Do GnRH neurons express the gene for the NMDA receptor?

BRAIN RESEARCH ELSEVIER

Brain Research 690 (1995) 117-120

Short communication

Do GnRH neurons express the gene for the N M D A receptor? Rula Abbud

*'~,

M. Susan Smith 2

Department of Physiology, School of Medicine, UniL'ersity of Pittsburgh, Pittsburgh, PA 15261, USA Accepted 16 May 1995

Abstract Previous studies have revealed that in several animal models, N-methyl-D,L-Aspartate (NMA) stimulates LH secretion by acting at a suprapituitary site. In addition, NMDA receptor antagonists appear to block GnRH neuronal activation on the afternoon of proestrous as evidenced by the lack of c-Fos expression in the neurons and by the absence of an ovulatory LH surge. However, administration of NMA does not induce c-Fos or c-Jun expression in GnRH neurons. To better understand the effects of NMDA receptor activation on GnRH neuronal function, we examined whether GnRH neurons express the NMDA receptor in male rats, and in female rats during diestrus and proestrus, by performing double label in situ hybridization. An 35S-labeled cRNA probe for the NMDA receptor subunit (NMDAR1) was used to quantify NMDAR1 mRNA and a digoxigenin-labeled cRNA probe for GnRH was used to identify GnRH neurons. The data were quantified and expressed as grains/average cell area. In male and female rats, less than 5% of GnRH neurons expressed grain levels twice the minimum detectable level and were considered double-labeled. However, many non-GnRH neurons in the same areas as GnRH neurons expressed high levels of NMDAR1 mRNA. These results suggest that the effects of NMA on GnRH secretion are unlikely to be mediated solely by the activation of NMDA receptors on GnRH neurons. Given the widespread expression of NMDAR1 mRNA in the hypothalamus, it is possible that the stimulatory effects of NMA on GnRH neurons are indirect through activation of other neurons. Keywords: GnRH neurons; NMDA receptors; Gene expression; N-Methyl-D,L-aspartate

The evidence that N M D A receptors are involved in the regulation of GnRH neuronal function is several-fold. Firstly, N M A can stimulate LH secretion in male and female rats by stimulating GnRH release from the hypothalamus, since the LH response to N M A can be blocked by GnRH antagonists [6]. Secondly, N M A can stimulate GnRH release from the mediobasal hypothalamus and arcuate nucleus-median eminence in vitro [4,5]. Thirdly, N M A increases GnRH m R N A levels within 1 5 - 6 0 min after administration [11]. Fourthly, N M D A receptor antagonists, such as AP-5 [3], can suppress the elevated levels of LH observed in castrated male rats. Finally, administration of N M D A receptor antagonists, such as MK-801, AP-5, and AP-7, blocks the proestrus LH surge [8,13]. Recently, we reported some seemingly contradictory results [7]. On the one hand, administration of MK-801

* Corresponding author. Fax: (1) (216) 368-3395. Present address: Department of Pharmacology, Case Western Reserve University, 2109 Adelbert Rd., Cleveland, OH 44106, USA. Fax: (1) (216) 368-3395. 2 Present address: Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006, USA. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 6 1 0 - 9

blocked the induction of c-Fos in GnRH neurons and the LH surge on the afternoon of proestrus, suggesting that GnRH neuronal activation was blocked [7]. However, administration of N M A did not induce c-Fos or c-Jun expression in GnRH neurons, although it did in many other neuronal populations in the hypothalamus [2,7]. These observations led us to ask whether the activation of GnRH neurons in response to N M D A receptor activation results from direct effects on GnRH neurons or indirect effects through activation of afferent neuronal input. To address this question, we determined whether GnRH neurons express the N M D A receptor m R N A by performing double label in situ hybridization using a digoxigenin-labeled c R N A probe for GnRH and 3sS-labeled probe for the N M D A receptor subunit N M D A R 1 . Brain tissue for double label in situ hybridization was obtained from female rats on diestrus day 1 (n = 4), morning (n = 2) and afternoon ( n = 4, animals sacrificed during the LH surge) of proestrus, and male rats (n = 4). Animals were sacrificed by decapitation and the brain dissected out of the skull under RNAase-free conditions and then rapidly frozen on dry-ice. Brains were stored at - 8 0 ° C until the tissue was sectioned.

