Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Ede Marie Apostolakis, Ph.D., Department of Molecular and Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: edea{at}bcm.tmc.edu.
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ABSTRACT |
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INTRODUCTION |
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PACAP exerts its biological action by binding to G protein-coupled membrane receptors (4). Three receptors have been cloned including cognate PAC1 receptor and two receptors with similar high affinity for PACAP and vasoactive intestinal peptide (VIP) (VPAC1 and VPAC2 receptors) (8). All three receptors are expressed in the hypothalamus (6, 9, 10). Of interest, PAC1 consists of at least five splice variants depending on the presence or absence of either one or two 84-bp cassettes named "hip" or "hop" in the third intracellular loop (11). These splice variants are coupled to different intracellular signaling cascades including cAMP, protein kinase A (PKA) (9, 12), protein kinase C (13), phospholipase C (PLC) (8, 11), and calcium signaling (8). Moreover, PACAP can induce a cascade of intracellular signaling independent of cAMP/PKA in cultured astrocyctes (14). To elucidate the membrane-bound receptor(s) and the signaling cascade activated in the hypothalamic VMN by PACAP for PR-dependent receptivity, we undertook the present study. Here, we show that the biological effect of PACAP and P on receptivity in EB-primed rodents is mediated by the PAC1 receptor and that its expression and the expression of two PAC1 splice variants is regulated by steroids in individual PR-expressing neurons of the VMN. Finally, we report that PACAP induces behavior through the intracellular cAMP/PKA/ cAMP-regulated phosphoprotein of 32 kDa (DARPP32) pathway. Collectively, our current study supports the hypothesis that PACAP acts through the PAC1 membrane receptor to reenforce steroid receptor-dependent function in the female VMN.
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RESULTS |
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Consistent with our previous report on the dose-response curve for PACAP (7), an optimal PACAP dose of 2 ng intracerebroventricularly (icv) induced receptivity at 30120 min in EB-primed females (Fig. 1A; ANOVA, P < 0.01) compared with vehicle-, EB-, and PACAP-only controls. Likewise, PACAP given bilaterally onto the VMN (i.n., 1 ng/side) also facilitated sex behavior in EB-primed rats (Fig. 1A
, P < 0.01). In contrast to this, VIP failed to induce sex behavior regardless of dose (01.33 x 106 ng/side, i.n. or icv) and EB-priming (Fig. 1A
, data not shown for icv). A repertoire of proceptive behaviors (hopping, darting, ear wiggling) was observed in conjunction with PACAP-induced lordosis in all females whereas in its absence, rejection behaviors (kicking, biting, standing, rolling over, and running away) were noted. In all rats, VIP was associated with rejection behaviors.
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P-Induced Lordosis Is Also Inhibited by PAC1 Receptor AS
Next, we ascertained whether PAC1 receptors are important for P-facilitated receptivity. Pretreatment with AS oligo to PAC1 receptor (Fig. 1C) also blocked the facilitation of sex behavior by P in EB-primed rats compared with controls (Fig. 1C
). As expected, P induced sex behavior when EB-primed females were given RS oligos (Fig. 2C
) compared with those control animals administered vehicle-, EB-, P-, or oligo-only and compared with EB+oligos. Collectively, the findings implicate PAC1 receptors in the modulation of progestin-induced sexual behavior in rats and provide further support for the hypothesis that PAC1 is a critical requirement for receptivity.
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To verify that VPAC1 receptors do not effect receptivity, the VPAC1 receptor antagonist acetyl-His1[D-Phe2, K15, R16, L27] VIP(17)/GH-releasing factor (GFR)(827) was administered 30 min before PACAP to EB-primed females. As with the VPAC2 receptor antagonist, VPAC1 antagonism failed to inhibit PACAP induction of receptivity in EB-primed females (Fig. 2A, P > 0.01). As expected, significant lordosis was not observed with either the VPAC1 antagonist acetyl-His1[D-Phe2, K15, R16, L27] VIP(17)/GFR(827) or EB+acetyl-His1[D-Phe2, K15, R16, L27] VIP(17)/GFR(827) (Fig. 2A
). Collectively, the data support the hypothesis that PACAP-induced sex behavior is mediated by PAC1 receptors in the female VMN.
