes. Replacement of sodium chloride by sodium bicarbonate caused different results when emf was measured with commercial analyzers, owing to their short measuring time of about 20 s.

Direct potentiometric determination of sodium ion in blood. II. Influence of cations.

We measured the emf of NaCl solutions (120-160 mmol/L), with and without the addition of KCl (5-20 mmol/L), CaCl2 (2-8 mmol/L), or MgCl2 (1-4 mmol/L). Measurements were made with a home-built cell in steady-state and with two commercial direct potentiometric analyzers about 20 s after the sample was introduced. We calculated the sodium ion activity in the mixed NaCl-KCl solutions according to different thermodynamic theories and found almost the same results. We conclude that the influence on the emf of physiological concentrations of these cations was negligible when emf measurements were made in steady-state with the home-built cell. Of the three added cations, K+ caused the greatest increase in apparent sodium ion activity (up to about 4%) when emf measurements were made with commercial analyzers, owing to low salt-bridge concentration and the short measuring time of about 20 s.

Direct potentiometric determination of sodium ion in blood. I. Potentiometric response in simple sodium chloride solutions.

We measured the emf of NaCl solutions (120-160 mmol/L) with a home-built cell in steady-state and with some commercial direct potentiometric analyzers about 20 s after the sample is introduced into the instrument. We compared the results with the theoretical sodium ion activity calculated according to different thermodynamic theories. The slope of the calibration graph was calculated with and without correction for the influence of NaCl concentration on the liquid junction potential of the calomel reference electrode. We conclude that different theories used to calculate the sodium ion activity in the concentration range investigated give almost the same results; furthermore, introduction of the liquid junction potential leads to a more nernstian-like slope of the calibration graph. Measurement of identical NaCl solutions with various commercial analyzers showed different displayed concentrations, presumably because of differences in junction structure, measuring time, and concentration of calibration solutions.