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The cRNA probes used for in situ hybridization were as follows: an 3sS-labeled cRNA probe for the NMDA receptor subunit (NMDAR1) described previously [1] (the pN60 clone for the NMDA receptor subunit NMDAR1 was kindly provided by Dr. S. Nakanishi at Kyoto University) and a digoxigenin-labeled cRNA probe for GnRH (the 387 bp cDNA was provided by Dr. James L. Roberts, Mount Sinai Medical Center, NY). The digoxigenin-labeled GnRH cRNA probe was synthesized using T3 polymerase, after linearization of the pBS ÷ plasmid with EcoRI. The transcription reaction was performed as described earlier [1] in the presence of 40% digoxigenin-ll-UTP (Boehringer Mannheim, Indianapolis, IN). After DNA digestion, the reaction mixture was loaded on a Quick Spin column (Boehringer Mannheim, Indianapolis, IN) to purify the newly synthesized cRNA. Twenty micron brain sections were cut using the cryostat (CMT 955A, RMC Inc., Tucson, AZ) and thawmounted on gelatin-coated slides. The tissue was fixed in 4% paraformaldehyde, treated with acetic anhydride in TEA (triethanolamine) buffer, washed with 2 x SSC, dehydrated in a series of alcohols, delipidated in chloroform, rehydrated in alcohol, and air-dried. Rat NMDAR1 3SSlabeled cRNA probe (0.3 / z g / m l . kb) and digoxigeninlabeled GnRH cRNA probe were applied to each slide in 100/xl hybridization buffer and incubated for 8 h at 60°C. At the end of this incubation period, slides were washed twice in 4 X SSC containing DTT, treated with RNAse and washed with 0.1 × SSC at 65°C. Slides were then incubated in 2 X SSC containing 2% normal sheep serum (NSS) and 0.05% Triton X-100, overnight. On the next day, the anti-digoxigenin-alkaline phosphatase conjugate (diluted 1:1000 in buffer containing 1% NSS and 0.3%

Triton X-100) was applied to each slide and allowed to incubate for 3 h at 37°C. The color reaction was performed using nitroblue tetrazolium salt, 5-bromo-4-chloro-3-indole phosphate toluidinum salt, and levamisol, which stains GnRH neurons blue or brown. Afterwards, the slides are allowed to dry, dipped in 3% parlodion in isoamyl acetate and exposed to emulsion for 7 days. The data were analyzed using the Bioscan Optimas Image Analysis system. Each GnRH neuron (blue-brown stain) was identified under bright field using a 40X objective and the silver grains covering it were counted under dark-field epi-illumimation (Mercury light, 100W, Nikon). An average of 98 + 10 GnRH cells were analyzed per animal. In addition, the NMDAR1 signal was analyzed in other non-GnRH containing cells in the cortex and hypothalamus. The data were normalized to cell area and expressed as grains per average cell area. The minimum detectable level was considered to be 20 grains per average cell area (after subtraction of background). Cells expressing grain levels twice this level were considered to be double-labeled. The use of double-label in situ hybridization has allowed us to determine quantitatively whether GnRH neurons express the NMDA receptor. The non-radioactive digoxigenin-labeled antisense cRNA for GnRH identified GnRH neurons, while the 3sS-labeled NMDAR1 probe permitted the quantification of the NMDA receptor signal. Fig. 1 shows an area of the hypothalamus containing GnRH neurons. The image was obtained by double exposure of bright and dark field images, so as to show simultaneously both the NMDAR1 signal (silver grains; Fig. 1) and the GnRH signal (dark stain, arrows; Fig. 1). The NMDAR1 signal was very abundant in the hypothala-

Fig. 1. Double label in-situ hybridization for NMDAR1 and GnRH mRNAs. The photomicrograph of the hypothalamus was obtained from a female rat on the afternoon of proestrus. Five GnRH neurons (arrows) and many NMDAR1 containing neurons (silver grains) could be observed. Note the row level of silver grains associated with GnRH neurons. Scale bar is 50 /.*m.

R. Abbud, M.S. Smith/Brain Research 690 (1995) 117-I20

mus. The amount of grains overlying the GnRH neurons was quantified and compared to background levels to determine whether a neuron was double-labeled. In addition, grain counts were obtained for non-GnRH containing neurons in the hypothalamus and cortex to compare the NMDAR1 signal in these neurons with that in GnRH neurons. An example of such neurons in the hypothalamus is shown in Fig. 2. Fig. 3 summarizes the data for the male and for the female on diestrus and afternoon of proestrus (expressed as average grains per average cell area). The average NMDAR1 signal in GnRH neurons was negligible when compared to the signal in other hypothalamic neurons or to neurons in the cortex. In fact, in both male and female rats, fewer than 5% of GnRH neurons were considered to be double-labeled (i.e. expressed 40 grains per average cell area or more). In the non-GnRH neurons, NMDAR1 mRNA expression averaged 51 +__3 grains/average cell in the OVLT-MPOA area and 88 + 10 grains/average cell area in the cortex. Because of the very low level of NMDAR1 expression in GnRH neurons, it was difficult to determine whether there were any differences among the groups examined. These data suggest that the NMDAR1 signal in GnRH neurons is extremely small. In the vast majority of neurons ( > 95%), the NMDARI signal was at or near background levels. The very small number of GnRH neurons that appeared to be double-labeled in either male or female rats ( < 5%) may possibly be false-positive determinations. This could occur if a high level of silver grains associated with a non-GnRH neuron were in very close proximity to a GnRH neuron, a condition occurring very frequently in the hypothalamus. In this study, we chose to examine the expression of the

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Fig. 3. Quantitative analysis of the NMDAR 1 mRNA signal, expressed in terms of average grains per average cell area, in GnRH neurons (open bar) and in other hypothalamic (hatched bar) and cortical (solid bar) neurons. Data is shown for male and female (during diestrus and afternoon of proestrus) rats.