VPAC Receptor AS Oligonucleotides Do Not Attenuate PACAP-Facilitated Receptivity
To more conclusively substantiate our conclusions relative to the effects of VPAC2 and VPAC1 receptors, oligos to these receptors also were administered to females (see Ref.19 for oligo specificity). As with the inhibitors, neither VPAC2 nor VPAC1 AS nor concurrently given VPAC2 and VPAC1 AS blocked the facilitation of receptivity by PACAP in EB-primed females (Fig. 2B). As expected, lordosis was displayed by EB-primed, PACAP-treated control females [EB+PACAP (Fig. 2B
) and EB+RS+PACAP] but not by females treated with vehicle and only EB, PACAP, RS, and AS. Thus, no support for a role of VPAC receptors in PACAP-facilitated receptivity was found.
P-Induced Lordosis Is Not Inhibited by VPAC Receptor Blockade
Next, we ascertained whether VPAC receptors influenced P-facilitated receptivity. As with PACAP, neither pretreatment with VPAC2, VPAC1, nor both AS oligos (Fig. 2C) blocked the facilitation of sex behavior by P in EB-primed rats compared with controls (Fig. 2C
). Also of note, P induced sex behavior when EB-primed females were given RS oligos (Fig. 2C
) compared with those control animals administered vehicle-, EB-, P-, or oligo-only and compared with EB+oligos. Collectively, the findings implicate PAC1 receptors alone in the modulation of sexual behavior in rats and support the hypothesis that PAC-1 is a requirement for receptivity.
Steroids and PACAP Receptor mRNA Expression in the VMN
We reasoned that if the endogenous PAC1 receptor or any splice variant of it plays an important role in the modulation of sexual behavior, ovarian steroids might mediate feedback regulation of PAC1 receptor synthesis in the VMN. To test this hypothesis, we performed real-time quantitative RT-PCR (RT-qPCR) on total mRNA processed from punched-out VMN from rats and wild-type mice with whole brain tissue serving as positive control. The primers and probes used were designed to detect the N terminus for the whole PAC1 receptor and flanking nucleotides to the third intron. VMN from rats treated with EB, P, or EB+P expressed significantly high levels of PAC1 receptor mRNA relative to vehicle (control) rat tissue (Fig. 3A; P < 0.05, P <0.01, and P < 0.01, respectively). Thus, the data suggest that ovarian steroids can regulate the total levels of the PAC1 receptor mRNA in the female VMN.
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Next, we examined mRNA expression of VPAC2 and VPAC1 receptors (r) in the mouse VMN. Relative to PAC1 expression, VPAC2 and VPAC1 mRNA were significantly low (Fig. 3A, P < 0.01; not indicated in Fig). Compared with vehicle control for each of these receptors, steroids failed to change mRNA expression levels (Fig. 3A
, P > 0.05). These findings are consistent with the above behavior data and further support the hypothesis that neither VPAC2 nor VPAC1 receptors play a role in mediating the effect of PACAP on receptivity in female rodents.
Variants of PACAP Receptor and Steroids
In the brain, PAC1 receptors are expressed as 1) three variants containing one of three 28-amino acid cassettes (hip1, hop1, and hop2) in the third intracellular loop; 2) a variant with a double insert (PAC1hip1-hop1); or 3) a variant in the N-terminal [short form (sfPAC1)] (4, 8, 11). Such alternate splicing results in activation of different signaling cascades and different functional outcomes. For example, sfPAC1 only stimulates adenylate cyclase (11, 17, 21). In addition to adenylate cyclase, hop1 and hop2 variants can also activate phosphatidyl inositol, phospholipase C, protein kinase C, and/or L-type Ca channels (11, 21).
The above RT-qPCR study for PAC1 and VPAC receptors was not designed to probe for changes in specific splice variants within the total population of PAC1 receptors. To characterize changes in the expression of PAC1 splice variants, semiquantitative RT-PCR was performed using PAC1 receptor primers that detect sfPAC1 (290 bp), single-cassette insert (374 bp for Hip1, Hop1, or Hop2), and double insert (458 bp for Hip/Hop) variants. The results, as shown in Fig. 3B, indicate that two receptor isoforms [a single insert (374 bp) and sfPAC1 (290 bp)] were expressed in the VMN under all steroid conditions. No product corresponding to the double insert (458 bp) was detected (data not shown). Thus, sfPAC1 and single-cassette variant receptors (but not PAC1hip-hop1) are expressed in the female VMN regardless of steroid environment.
