Quantitative immunocytochemistry of hypothalamic and pituitary hormones: validation of an automated, computerized image analysis system.

A limiting factor in the use of immunocytochemistry in experimental endocrine studies has been the lack of a suitable procedure for quantification of immunoreactive hormones. The objective of the present study was the development of an automated, computerized image analysis system adapted to the quantitative analysis of light microscopic immunocytochemical reaction product. Reaction conditions that result in optimum, standardized, and quantitatively linear development of reaction deposit are described for H2O2 and diaminobenzidine concentrations, antiserum dilutions, and substrate incubation times. In addition, evaluation techniques, including the use of a standard control section to monitor variance and incorporate it into the statistical analysis of the results are documented. For each of the reaction variables, the immunostaining was linear over the range of specific staining. When the optimum conditions were exceeded, marked over-estimations of hormone levels occurred due to the detection of nonspecific background features reaching the detection threshold. Application of this quantitative immunocytochemical (QICC) method to the analysis of variations in hypothalamic and pituitary hormone levels was validated by comparing values obtained with QICC to those with radioimmunoassay (RIA). The relative changes in both hypothalamic gonadotropin-releasing hormone and pituitary luteinizing hormone induced by manipulation of gonadal steroid levels, as measured by RIA and QICC, were highly correlated. Two-way analysis of variance revealed that the two techniques were not significantly different in their detection of changes in either hormone. Thus, under optimally defined conditions, quantitative immunocytochemistry using computerized image analysis has been validated for the accurate measurement of pituitary and brain hormones in precise regions.

Left ventricular systolic torsion and early diastolic filling by echocardiography in normal humans.

This study describes a novel 2-dimensional echocardiographic technique to measure left ventricular (LV) systolic twist in humans and relates this measure to early ventricular filling. LV twist is the counterclockwise rotation of the left ventricle during systole when viewed from the apex. The effect of ventricular twist has been postulated to store potential energy, which ultimately aids in diastolic recoil, leading to ventricular suction. The generated negative early diastolic pressures may augment early ventricular filling. We measured ventricular twist in 40 patients with normal transthoracic echocardiograms. End-systolic twist was determined by measuring rotation of the anterolateral papillary muscle about the center of the ventricle. LV filling was assessed by analysis of transmitral Doppler flow velocities. The mean value obtained was 9 +/- 7 degrees of rotation. Twist measurements were highly reproducible with an intraobserver correlation coefficient of r = 0.881, p <0.001. The magnitude of ventricular twist was strongly correlated positively with acceleration of the mitral E-wave (r = 0.75; p <0.0001) and negatively with the mitral E-wave acceleration time (r = -0.83; p <0.0001).

Neuronal responsiveness to gonadotropin-releasing hormone and its correlation with sexual receptivity in the rat.

In the present study, an attempt was made to correlate the neuronal responsiveness of individual preoptic-septal (POA/S) units to iontophoretically applied GnRH with the onset of sexual receptivity. In both behavioral and electrophysiological studies, ovariectomized, estrogen-primed rats were used. In behaviorally tested rats, lordosis quotients (LQ) were determined at varying times following progesterone (P) injection. For electrophysiological studies, P was given 1 hr after the start of recording. GnRH was iontophoretically applied for 30 sec at 16 nA on spontaneously discharging cells. A unit was deemed excited or inhibited if a repeatable 30% change in discharge rate was observed. From 2-10 hours as the LQ increased from 17 to 90 the total number of GnRH sensitive cells did also. The majority of responsive cells were excited by the peptide. As receptivity displayed a sharp increase from 2 to 6 hours the mean responsiveness of cells excited by GnRH was significantly elevated over inhibitory responses. These findings confirm the E/P biasing effect on POA/S unit responses to GnRH. Moreover, they suggest that a dynamic relationship exists between GnRH responses at the cellular level and sexual behavior throughout the course of steroid-induced receptivity.

Gonadotropin-releasing hormone within the organum vasculosum lamina terminalis in the ovariectomized, estrogen/progesterone-treated rat: a quantitative immunocytochemical study using image analysis.

