Neuroscience Letters, 113 (1990) 101-106
101
Elsevier Scientific Publishers Ireland Ltd.
NSL 06864
Differential effect of neurointermediate lobectomy on central oxytocin and vasopressin Craig A. Johnston l, Katherine D. Fagin 2 and Andr6s Negro-Vilar l 1Reproductive Neuroendocrinology Section, Laboratory of Molecuar and Integrative Neuroscience, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (U.S.A.) and 2ALZA, Palo Alto, CA (U.S.A.) (Received 28 November 1989; Revised version received 24 January 1990; Accepted 27 January 1990)
Key words: Neurointermediate lobectomy; Neurointermediate pituitary; Oxytocin; Vasopressin; Discrete brain nuclei; Median eminence Removal of the neurointermediate pituitary (NIL) affects the secretion of anterior pituitary (AP) hormones. It is not known if these effects are due to changes in central neuropeptide neurons. Two neuropeptides implicated in the neuroendocrine regulation of AP hormone secretion, and which are located in the NIL, are oxytocin and vasopressin. The present study evaluated whether removal of the NIL affected oxytocin and vasopressin concentrations in discrete brain areas containing cell bodies (paraventricular nucleus), fibers (arcuate nucleus), and/or terminals (median eminence) of these central neurons. Adult male rats underwent removal (NIL-X) or visualization (SHAM controls) of the NIL using a parapharyngeal approach. Oxytocin levels increased in the paraventricular nucleus and median eminence following NILX, whereas vasopressin concentrations were relatively unaffected by NIL-X. The data suggest that at least part of the influence of the NIL on AP hormone secretion may result from the ability of feedback from the NIL to differentially affect central neuropeptide neurons.
Recent studies have demonstrated an influence of the neurointermediate pituitary lobe (NIL) on anterior pituitary (AP) hormone secretion (see review in [6]). In particular, evidence for an altered secretion of adrenocorticotropin (ACTH [3,14]), luteinizing hormone (LH [2,4]) and prolactin (PRL [5,10,11]) secretion following surgical removal of the NIL (NIL-X) has been shown in various experimental conditions. Although an acknowledged role of the NIL in the neuroendocrine regulation of AP hormone secretion is becoming more widely accepted, the mechanisms by which this influence is exerted are poorly understood. To date, most studies have concentrated primarily on the potential direct action of factors contained within the NIL to influence AP hormone secretion. The possible feedback influence of the NIL on cen.
Correspondence: C.A. Johnston. Present address: College of Pharmacy, Department of Pharmaceutical Sciences, Wegner Hall, Room 340D, Washington State University, Pullman, WA 99164-6510, U.S.A.
102
tral peptidergic neurons has been virtually unexplored. The present study examined whether NIL-X can influence central oxytocin- and arginine vasopressin (AVP)-containing neuronal processes. These two neuropeptide systems were chosen because they terminate in the NIL and median eminence (ME [20]) and have been implicated in the neuroendocrine regulation of AP hormone secretion [7,9, ! 7]. Male Sprague-Dawley rats (280-320 g BW; Charles River, Raleigh, NC) were housed in a temperature- (22+ I°C) and light- (lights on:8.00-20.00 h) controlled room. Following surgery, animals were housed individually, and were supplied with food and water ad libitum. Water intake and BW were monitored daily. Animals receiving NIL-X operations all demonstrated a significant elevation in daily water intake confirming removal of the NIL. NIL-X rats drank significantly more than sham-operated animals (2-3-fold more/day than the sham average of 22 + 2 to 31 + 3 ml/day) from day 3 to day 11 following surgery (performed on day I). The maximum NIL-X-induced increase in water intake occurred on day 5 (approximately 4-fold v. sham intake). No significant differences in body weight were observed between sham and NIL-X animals during the 13 days following surgery (data not shown). Following decapitation, the pituitary glands were examined in situ from the dorsal aspect under 15 X magnification for completeness of NIL removal and color indication of the remaining AP. Rats exhibiting AP tissue of less than the normal pink color, indicating infarction, were excluded from the studies. NIL-X or visualization (SHAM) of the NIL was performed 13 days prior to decapitation via a parapharyngeal exposure of the pituitary glands in rats as previously described [2] under halothane anesthesia in 95% O2/5%CO2. In NIL-X animals, the body of the posterior pituitary with the adhering intermediate lobe was removed, while the neural stalk stump of approximately 0.24).4 mm in length consistently remained within the sella. The skin incision was swabbed with polysporin ointment (Polymyxcin B/Bacitracin, Burroughs Welcome Co., Research Triangle Park, NC) and closed with a wound clip. Following decapitation, the brains were quickly removed and the ME was dissected from the hypothalamus. The remaining brain was cut caudal to the hypothalamus and the anterior portion was frozen on dry ice until it was sliced on a cryostat microtome. The paraventricular and arcuate nuclei were dissected using the Palkovits punch technique [13]. Each tissue was homogenized in 100 pl of 1.0 N acetic acid and boiled for 10 min to inactivate degradative enzymes. The homogenates were centrifuged at 10000 x g at 4°C to precipitate protein. The supernatants were removed and frozen at -70°C until assayed for oxytocin and AVP content using specific radioimmunoassays as previously described [12,16]. The protein content of the pellets resulting from the centrifugation step was analyzed using a modification of the method of Lowry et al. [8]. The effects of NIL-X on the concentration of oxytocin in the paraventricular nucleus (left panel), arcuate nucleus (central panel), and ME (right panel) are shown in Fig. 1. Oxytocin content doubled in the paraventricular nucleus and nearly tripled in the ME 13 days following NIL-X. The concentration of oxytocin in the arcuate nucleus was not altered.