mRNA for the NMDA receptor subunit NMDAR1, although there are many other subunits for the NMDA receptor [10]. The choice of NMDAR1 was important since its presence is mandatory for the other subunits to be able to form a functional NMDA receptor. In addition, none of the subunits cloned so far are expressed in the hypothalamus, except for NMDAR2D. The observations from our studies are in sharp contrast to recent studies in the GnRH neuronal cell line (GT-1, derived from a transgenically induced mouse tumor). This cell line expresses NMDAR1 transcripts and responds to NMDA receptor activation [9,12]. There are several possible explanations for the differences between the studies. First, the presence of NMDA receptors in the GnRH neuronal cell line may reveal an interesting phenotype that may be unique to GnRH neurons at a specific stage during development. It is possible that NMDA receptor expres-

Fig. 2. NMDAR1 gene expression in neurons of the anterior hypothalamus that do not express GnRH. In this region of the hypothalamus, there are no GnRH neurons. The NMDAR1 signal appears as clusters of silver grains in individual neurons. Scale bar is 100 /xm.

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sion occurs in GnRH neurons at a certain stage during embryonic development and becomes suppressed as GnRH neurons become differentiated. Second, there may be a species difference between the mouse (source of the neuronal cell line) and the rat. Finally, GnRH neurons may express extremely low levels of NMDAR mRNA compared to other neurons, levels too low to be detected by in situ hybridization. From previous studies, it is clear that the NMDA receptor-mediated signal is required for GnRH neuronal activation [3,6,8,13]. In view of the low level of NMDAR1 gene expression in GnRH neurons in adult male and female rats, it is unlikely that direct effects of NMDA on GnRH neurons account for the stimulation of GnRH release. These data suggest that the effects of N M D A on GnRH release are mediated by neurons which express the N M D A receptor and have projections on GnRH neurons.

Acknowledgements The authors wish to thank Dr S. Nakanishi at Kyoto University for providing the NMDAR1 cDNA and Dr. James L. Roberts at the Mount Sinai Medical Center for providing the GnRH cDNA. This work was supported by NIH Grant HD14643. Special thanks to Dr. Gloria Hoffman, Dr. Barbara Attardi, Hui-Ju Wang, Deborah Hollingshead, Carolyn Phalin and Thomas C. Waters for providing technical advice and support.

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[2] Abbud, R., Lee, W.-S., Smith M.S. and Hoffman, G.E.. Expression of cJun protein in LHRH neurons: similarity to the pattern of cFos activation, 74th Annual Endocrine SocieO,, San Antonio, 1992. [3] Arslan, M., Pohl, C. and Plant, T., DL-2-amino-5-phosphonopentanoic acid, a specific N-methyl-~aspartic acid receptor antagonist, suppresses pulsatile LH release in the rat, Neuroendocrinology, 47 (1988) 465-468. [4] Bourguignon, J-P., Gerard, A., Debougnoux, G., Rose, J. and Franchimont, P., Pulsatile release of gonadotropin-releasing hormone (GnRH) from the rat hypothalamus in vitro: calcium and glucose dependency and inhibition by superactive GnRH analogs, Endocrinology, 121 (1987) 993-999. [5] Bourguignon, J-P., Gerard, A. and Franchimont, P., Direct activation of gonadotropin-releasing hormone secretion through different receptors to neuroexcitatory amino acids, Neuroendocrinology, 49 (1989) 402-408. [6] Brann, D.W. and Mahesh, V.B., Excitatory amino acid neurotransmission: evidence for a role in neuroendocrine regulation, Trends Endocrinol. Metab., 3 (1992) 122-126. [7] Lee, W-S., Abbud, R., Hoffman, G.E. and Smith, M.S., Effects of NMDA receptor activation on cFos expression in LHRH neurons in female rats, Endocrinology, 133 (1993) 2248-2254. [8] Lopez, F.J., Donoso, A.O. and Negro-Vilar, A., Endogenous excitatory amino acid neurotransmission regulates the estradiol-induced LH surge in ovariectomized rats, Endocrinology, 126 (1990) 17711773. [9] Mahachoklertwattana, P., Sanchez J., Kaplan, S.L. and Grumbach, M.M., NMDA receptors mediate the release of GnRH by NMDA in a hypothalamic GnRH neuronal cell line (GT 1-1), Endocrinology, 134 (1994) 1023-1030. [10] Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev, N., Sakmann, B. and Seeburg, P.H., Heteromeric NMDA receptors: molecular and functional distinction of subtypes, Science, 256 (1992) 1217-1221. [11] Petersen, S.L., McCrone, S., Keller, M. and Gardner, E., Rapid increase in LHRH mRNA levels following NMDA, Endocrinology, 129 (1991) 1679-1681. [12] Spergel, D.J., Krsmanovic, L.Z., Stojilkovic, S.S. and Catt, K.J., Glutamate modulates [Ca2 + ] and gonadotropin-releasing hormone secretion in immortalized hypothalamic GT1-7 neurons, Neuroendocrinology, 59 (1994) 309-317. [13] Urbanski, H.F. and Ojeda, S.R., A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty, Endocrinology, 126 (1990)1774-1776.