To verify expression and to identify the specific cassette in the single isoform population of PAC1 receptors in the female VMN, the cDNA was cut out of the gel, purified, and then incubated overnight without restriction enzymes (U) or with the enzyme (Fig. 3C). For the purified sfPAC1 product in Fig. 3C
(left panel), EB and EB+P generated bands at 170 bp with HinfI (Hin) and 251 bp with PstI (Pst), respectively, thereby confirming an enhancing effect of EB and EB+P on the expression of sfPAC1 within the total population of VMN PAC1 receptors.
Digest of the longer single cassette product with enzymes specific for the different cassettes showed less definitive results (Fig. 3C, right panel), likely due to limited sensitivity of gel analyses. No bands were detected at 257 bp for either AvaII (Ava; Hip1-specific; Fig. 3C
, right panel) or PvuII (Pvu; Hop1-specific); thus neither Hip1 nor Hop1 variants were detected in the female VMN regardless of steroid milieu. In contrast, a faint but consistent 257 fragment was generated using CelII (Cel; hop1/2 specific enzyme; Fig. 3C
, right panel) in VMN treated with EB alone and EB+P, confirming the presence of the Hop2 isoform. Hence, EB changes the ratio of PAC1hop2 expression to total PAC1 receptor mRNA expression in the VMN, raising the possibility that EB influences signaling pathways through PAC1 receptors in the VMN.
Steroids and mRNA Expression in the Arcuate Nucleus
We have previously shown that, within rodent medial basal hypothalami, PACAP is released after EB+P treatment whereas, in punched-out rat VMN, PACAP mRNA is enhanced by EB, P and EB+P (7). Because the arcuate nucleus sends long projections that surround and penetrate the VMN (34) and serves to facilitate lordosis [Ref.3 and references therein], we questioned the influence of steroids on PACAP and PACAP receptor mRNA expressions in the arcuate nucleus. Overall receptor expression in the mouse arcuate nucleus was less than that in the VMN (Fig. 3, panel D vs. panel A; note scale change). As in the VMN, P and EB+P induced significant changes in PAC1 expression in the mouse arcuate nucleus when compared with vehicle PAC1 control (Fig. 3D
, P < 0.01). Likewise, steroids had no effect on the expression of VPAC2 and VPAC1 compared with VPAC2 and VPAC1 vehicle controls (Fig. 3D
, P > 0.05). Because the expression of VPAC receptor mRNAs failed to change with steroid treatment, the data also suggest it is unlikely that VPAC receptors play a role in regulating sex behavior. In contrast, the data for PAC1 support the hypothesis that PAC1 receptor activation through long arcuate nucleus projections around and into the VMN are available for activation by extracellular release of PACAP within the VMN.
To determine whether the synthesis of PACAP by the arcuate nucleus may be available for release into the VMN and to compare the results relative to previous VMN data (7), we determined the influence of steroids on PACAP mRNA in the rat arcuate nucleus. Both EB- and P-alone but not EB+P enhanced arcuate nucleus PACAP expression (Fig. 3E, P < 0.01). These findings suggest that transcription of PACAP in rat arcuate nucleus does not change under the EB+P steroid milieu associated with lordosis, making it unlikely to be a paracrine factor associated with VMN PAC1 activation.
PACAP-Induced Sex Behavior Is Suppressed by Antagonism of cAMP Action
The third intron of the PAC1 receptor is a region widely accepted to be a determinant of the selectively of G protein coupling (20). In cells transfected with rat sfPAC1, hop1 and hop2 receptor variants, adenylate cyclase, and phospholipase C are enhanced (11, 21). Likewise in most tissue, PACAP initiates the cAMP signaling cascade through PKA (12). However, in cultured astrocytes, PACAP induces a cascade of intracellular signaling independent of cAMP-dependent protein kinase A (14). In the EB-primed VMN after P treatment, cAMP and PLC are stimulated in association with receptivity (see Fig. 7.13 in Ref.22). Therefore, we determined whether inhibition of cAMP action suppressed PACAP-induced receptivity by administering the competitive Rp isomer of cAMP (Rp-cAMP) (23) 30 min before agonist challenge with either P or PACAP in EB-primed rats. Of significance, Rp-cAMP pretreatment blocked PACAP-induced receptivity (Fig. 4A). As expected, control treatments (vehicle-, EB-, P-, PACAP-, and Rp-cAMP-alone, and EB+Rp-cAMP) failed to facilitate lordosis whereas EB+P and EB+PACA did. Thus, the data suggest that permissive levels of cAMP-dependent target proteins are essential to both P- and PACAP-facilitated receptivity.