103
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F i g . 1. Effect of NIL-X on oxytocin concentrations in the paraventricular nucleus (left panel), arcuate nucleus (central panel), and median eminence (right panel). Each column represents the mean concentration and vertical lines indicate 1 S E M obtained from 8-9 animals 13 days following either NIL-X or sham operations. **, significantly different from sham-operated control value (p < 0.01).
The concentration of AVP in various discrete rat brain areas from animals subjected to NIL-X or sham operations is shown in Fig. 2. AVP content was significantly decreased only in the arcuate nucleus following NIL-X. The results demonstrate the first evidence that NIL-X differentially affects central peptidergic neurons, specifically central oxytocin- and AVP-containing neurons. In the past few years, evidence has been accumulating to support a potentially physiologic role for oxytocin in the neuroendocrine regulatory control of PRL [7,9], LH [7], and possibly ACTH [17] secretion from the AP. Therefore, the NIL-X-induced changes in central oxytocin-containing neurons may be at least partly responsible for the previously reported alterations in the secretion of LH [2,4], ACTH [3,14] and PRL [5,10,11] which accompany NIL-X. The large increases in the concentration of oxytocin occurring in central cell bodyand terminal-containing areas following NIL-X suggest that synthesis and/or release may be altered by the removal of the NIL. Studies are now being directed toward determining if NIL-X alters messenger RNA levels or release from central oxytocinand/or AVP-containing neurons. The large, selective increase in oxytocin levels occurring in both the cell body (paraventricular nucleus)- and terminal (ME)-contain° ing areas following NIL-X, together with results of a previous study by Wetsel and Fernstrom [19] indirectly support the hypothesis that oxytocin synthesis may be stimulated following NIL-X. In that study the authors clearly demonstrated that hypophysectomy (which would remove the AP as well as the NIL) strongly stimulated oxytocin synthesis in the face of a modest reduction in hypothalamic AVP synthesis. Circulating levels of oxytocin in peripheral plasma have also been reported to increase following hypophysectomy [1]. These results, together with the present data,
104
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101
Elsevier Scientific Publishers Ireland Ltd.
NSL 06864
Differential effect of neurointermediate lobectomy on central oxytocin and vasopressin Craig A. Johnston l, Katherine D. Fagin 2 and Andr6s Negro-Vilar l 1Reproductive Neuroendocrinology Section, Laboratory of Molecuar and Integrative Neuroscience, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 (U.S.A.) and 2ALZA, Palo Alto, CA (U.S.A.) (Received 28 November 1989; Revised version received 24 January 1990; Accepted 27 January 1990)
Key words: Neurointermediate lobectomy; Neurointermediate pituitary; Oxytocin; Vasopressin; Discrete brain nuclei; Median eminence Removal of the neurointermediate pituitary (NIL) affects the secretion of anterior pituitary (AP) hormones. It is not known if these effects are due to changes in central neuropeptide neurons. Two neuropeptides implicated in the neuroendocrine regulation of AP hormone secretion, and which are located in the NIL, are oxytocin and vasopressin. The present study evaluated whether removal of the NIL affected oxytocin and vasopressin concentrations in discrete brain areas containing cell bodies (paraventricular nucleus), fibers (arcuate nucleus), and/or terminals (median eminence) of these central neurons. Adult male rats underwent removal (NIL-X) or visualization (SHAM controls) of the NIL using a parapharyngeal approach. Oxytocin levels increased in the paraventricular nucleus and median eminence following NILX, whereas vasopressin concentrations were relatively unaffected by NIL-X. The data suggest that at least part of the influence of the NIL on AP hormone secretion may result from the ability of feedback from the NIL to differentially affect central neuropeptide neurons.
Recent studies have demonstrated an influence of the neurointermediate pituitary lobe (NIL) on anterior pituitary (AP) hormone secretion (see review in [6]). In particular, evidence for an altered secretion of adrenocorticotropin (ACTH [3,14]), luteinizing hormone (LH [2,4]) and prolactin (PRL [5,10,11]) secretion following surgical removal of the NIL (NIL-X) has been shown in various experimental conditions. Although an acknowledged role of the NIL in the neuroendocrine regulation of AP hormone secretion is becoming more widely accepted, the mechanisms by which this influence is exerted are poorly understood. To date, most studies have concentrated primarily on the potential direct action of factors contained within the NIL to influence AP hormone secretion. The possible feedback influence of the NIL on cen.
Correspondence: C.A. Johnston. Present address: College of Pharmacy, Department of Pharmaceutical Sciences, Wegner Hall, Room 340D, Washington State University, Pullman, WA 99164-6510, U.S.A.
