Etsuko Kasuya

Effects of pulsatile infusion of the GABA(A) receptor blocker bicuculline on the onset of puberty in female rhesus monkeys.

In order to test the hypothesis that GABA is an inhibitory neurotransmitter restricting the release of LHRH before puberty, we examined the effects of pulsatile infusion of the GABA(A) receptor blocker, bicuculline, on the timing of puberty. Eleven female monkeys at 14-15 months of age were implanted with a stainless steel cannula into the base of the third ventricle above the median eminence. Five monkeys received bicuculline infusion every 2 h at a dose of 1 microM with a gradual increase to 100 microM in 10 microl using a portable infusion pump. The remaining 6 monkeys received similar infusions of saline. An additional 11 colony monkeys without cannula implantation were used for controls. Results indicate that bicuculline infusion advances the timing of puberty. The age of menarche (17.8+/-0.5 months) in the bicuculline infusion animals was significantly earlier than that in the saline controls (28.2+/-2.3, P < 0.001) as well as in colony controls (30.6+/-0.9, P < 0.001). The age of first ovulation (30.5+/-3.3 months) in bicuculline-treated animals was much younger (P < 0.001) than that in both controls (44.8+/-1.8 and 44.7+/-1.2, respectively). Bicuculline also accelerated the growth curve. These results suggest that the reduction of tonic GABA inhibition of LHRH neurons advances the onset of puberty.

N-methyl D,L-aspartate induces the release of luteinizing hormone-releasing hormone in the prepubertal and pubertal female rhesus monkey as measured by in vivo push-pull perfusion in the stalk-median eminence.

The role of the excitatory amino acid glutamate, N-methyl d-aspartate (NMDA) receptor agonist, in stimulating in vivo luteinizing hormone-releasing hormone (LHRH) release in the stalk-median eminence of conscious prepubertal and pubertal female rhesus monkeys was evaluated using push-pull perfusion. In Exp 1, the effects of iv bolus injection of N-methyl d,l-aspartate (NMA) on LHRH release were examined. Injection of NMA induced an increase in LHRH release in all maturational stages of monkeys. Although the LHRH response to NMA tended to be larger in the older groups, only the duration of the LHRH response in the midpubertal group was significantly longer than that in the prepubertal group. In Exp 2, the effects of direct infusion of NMA (0.1, 1, and 100 μm) into the stalk-median eminence on LHRH release were similarly examined. NMA infusion stimulated LHRH release in pubertal monkeys, whereas it did not induce any consistent changes in LHRH release in prepubertal monkeys except for the highest dose. Thes...

A Role of γ-Amino Butyric Acid (GABA) and Glutamate in Control of Puberty in Female Rhesus Monkeys: Effect of an Antisense Oligodeoxynucleotide for GAD67 Messenger Ribonucleic Acid and MK801 on Luteinizing Hormone-Releasing Hormone Release1

Previously we have shown that γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter restricting the pubertal increase in LHRH release in juvenile monkeys, and that interfering with GABA synthesis with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD67) mRNA results in an increase in LHRH release in prepubertal monkeys. GAD67 is a catalytic enzyme that synthesizes GABA from glutamate. To further clarify the role of GABA in puberty, we examined whether the inhibition of LHRH release by GABA continues after the onset of puberty and whether input from glutamatergic neurons plays any role in the onset of puberty when GABA inhibition declines, using a push-pull perfusion method. In Study I, the effects of the AS GAD67 mRNA on LHRH release in pubertal monkeys (34.3 ± 1.5 months of age, n = 8) were examined, and the results were compared with those in prepubertal monkeys (18.5 ± 0.4 months, n = 12). Direct infusion of AS GAD67 (1 μm) into the stalk-median eminence (S-ME) for 5 h ...

Effects of ghrelin on growth hormone secretion in vivo in ruminants

Ghrelin, a novel endogenous growth hormone (GH) secretagogue, has been shown to exert very potent and specific GH-releasing activity in rats and humans. However, little is known about its GH-releasing activity and endocrine effects in domestic animals. To clarify the effect of ghrelin on GH secretion in vivo in ruminants, plasma GH responses to intra-arterial and intra-hypothalamic injections of rat ghrelin (rGhrelin) were examined in goats and cattle. The intra-arterial injection of 1 microg/kg BW of rGhrelin in ovariectomized goats failed to stimulate GH release, however, a dosage of 3 microg/kg BW significantly increased plasma GH concentrations (P<0.05). GH levels peaked at 15 min after the injection, then decreased to basal concentrations within 1 h after the injection. However, the secretory response to 3 microg/kg BW of rGhrelin was weaker than that of growth hormone-releasing hormone (GHRH) (0.25 microg/kg BW) (P<0.05). An infusion of 10 nmol of ghrelin into the medial basal hypothalamus (arcuate nucleus) significantly stimulated the release of GH in male calves (P<0.05). GH levels began to rise just after the infusions and peaked at 10 min, then decreased to the basal concentrations within 1 h after the injection. The present results show that ghrelin stimulates GH release in ruminants.