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DARPP32 AS Suppresses PACAP-Induced Lordosis
DARPP32, a key regulator of PKA kinase-phosphatase signaling cascades modulated by dopaminergic, serotonergic, and glutamatergic neurotransmission (25), is known to mediate P-facilitated sex behavior in female rodents (26). Therefore, we tested the effect of DARPP32 AS on PACAP-induced behavior in EB-primed females by administering DARPP32 oligos (4 nM i.n.) concurrently with EB and 24 h later. When challenged with PACAP (at 44 h after EB), females given AS to DARPP32 AS demonstrated significantly decreased lordosis (Fig. 4C). This was in contrast to those EB-primed females treated with either PACAP or RS+PACAP (Fig. 4C
). As expected for negative controls, neither vehicle-, EB-, PACAP-, nor oligo-only, or EB+oligo facilitated lordosis. Together with that for cAMP and PKA, the data provide further support for the hypothesis that the cAMP/PKA pathway is essential for reenforcement of receptivity by PACAP.
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DISCUSSION |
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In the present study, we examined the role of PACAP receptors in the female VMN. Because PACAP acts through membrane receptors that also bind VIP, we first determined whether VIP stimulated receptivity in females. Previous studies demonstrated that PACAP and VIP serve similar biological functions including that of stimulating the release of prolactin and other pituitary hormones (10). Other studies (34, 35) have shown that VIP conveys time of day from the suprachiasmic nucleus to neurons synthesizing GnRH in the medial preoptic area through activation of VPAC2 receptors. Herein, we show that, when administered directly onto the VMN, PACAP but not VIP facilitated sex behavior (and proceptivity) in rats primed with EB. Therefore, it is not surprising that, compared with wild-type counterparts, PACAP null female mice displayed a significant reduction in mating frequency (as measured by the presence of vaginal plugs) with no remarkable changes in the estrous cycle (as measured by vaginal cytology) (36). Although, PACAP null mice have impaired lipid and glucose metabolism (37, 38), which may contribute to their reproductive impairments (39), the present findings for VMN PACAP and VIP do not exclude the possibility that PACAP and VIP may act together to induce an array of coordinated functions that ultimately could influence select aspects of reproduction.
The biological membrane effects of PACAP are mediated by seven-transmembrane receptors coupled to G proteins; PACAP receptors are members of the secretin/glucagons subfamily of receptors (40). Both PACAP and VIP have equal affinity for hypothalamic VPAC1 and VPAC2 receptors and stimulate adenylate cyclase with equal potency. Here, EB-primed females treated with PACAP displayed positive lordosis responses in spite of pretreatment with antagonists or AS to VPAC1 and/or VPAC2 receptors. Because the EB-primed females, pretreated with RS icv or directly on the VMN, exhibited sex behavior when challenged with PACAP, the effects of the AS oligos were specific for the respective VPAC receptor in the VMN. Our data suggest that neither VPAC1 nor VPAC2 receptors mediate the effect of PACAP on sex behavior. Likewise, AS oligonucleotides to VPAC1 and/or VPAC2 receptors failed to inhibit P-facilitated lordosis, thus providing additional evidence that the two VPAC receptors have little if any role in mediating the behavioral effect of endogenous PACAP. In contrast, AS to the cognate PAC1 receptor inhibited the display of receptivity in those EB-primed females given PACAP, an effect that also was observed in EB-primed females challenged with P. These findings are consistent with that observed in null PAC1 receptor mice, which had a significantly lower mating frequency than wild-type counterparts (41). Thus, it is likely that VMN PAC1 receptors play an important role in P-facilitated receptive behavior in female rats.