No specific function has been ascribed to the high gonadotropin-releasing hormone (GnRH) content of the organum vasculosum lamina terminalis (OVLT). The objective of this study was to determine whether levels of GnRH within the OVLT are altered during cyclic gonadotropin secretion. GnRH levels were determined at various times during an estrogen/progesterone (E/P)-induced LH surge. Groups of E/P and sesame oil-treated animals were decapitated at 12.00 h, 14.00 h, 16.00 h, 18.00 h, and 22.00 h following the P or oil treatment. Morphological localization, as well as quantitation of immunoreactive GnRH within discrete regions of the brain was achieved by combining unlabeled antibody immunocytochemistry with computerized image analysis. Analysis of GnRH levels in the OVLT revealed that at any of the 5 times examined, there was: (1) no significant difference among controls, (2) no significant difference among E/P-treated animals, and (3) no significant difference between E/P-treated versus control animals. In contrast, ME GnRH levels in E/P-treated rats showed the expected decrease prior to the onset of the LH surge. These findings suggest that the level of GnRH detected in axon terminals within the OVLT cannot be related directly to the serum LH status of ovariectomized, E/P-treated rats. It is therefore possible that GnRH within the OVLT might function in a neuromodulatory role, rather than as a direct regulator of cyclic gonadotropin secretion.

Sexual responsiveness and its relationship to vaginal stimulation-produced analgesia in the rat.

Increasing amounts of pressure applied to the cervix produce a dose-response-like elevation of pain threshold in rats. This vaginal stimulation-produced analgesia (VSPA) is facilitated in animals given estrogen (E) doses sufficient to induce high levels of sexual receptivity. It has been proposed that enhancement of VSPA may serve to decrease any noxious input associated with multiple intromissions by the male. In this study, the anti-nociceptive effect of VSPA was compared in animals given E doses insufficient to increase receptivity with animals made receptive using subthreshold E levels + progesterone (P) in an attempt to determine if enhancement of VSPA is associated with the receptive state of the animal or the dose of E used. Tail flick latencies and tail shock vocalization thresholds were measured in groups of E, E + P and oil-treated rats during application of 0, 100 and 200 g of force on the cervix. Within oil, E and E + P-treated animals, significant increases in tail flick latencies were observed at 100 and 200 g with respect to baseline (0 g). Moreover, at 100 g of force E treated animals displayed a significant increase in tail flick latency over oil and E + P treated rats. In contrast, tail shock vocalization was increased at 100 and 200 g levels of probing in oil and E + P groups but was not facilitated by E. In the present study, as in previous work, VSPA was potentiated by E; however, this potentiation was not correlated with steroid-induced receptivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical stimulation of the rat ventral midbrain elicits antinociception via the dorsolateral funiculus.

The pain-suppressive effects of focal electrical stimulation of sites throughout the ventral midbrain were examined in awake rats. Chronic bipolar electrodes were implanted in medial and lateral regions of the midbrain. Current thresholds for suppression of the tail-flick reflex in response to noxious heat were determined for both a biphasic and a monophasic stimulation parameter at each site. Stimulation of areas throughout the ventral midbrain produced tail-flick suppression (TFS), but no one area was consistently effective in all animals. Monophasic and biphasic stimulation were qualitatively equal in the duration of TFS and the distribution of effective sites. The production of TFS was not correlated with other behavioral reactions to brain stimulation. TFS appeared to be mediated by non-opiate pathways since naloxone administration (10 mg/kg) had no discernible effect on the production of TFS. The current threshold for producing TFS was extremely variable over both short (one half hour) and long (one week) intervals. The incidence of TFS from previously effective sites was significantly less following bilateral dorsolateral funiculus (DLF) lesions, indicating that the antinociceptive effects of ventral midbrain stimulation are mediated by this spinal pathway.

Phorbol esters and forskolin infused into midbrain central gray facilitate lordosis.