Effects of an antisense oligodeoxynucleotide for neuropeptide Y mRNA on in vivo luteinizing hormone-releasing hormone release in ovariectomized female rhesus monkeys

The present study examines the effects of an antisense oligodeoxynucleotide (AS) for human neuropeptide Y (NPY) mRNA on in vivo LHRH release using the push-pull perfusion method in female ovariectomized monkeys. After 6 h of control perfusion, 10 microM of the AS NPY was infused for 8 h, which was followed by an additional 4 h of control perfusion. As a control for AS, an oligodeoxynucleotide containing the same bases in a scrambled sequence (SC) was similarly examined. LHRH and NPY levels in perfusate samples, collected in 10-min fractions, were measured by RIA. AS NPY infusion resulted in a significant decrease in mean NPY release starting 2 h after the initiation of infusion, and continuing until shortly after the end of AS infusion (P < 0.05, n = 7). AS NPY also suppressed mean LHRH release significantly (P < 0.05, n = 7): the AS NPY-induced LHRH suppression started 2 h after the initiation of AS infusion, and continued throughout AS infusion, lasting for the entire period of the experiment. In contrast, SC NPY resulted in neither significant changes in NPY release nor LHRH release. These data suggest that NPY release in the stalk-median eminence plays an important role in the control of pulsatile release of LHRH in vivo in the rhesus monkey.

Methodology for the study of the hypothalamic-pituitary hormone secretion in cattle

Studies on the neuroregulatory mechanisms on the secretion of anterior pituitary (AP) hormones in domestic animals are important because nearly all complex physiological and metabolic processes are regulated by the AP hormones. To examine them, this article considers in vivo approaches such as the techniques of intrahypothalamic injection, intracerebroventricular injection, push-pull perfusion, and microdialysis, which have been employed in our own research group for the study in cattle. Also, in vitro approaches such as bovine AP cell culture and the AP explants superfusion system are described. This article clarifies the potential of neuroendocrine study techniques in cattle.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.

Development of a stereotaxic instrument for study of the bovine central nervous system

We have developed a bovine stereotaxic instrument in this study to be used for Holstein cattle from weanling to adult age. The bovine stereotaxic instrument is derived from the Horsley-Clarke principle. A pair of straight ear-bars and orbital ridge pieces with their carriers, head clamps, AP bars are symmetrically arranged across the head pace. For holding the heavy heads, a hard palate and a mandibular support are designed to be placed on the surface of the base frame. A new manipulator with long working distance has also been made. For ventriculography, a holder inserting an X-ray film cassette is placed just next to the AP bar. Finally, the instrument was combined together with an oil-operated lift, so that it can be adjusted to a proper height. Details of this instrument are described in this paper.

Radiotelemetry recording of electroencephalogram in piglets during rest

A wireless recording system was developed to study the electroencephalogram (EEG) in unrestrained, male Landrace piglets. Under general anesthesia, ball-tipped silver/silver chloride electrodes for EEG recording were implanted onto the dura matter of the parietal and frontal cortex of the piglets. A pair of miniature preamplifiers and transmitters was then mounted on the surface of the skull. To examine whether other bioelectrical activities interfere with the EEG measurements, an electrocardiogram (ECG) or electromyogram (EMG) of the neck was simultaneously recorded with the EEG. Next, wire electrodes for recording movement of the eyelid were implanted with EEG electrodes, and EEG and eyelid movements were simultaneously measured. Power spectral analysis using a Fast Fourier Transformation (FFT) algorithm indicates that EEG was successfully recorded in unrestrained piglets, at rest, during the daytime in the absence of interference from ECG, EMG or eyelid movements. These data indicate the feasibility of using our radiotelemetry system for measurement of EEG under these conditions.

A possible role of central serotonin in L‐tryptophan‐induced GH secretion in cattle

To clarify the role of serotonin (5-HT) in the regulatory mechanism of L-tryptophan (TRP)--induced growth hormone (GH) secretion in cattle, changes in 5-HT concentrations in the cerebrospinal fluid (CSF) in the third ventricle (3V) and GH in plasma before and after the peripheral infusion of TRP were determined simultaneously. The direct effect of TRP on GH release from the dispersed anterior pituitary cells was also assessed. A chronic cannula was placed in 3V by stereotaxic surgery, then CSF and blood were withdrawn under physiological conditions. TRP (38.5 mg/kg BW) was infused through an intravenous catheter from 12.00 to 14.00 hours and CSF and blood sampling were performed from 11.00 to 18.00 hours at 1-h intervals. The concentration of 5-HT in CSF was determined by high-performance liquid chromatography with electrochemical detection. GH, melatonin (MEL), and cortisol (CORT) concentrations were measured by radio-immunoassay and enzyme-immunoassay. Concentrations of 5-HT were increased by TRP infusion. The TRP infusion significantly increased GH release. On the other hand, TRP did not stimulate GH release from the bovine pituitary cells. MEL and CORT concentrations were not altered by TRP infusion. These results suggest that TRP induced GH release via the activation of serotonergic neurons in cattle.