Unlike VPAC receptors, PAC1 receptors are produced from alternative splicing of the transcript from a single gene for the inclusion or exclusion of one or two cassettes (Hip and Hop cassettes). In the total medial basal hypothalamus (MBH), the ratio of PAC1-Hop mRNA to those of other PAC1 variant receptors changes with different steroid environments (16). This suggests that EB and P could differentially regulate alternative splicing at the third intracellular loop of PAC1 receptor in the VMN. In the present study, we tested this hypothesis using punched-out VMN, RT-qPCR, RT-PCR, and restriction enzyme digestion. Consistent with a previous study of the whole MBH (16), the present PCR data from the VMN show that the ratio of sfPAC1 and PAC1hop2 mRNA to total PAC1 mRNA levels appears to be dynamic, changing with the different steroid environments in the VMN. Alternate usage of the hip and/or hop variants is important for receptor-G protein interaction in the third intron of the PAC1 receptor. Indeed, steroid-dependent changes in expression of PAC1 receptor variants are known to influence receptor signaling in other tissues (11, 42). Thus, steroid-regulated changes in PACA1 variant expression in the present study may contribute to the fine-tuned regulation of receptor function in the VMN and the feed-forward effect of PACAP on female receptivity (7).
EB activates the cAMP/PKA pathway but not sufficiently to facilitate receptivity in female rodents (Refs.2, 3, 7, 26, 28, 29, 30, 31 , and 33 and see Fig. 7.13 in Ref.22). Such activity influences the effects of PACAP on functions of the VMN as EB stimulates the synthesis of PR in the VMN, an effect critical to both PACAP- (7) and P-facilitated receptivity (3, 29, 30). EB also induces the formation of cAMP in the MBH and receptivity in intact, proestrous and ovariectomized (ovx), steroid-treated females (Refs.3, 22, 26, 28 and 29 and see Fig. 7.13 in Ref.22). The sfPAC1 receptor also potently activates cAMP/PKA and PLC (11), whereas PAC1hop variants activate PLC (11) and protein kinase C (42). Here we show that receptivity is associated with EB-mediated induction of sfPAC1 and PAC1hop2 variants, both of which are known to activate cAMP/PKA signaling. We go on to report that the suppression of cAMP/PKA/DARPP32 pathway inhibited sex behavior facilitated by either PACAP or P, substantiating the role of PAC1 receptors in mediating VMN intracellular PKA signaling associated with receptivity. EB also mediates the synthesis of PACAP protein in the female MBH and, in combination with P, regulates release of PACAP into the cerebral spinal fluid (7). In part, this scenario may also account for effects of other agents that activate cAMP and lordosis [including dopamine and D1-like agonists (2, 7, 29)] as blockade of PACAP inhibits D1 agonist-induced sex behavior (7). Thus, one may envision a physiological system whereby EB sets the stage for optimizing receptivity through induction of PR, cAMP, PAC1 receptors, and PACAP in the VMN. Adding P to the steroid milieu provides for fine tuning of intracellular PACAP levels and subsequent secretion of PACAP for extracellular binding to PAC1 receptors. In turn, extracellular PACAP coordinately stimulates additional, relevant second messenger pathways for reinforcement of the PR-dependent genomic response required for reproductive behavior. Hence, the present data provide further support for the hypothesis that a complex but elegant, feed-forward autocrine loop for receptivity exists within the female VMN.
PAC1 receptors are stimulated by PACAP in an ultrashort, autocrine, and/or paracrine fashion, depending on the origin of the synaptic PACAP. Long fibers from the arcuate nucleus project to the VMN (34), making it possible for PAC1 receptors from the arcuate nucleus to play a role in PACAP signaling. In our present study, P and EB+P enhanced levels of PAC1 receptor mRNA, raising the possibility that the arcuate nucleus PAC1 receptors modulate intracellular VMN signaling by regulating extracellular synaptic levels of PACAP in the VMN. This hypothesis is consistent with data in the pituitary where PACAP concentrations are critical for PACAP-mediated bidirectional intracellular signaling, which regulates sensitization of gonadotropes for the initiation of the LH surge (45, 46). Alternately, steroid-induced activation of PAC1 receptors of arcuate nucleus origin may contribute to sensitization of the arcuate nucleus for behavioral receptivity. Because EB- and P-only but not EB+P changed PACAP mRNA levels in the arcuate nucleus, it is likely that PACAP could provide only a positive feed-forward signaling via an autocrine rather than paracrine mechanism (7). Our unpublished studies of mRNA expression in individual neurons of the VMN support this hypothesis (47).