102
tral peptidergic neurons has been virtually unexplored. The present study examined whether NIL-X can influence central oxytocin- and arginine vasopressin (AVP)-containing neuronal processes. These two neuropeptide systems were chosen because they terminate in the NIL and median eminence (ME [20]) and have been implicated in the neuroendocrine regulation of AP hormone secretion [7,9, ! 7]. Male Sprague-Dawley rats (280-320 g BW; Charles River, Raleigh, NC) were housed in a temperature- (22+ I°C) and light- (lights on:8.00-20.00 h) controlled room. Following surgery, animals were housed individually, and were supplied with food and water ad libitum. Water intake and BW were monitored daily. Animals receiving NIL-X operations all demonstrated a significant elevation in daily water intake confirming removal of the NIL. NIL-X rats drank significantly more than sham-operated animals (2-3-fold more/day than the sham average of 22 + 2 to 31 + 3 ml/day) from day 3 to day 11 following surgery (performed on day I). The maximum NIL-X-induced increase in water intake occurred on day 5 (approximately 4-fold v. sham intake). No significant differences in body weight were observed between sham and NIL-X animals during the 13 days following surgery (data not shown). Following decapitation, the pituitary glands were examined in situ from the dorsal aspect under 15 X magnification for completeness of NIL removal and color indication of the remaining AP. Rats exhibiting AP tissue of less than the normal pink color, indicating infarction, were excluded from the studies. NIL-X or visualization (SHAM) of the NIL was performed 13 days prior to decapitation via a parapharyngeal exposure of the pituitary glands in rats as previously described [2] under halothane anesthesia in 95% O2/5%CO2. In NIL-X animals, the body of the posterior pituitary with the adhering intermediate lobe was removed, while the neural stalk stump of approximately 0.24).4 mm in length consistently remained within the sella. The skin incision was swabbed with polysporin ointment (Polymyxcin B/Bacitracin, Burroughs Welcome Co., Research Triangle Park, NC) and closed with a wound clip. Following decapitation, the brains were quickly removed and the ME was dissected from the hypothalamus. The remaining brain was cut caudal to the hypothalamus and the anterior portion was frozen on dry ice until it was sliced on a cryostat microtome. The paraventricular and arcuate nuclei were dissected using the Palkovits punch technique [13]. Each tissue was homogenized in 100 pl of 1.0 N acetic acid and boiled for 10 min to inactivate degradative enzymes. The homogenates were centrifuged at 10000 x g at 4°C to precipitate protein. The supernatants were removed and frozen at -70°C until assayed for oxytocin and AVP content using specific radioimmunoassays as previously described [12,16]. The protein content of the pellets resulting from the centrifugation step was analyzed using a modification of the method of Lowry et al. [8]. The effects of NIL-X on the concentration of oxytocin in the paraventricular nucleus (left panel), arcuate nucleus (central panel), and ME (right panel) are shown in Fig. 1. Oxytocin content doubled in the paraventricular nucleus and nearly tripled in the ME 13 days following NIL-X. The concentration of oxytocin in the arcuate nucleus was not altered.
103
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F i g . 1. Effect of NIL-X on oxytocin concentrations in the paraventricular nucleus (left panel), arcuate nucleus (central panel), and median eminence (right panel). Each column represents the mean concentration and vertical lines indicate 1 S E M obtained from 8-9 animals 13 days following either NIL-X or sham operations. **, significantly different from sham-operated control value (p < 0.01).
The concentration of AVP in various discrete rat brain areas from animals subjected to NIL-X or sham operations is shown in Fig. 2. AVP content was significantly decreased only in the arcuate nucleus following NIL-X. The results demonstrate the first evidence that NIL-X differentially affects central peptidergic neurons, specifically central oxytocin- and AVP-containing neurons. In the past few years, evidence has been accumulating to support a potentially physiologic role for oxytocin in the neuroendocrine regulatory control of PRL [7,9], LH [7], and possibly ACTH [17] secretion from the AP. Therefore, the NIL-X-induced changes in central oxytocin-containing neurons may be at least partly responsible for the previously reported alterations in the secretion of LH [2,4], ACTH [3,14] and PRL [5,10,11] which accompany NIL-X. The large increases in the concentration of oxytocin occurring in central cell bodyand terminal-containing areas following NIL-X suggest that synthesis and/or release may be altered by the removal of the NIL. Studies are now being directed toward determining if NIL-X alters messenger RNA levels or release from central oxytocinand/or AVP-containing neurons. The large, selective increase in oxytocin levels occurring in both the cell body (paraventricular nucleus)- and terminal (ME)-contain° ing areas following NIL-X, together with results of a previous study by Wetsel and Fernstrom [19] indirectly support the hypothesis that oxytocin synthesis may be stimulated following NIL-X. In that study the authors clearly demonstrated that hypophysectomy (which would remove the AP as well as the NIL) strongly stimulated oxytocin synthesis in the face of a modest reduction in hypothalamic AVP synthesis. Circulating levels of oxytocin in peripheral plasma have also been reported to increase following hypophysectomy [1]. These results, together with the present data,
104
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