Several peptides, when infused into the MCG, facilitate lordosis in estrogen-primed female rats. Since these peptides can act through cAMP and/or protein kinase C, and these second messenger systems have been implicated in neuromodulation, this study examined if pharmacological agents which stimulate these systems would facilitate lordosis. Ovariectomized female Fisher rats were given bilateral cranial cannulae targeted to the MCG, or cortex dorsal to MCG, and allowed at least a week to recover. Forty-eight hours after injection of 1.25 micrograms estradiol benzoate (EB), 1 microliter of each of the following was infused into the MCG (n=8-12): 1) forskolin (5 micrograms/microliter 50% DMSO); 2) phorbol-20-oxo-20-deoxy-12,13-dibutyrate (PBu; 5 micrograms/microliter 50% DMSO); 3) both forskolin and PBu (2.5 micrograms of each/microliter); 4) vehicle (50% DMSO). In a separate study of identical design, 1 microliter of another phorbol ester (12-myristate 13-acetate) was infused into the MCG of EB-primed rats. Forskolin and phorbol esters each facilitated lordosis maximally at 60-90 minutes after infusion. Combining both agents also facilitated lordosis, and vehicle had no effect. These results suggest that infusing agents which stimulate cAMP and protein kinase C into the MCG can facilitate lordosis in estrogen-primed female rats.

Reversible disruption of lordosis via midbrain infusions of procaine and tetrodotoxin.

Behavioral effects of bilateral intracranial infusions of tetrodotoxin (1, 3.3 or 10 ng/rat), 50% procaine (2 microliters/rat) or phosphate-buffered saline (PBS-2 microliters/rat) into the dorsal midbrain of conscious, lightly-restrained female rats were evaluated. High levels of lordotic responsiveness were induced in ovariectomized animals treated with estradiol (E2) capsules or subcutaneous injections of estradiol benzoate (EB) followed by progesterone (P). The effect of each of the 3 infusates on lordosis was determined using manual stimulation and lordosis quotient determinations. In addition, the vocalization by an animal during lordosis measurements, paw withdrawal to pinch, righting reflex latency and recognition of a platform edge were also monitored. Within 2 minutes following procaine or tetrodotoxin (TTX) infusions in E2 implanted rats, lordotic responsiveness declined sharply. Whereas procaine-treated animals returned to control levels of responsiveness within 20 minutes, TTX infusions induced a more prolonged depression of lordosis lasting up to 8 hours. Infusions of PBS had no effect on any of the behaviors. In a separate group of animals treated with either E2 or EB + P and infused with 10 ng TTX the time course of the decline in lordotic responsiveness was identical for both steroid treatments. Paw withdrawal was unaffected by TTX while all other measured behaviors were disrupted along the same time course as lordosis. Collectively the above results implicate the requirement of sodium-dependent neuronal activity within dorsal midbrain for the maintenance of the lordosis reflex, along with other behavioral responses influenced by this brain region.

LHRH messenger RNA in neurons in the intact and castrate male rat forebrain, studied by in situ hybridization.

The purpose of the present study was to localize luteinizing hormone-releasing hormone (LHRH) mRNA within the male rat forebrain using an in situ hybridization approach. The expression of LHRH mRNA was compared in castrate and intact males to approach questions on the chronic influences of circulating testicular steroids on the gene expression of the peptide. Frozen 10 micron sections fixed in paraformaldehyde were obtained from the forebrain region of intact and 2 week post-castrate adult male rats. LHRH mRNA was autoradiographically detected using an oligomer (59mer) complementary to the mRNA coding for amino acids -5 to 15 of the human LHRH preprohormone. Individual brain sections were incubated in prehybridization buffer for 2 h to reduce nonspecific binding. Following this, 20 microliter of hybridization buffer containing 65,000-120,000 cpm of the 59mer were applied to sections and hybridized at 37 degrees C for 3 days. The sections were then rinsed over a 48 h period, dehydrated, dipped in Kodak NTB2 liquid emulsion and exposed for 22 days. Autoradiograms were developed and counterstained with fast green and cresyl violet. As reported in the female, LHRH message-containing cells were localized in ventral septal regions, the diagonal bands of Broca, preoptic area and anterior hypothalamus. On occasion, LHRH gene expressing cells were found to appear in loose clusters. Labeled cells were never found in control sections treated with hybridization buffer lacking the 59mer. The total number of LHRH mRNA-containing cells localized in intact rats did not differ significantly from the castrate group.(ABSTRACT TRUNCATED AT 250 WORDS)