Biologically, our results for PAC1 receptor and PACAP (7) suggest that dynamic changes in cellular signaling within relevant hypothalamic cells set the stage for a switch that allows initiation of sexual behavior. Clearly, steroid alone induces neither receptivity nor coordinate changes in PACAP levels. Likewise, neither PACAP alone (7) nor changes in cAMP levels are sufficient for facilitation of sex behavior in the absence of EB priming (3). However, when P is given in combination with EB priming, cerebrospinal fluid PACAP protein levels (7) are detected coincident with the exhibition of sex behavior in ovx rats. Moreover, administering PACAP onto the VMN or into the third ventricle of EB-primed females mimics the functional response of EB+P in that receptivity is displayed. Yet, PACAP does not rescue receptivity in PRKO mice primed with EB (7), suggesting that all specific downstream targets of PR essential for receptivity are not sufficiently activated by the cellular signaling pathway(s) triggered by PACAP stimulation of PAC1 receptors in the absence of PR. Thus, the response is mediated by, and dependent on, PR.
The effect of PACAP and its cognate receptor in the VMN appears complex and may involve several target genes. Our data implicate PAC1 receptor activation of adenylate cyclase/PKA signaling in the reproductive function of VMN cells. The present data suggest PACAP also activates the third messenger DARPP32, a downstream protein in the adenylate cyclase/PKA pathway (48) that mediates PR-dependent reproductive behavior in E-primed ovx rodents administered either P or the dopamine agonist SKF38393 (26). Interestingly, phosphorylation of DARPP32 can amplify dopaminergic signaling by a positive feedback loop (49). Moreover, several other genes induced by EB and PACAP, including preproenkephalin, are critical for receptivity (3, 49). Morphological spine formation (51, 52, 53), a process in the female VMN associated with reproductive behavior and ovulation (3), is induced by EB (51, 52), cAMP (52), and PACAP (53). Likewise, both EB and PACAP mediate neuroprotective actions after ischemic insult to the brain (54, 55). Although we believe PKA and other kinases play an important role for steroid receptor-mediated events, our results again confirm the requirement for receptor-mediated transcription as a component of steroid-induced reproductive behavior and argue against a direct and sufficient membrane or nongenomic explanation for the response.
In summary, the ability of PR to modulate the release of PACAP concomitant with receptivity suggests that both PACAP and PAC1 receptors are integral upstream participants in neuroendocrine function and synchronization of reproductive behavior with ovulation. PACAP represents a feed-forward mechanism for reinforcement of the genomic activation that produces PR-dependent behavior. This mechanism is not unexpected in concept. It is akin to the rapid effects of steroids in peripheral tissues that result in kinase activations, leading to phosphorylation and activation of coactivators used in the genomic activations induced by steroid receptors. The PACAP pathway is simply the brains choice as a mechanism to produce this important positive reinforcement.
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MATERIALS AND METHODS |
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Age-matched wild type mice (C57B/129SV) were obtained from the breeding colony at Baylor College of Medicine (7, 28, 31) and used to verify expression changes in receptor mRNA. Female mice were maintained in accordance with federal guidelines on 12-h light, 12-h dark cycle, and food and water were available ad libitum. Females were ovariectomized under anesthesia and 7 d later primed for 3 consecutive weeks with EB (0.5 µg sc) followed by P (100 µg sc at 40 h).
