| Summary: | 博士 === 國立陽明大學 === 生理學研究所 === 89 === Abstract
It is well known that dopamine acting through D2-like autoreceptors plays an important role in the regulation of midbrain dopaminergic (DA) neuronal activity. Whether a similar type of autoreceptor exists on tuberoinfundibular (TI) DA neurons and what roles it may possess have not been ascertained. We have previously shown that dopamine and 7-OH-DPAT, a D3 agonist, exhibit potent inhibitory effects (74.2% and 82.5%, respectively) on the firing rates of neurons recorded in dorsomedial arcuate nucleus (dmARN) in rat brain slices, where the TIDA neurons reside. Moreover, intracerebroventricular (icv) injection of either dopamine or 7-OH-DPAT, a presumed D3 receptor agonist, inhibits the TIDA neuronal activity as determined by neurochemical indices. To further explore whether the D2-like autoreceptors exists on TIDA neurons and what roles it may possessed, more specific agonists and antagonists against D2 or D3 receptors as well as antisense oligodeoxynucleotides (ODNs) against these receptor mRNAs were used in the study. Both electrophysiological activity of dmARN neurons in brain slices and neurochemical activity of TIDA neurons were determined.
Adult Sprague-Dawley rats ovariectomized (OVX) for 1 week and implanted with capsules containing estradiol-17b (E2, 150 mg /ml corn oil) for another 6 days were used. Fresh brain slices were prepared from these rats and used for extracellular single-unit recording. The dmARN neurons were first identified by their inhibitory responses to dopamine and then tested with PHNO and/or PD128907, selective D2 and D3 agonists, respectively. PD128907 in 5-50 nmole doses significantly inhibited the majority of dopamine-responsive dmARN neurons (86.3% of 44 units). Moreover, PHNO in 5-25 nmole doses inhibited all dopamine-responsive neurons tested (100% of 34 units). The inhibitory effects of PHNO and PD128907 were not only prominent; but also persisted in low Ca2+, high Mg2+ medium, indicating that they were acting directly on the recorded neuron. Pretreatment of either raclopride or U99194A, D2 and D3 receptor antagonist respectively, reversed the effects of dopamine in a few trials. In contrast, SKF81297, a D1 receptor agonist, induced variable responses in dmARN neurons. These results clearly indicate that dopamine may act through D2 and/or D3 receptors to exhibit an inhibitory effect on neurons in dmARN.
To further delineate the dopamine receptor types involved, we used OVX+E2-primed sprague-dawley rats pretreated with antisense ODN (10 mg/3 ml, icv) against D2 or D3 receptor mRNA for 2 days for brain slices preparation. Both artificial cerebrospinal fluid (aCSF)-, random antisense ODN-, and sense ODN-pretreated rats were used as controls. Dopamine (5-50 nmole) inhibited a majority of dmARN neurons in slices prepared from rats pretreated with aCSF (71.4% of 35 units), random antisense ODN-, and sense ODN for D2 (67.6%, n=34), D3 (59.5%, n=42), or D2 plus D3 (60.5%, n=38) mRNAs. In contrast, dopamine only inhibited 43.6% (n=39) of dmARN neurons in slices prepared from D2 antisense ODN-pretreated rats, and 38.5% (n=39) of dmARN neurons in slices prepared from D3 antisense ODN-pretreated rats. Furthermore, in slices prepared from rats pretreated with combination of D2 and D3 antisense ODNs, dopamine only inhibited 18.4% (n=38) of dmARN neurons, indicating that both D2 and D3 receptors may indeed be involved in the action of dopamine on dmARN neurons in ovariectomized, estrogen-treated rats.
We also calculated and compiled the basal firing rates of dopamine-responsive dmARN neurons in brain slices recorded from 1100 to 1900 h (n=83). The firing rates of neurons recorded within an hour were averaged and plotted. A significant drop in the firing rates was observed from 2.3 Hz in average at 1100-1300 h to 1.19 Hz in average from 1400 to 1900 h. A significant example of dmARN was continuously recorded from 1100 to 2000 h. Not only a significant drop of its firing rate was observed from 1300 to 1500 h, its firing was also inhibited by dopamine and PHNO, both in normal aCSF-and in low Ca2+, high Mg2+ aCSF-perifused media. In contrast, the firing rate compiled from dopamine stimulating and dopamine non-responsive dmARN neurons show no significant change from 1100 to 1900 h (2.06 Hz in average from 1100-1300 h to 1.63 Hz in average from 1300-1900 h) (N=41). This is the first study reporting the existence of a diurnal rhythm of the firing rates of dmARN neurons, which is similar to the rhythmic change of TIDA neuronal activity in vivo.
A diurnal change in TIDA neuronal activity, i.e., high in the morning and low in the afternoon, is essential for the estrogen-induced prolactin (PRL) surge in female rats. In adult OVX+E2-prime rats, pretreatment with an antisense ODN (10 mg/3 ml, icv) against either D2 or D3 receptor mRNA for 2 days had no significant effect on basal TIDA neuronal activity in the morning. Nevertheless, the treatment significantly elevated the lowered levels of TIDA neuronal activity in the afternoon as determined either by 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence and inhibited the PRL surge. Moreover, icv injection of raclopride, U99194A or S14297 (0.1-10 mg/3 ml), D2 or D3 receptor antagonist, at 1200 h also significantly elevated the lowered levels of TIDA neuronal activity in the afternoon. Icv injection of PHNO and PD128907 in the morning inhibited the TIDA neuronal activity and stimulated PRL secretion in a time- and dose-dependent manner. Furthermore, the inhibitory effect of PD128907 could be blocked by co-administration of U99194A, but not by raclopride.
Our findings are summarized below
1.Both D2 and D3 receptors are involved in the action of dopamine on dmARN neurons in brain slices obtained from OVX+E2 rats.
2.Spontaneous firing rates of dopamine-responsive dmARN in brain slices may possess a diurnal rhythm just as the TIDA neurons do in female rats.
3.Both PHNO and PD128907, D2 and D3 agonist respectively, can have an inhibitory effect on TIDA neuronal activity and stimulate PRL secretion. They may act on specific receptors.
4.An endogenous DA tone acting on D2 and D3 receptors may be involved in the diurnal changes of TIDA neuronal activity and serum PRL secretion in OVX+E2 rats.
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