Experimental Treatments
For rat experiments, females were randomly assigned to treatment groups and, after experimental treatment, were randomly given identification numbers. Experiments began 10 d after screening for the untoward effects of cannula placement. Females were given selective agonists (including P) and/or antagonists ivc, or i.n. 44 h after EB-priming (1 µg sc), and behavior was measured within 30120 min after agonist challenge (3, 31). Antagonists (ivc or i.n.) were given 30 min before agonist challenge. For false positive behavior, all rats were tested before EB priming and excluded if they displayed responses (>20% LQ). Also for false positive responses, rats were tested 1 h before antagonist treatment. This allowed animals to serve as their own control in some experiments. In other experiments, groups of animals were tested by receiving either EB, sesame oil (vehicle control for EB), and/or agonists, antagonists, or oligos (vehicle control; ivc or i.n.) dissolved in normal saline. Data were statistically analyzed using one-way ANOVA by Mann-Whitney U test and Students t test for significance (P < 0.05) of behavioral results when groups of animals were compared. Two-way ANOVA with repeated measures was used to assess significant changes in reproductive behavior when females served as their own control. Duncans multiple-range test was used for individual comparisons. Proceptive behavior was not statistically reported here, because it was altered in parallel with lordotic activity. Except for Fig. 1A, data are presented together because Duncans range test for individual comparisons failed to detect differences in LQ% due to method of agonist administration (icv vs. i.n.).
RT-qPCR for Quantitative Analysis
Using the time schedule for appearance of positive lordosis (60 min after P), brain tissue samples (n = 3 animals per treatment group repeated thrice) were collected by micropunch technique using anatomical markers as described previously (31, 56) at 44 h after EB sc (or sesame oil), and 1 h after P (icv). Tissue samples from all experiments were processed at the same time for RNA extraction and analysis. After extraction using RNeasy Lipid Tissue Mini-kit (QIAGEN, Inc., Valencia, CA) per the manufacturers protocol, total RNA (50 ng) was analyzed by RT-qPCR using a one-step RT-PCR procedure (Taqman One-Step RT-PCR Master Mix reagents Kit, Applied Biosystems, Foster City, CA) and ABI Prism PE7700 Sequence Analyzer (Applied Biosystems) for receptor and 18S RNA expression. All primers and probes were designed using Primer Express software (Applied Biosystems) following Applied Biosystems guidelines and are presented in Table 1. Primers (300 nM each) and labeled probe (250 nM; 5'-label, 6-carboxy-fluorescein; 3' label, 6-carboxy-tetramethyl-rhodamine) were used in 25 µl reaction volume in MicroAmp 96-well plates. The specificity of each set of primers and probes was confirmed by blast search against the gene bank. Thermal cycling conditions included a reverse transcriptase step for 30 min at 48 C and 10 min at 95 C followed by 40 cycles of 15 sec at 95 C and 1 min at 60 C. Singleplex quantities were normalized against 18S RNA amplification (primer/probe set by Applied Biosystems), for which input mRNA was diluted 100-fold. Cycle threshold values (Ct) were analyzed using the SDS1.9 software (Applied Biosystems), and relative quantification of receptor expression was determined using the comparative Ct method (ABI Prism 7700, SDS User Bulletin 2; Applied Biosystems). The range of slopes of log input amounts vs.
Ct for each receptor was 3.398 in Fig. 3
, A and D, and 3.276 in Fig. 3E
, indicating similar amplification efficiency of each target cDNA and 18S RNA. Thus, 2-
Ct gives the amount of receptor, normalized to endogenous 18S RNA and relative to vehicle-treated tissue probed for VPAC1. All receptor data were analyzed together. ANOVA (P < 0.05) was used to statistically compare data from steroid-treated tissue vs. vehicle-treated tissue. After PCR amplification of each transcript, the size of each amplicon was confirmed by electrophoresis; specificity was evaluated by subcloning and performing sequence analysis (Baylor College of Medicine Sequencing Core). 18S RNA was used an internal calibrator because we have been unable to detect a relationship between vehicle and steroid treatments for 18S Ct values in the VMN (n = 32 per treatment), medial preoptic area (n = 24 per treatment), arcuate nucleus (n = 24 per treatment), and ventral tegmental area (n = 20 per treatment) of wild-type mice and VMN (n = 24) of rats (one-way ANOVA followed by least-squares linear regression analysis with change in Ct value as the dependent variable).
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RT-PCR for Third Intron PAC1 Receptor Splice Variants in the Female VMN
Tissue was collected 60 min after P (icv) was given. VMNs (n = 3 rats for each treatment group and repeated twice for a total of nine rats per treatment group) were collected by punch-out technique after deep anesthesia and dissection (31, 56). Whole brain was used as positive controls. Total RNA was extracted using RNeasy Lipid Tissue Mini-Kit (QIAGEN) in accordance with the manufacturers protocol. Total RNA (100 ng) was reverse transcribed with a random hexamer primer and Superscript II RT polymerase (Invitrogen Life Technologies, Carlsbad CA) for 50 min at 42 C in 20 µl reaction volume. RT product (2 µl) was amplified using primers previously described (57) in a Hot Start reaction with Platinum DNA Taq (Invitrogen), 1.5 mM Mg, 0.5 mM of each primer, 0.4 mM deoxynucleotide triphosphate, and 3 µl cDNA in a 50 µl reaction volume. Thermal cycling conditions for amplification included 2 min at 94 C, and 40 cycles of 1 min at 94 C, 1 min at 60 C, and 2 min at 72 C. After gel electrophoresis in a 1.5% agarose gel, fragments were excised and purified using QIAquick Gel Extraction Kit (QIAGEN). Using 5 µl of purified product, a restriction digest (incubated overnight at 37 C) was performed for detection of Intron 3 (hip/hop) inserts and N-terminal variants in a 1.5% agarose gel.
Compounds and Oligonucleotides
For all injections, doses were based on published studies for effective use concentrations whenever possible or verified when appropriate. Steroids for sc injections were dissolved in sesame oil, and all other compounds for icv or i.n. injections were dissolved in sterile normal saline. Vehicle controls received either sesame oil (sc steroids) or normal saline (all icv or i.n injections). At comparable doses, water-soluble P (Sigma Chemical Co., St. Louis, MO) + hydroxypropyl-ß-cyclodextrrin (HPB-C) + saline, P + saline, saline alone, HBP-C alone, EB + saline, and EB + HBP-C + saline given icv or i.n. have the same effect on female rodent behavior (LQ% = 2.2 ± 1% for those animals not primed with EB and 6.3 ± 1.8% for those EB primed), whereas both EB + water-soluble P + HBP-C + saline and EB + P (Sigma) + sesame oil induced positive lordosis (94.1 ± 4.8% in rats; 87.4 ± 4% in mice); hence, saline was used for preparation of P, and HBP-C + saline was used as vehicle treatment. Lyophilized phosphothiolated oligos (Invitrogen), PACAP38 (Calbiochem Laboratories, La Jolla CA), the VPAC1 receptor antagonist acetyl-His1[DPhe2, K15, R16, L27] VIP(17)/GFR(827) (Bachem, Torrance CA), the cAMP binding site antagonist Rp-cAMP (Calbiochem), the PKA inhibitor H89, the D1-like agonist SKF38393, VIP, and the VPAC2 receptor antagonist [4-Cl-D-Phe6, Leu17]VIP (Sigma Chemical Co., St. Louis, MO), were prepared on the experimental days. The AS oligo sequence for PAC1 was GGA-CTC-TGG-CCA-TGG-TGC-TTC-C, and the PAC1 RS was CGA-TCG-GAC-TCC-GGT-TGC-CTT-G; the biological effects of each are published (19). The properties of VPAC2 (TAC-TCC-CGC-AGC-CAC-CAC-GA-[AS] and ATG-AGG-GCG-TCG-GTG-GTG-CT [RS]) and VPAC1 (TAC-GCG-GGA-GGC-TCG-GGT-GG [AS] and ATG-CGC-CCT-AGC-CCA-CC [RS]) oligos have been published previously (19). Likewise, the sequence of rat DARPP-32 oligos and their functional effects have been published. Sterile normal saline was used as vehicle for all compounds dissolved in normal saline, and sesame oil was used for steroids dissolved in oil.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online June 23, 2005
Abbreviations: AS, Antisense; Ct, cycle threshold; EB, estradiol; GRF, GH-releasing factor; icv, intracerebroventricular(ly); i.n., onto the VMN; LQ, lordosis quotient; MBH, medial basal hypothalamus; ovx, ovariectomized; P, progesterone; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PLC, phospholipase C; PR, progesterone receptor; RS, random antisense sequence sense; RT-qPCR, real-time quantitative RT-PCR; sfPAC1, short form PAC1; VIP, vasoactive intestinal peptide; VMN, ventromedial nucleus.
Received for publication September 29, 2004. Accepted for publication June 17, 2005.
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