Anti-sense oligonucleotides, for progestin receptors in the VMH and glutamic acid decarboxylase in the VTA, attenuate progesterone-induced lordosis in hamsters and rats

Abstract

Immunocytochemical (ICC) staining for progesterone (P) receptors (PRs) and glutamic acid decarboxylase (GAD), the enzyme responsible for GABA production, reveal that there are many PRs in the ventral medial hypothalamus (VMH) and many GAD containing neurons in the ventral tegmental area (VTA). To investigate P’s action on lordosis in the VMH and VTA, anti-sense oligos specific to PRs and GAD_65&67 were intracerebrally infused into the VMH and VTA of 24 ovariectomized hamsters and 40 ovariectomized rats. Estradiol benzoate (2 μg) primed hamsters and rats were infused to the VMH and the VTA with either PR (250 ng/1.0 μl infusion) or GAD (500 ng/1.0 μl infusion) anti-sense oligos, their scramble controls, or saline vehicle at hour 0 and again at hour 24. At hr 44, rodents were subcutaneously injected with P (500 μg) and were tested for sexual receptivity with a male 4 h later. There were significant reductions in lordosis of hamsters and rats following PR anti-sense infusions to the VMH compared to scrambled or vehicle control infusions. Effects of PR anti-sense to the VMH were not different from combined VMH and VTA PR anti-sense infusions; however, VMH infusions reduced lordosis compared to VTA-only anti-sense infusions. GAD anti-sense infusions reduced lordosis when infused into the VTA, compared to scrambled or saline vehicle infusions. Lordosis responsiveness following VTA GAD anti-sense infusions was not different from combined VMH and VTA infusions, but VTA infusions of GAD anti-sense reduced lordosis compared to VMH-only anti-sense infusions. These data suggest that in the VMH, PRs are important for P-facilitated lordosis, whereas in the VTA, GABAergic neurons may be an important substrate for mediating P’s actions on lordosis of rodents.

Introduction

Estrous cycle variations in estradiol (E₂) and progesterone (P) result in ovulation and receptivity in rodents. Receptivity is characterized by females’ display of lordosis in response to appropriate environmental stimulation, which receptive females typically seek out from males. Because receptive behavior can be readily controlled, induced, observed, and quantified in ovariectomized (ovx) rodents by systemic administration of E₂ and P [7], receptivity is an ideal behavioral assay for investigating hormone action in the brain.

An important brain site involved in receptivity is the ventral medial hypothalamus (VMH): when E₂ and P are applied to the VMH of ovx rats [43], [46], [47], lordosis is readily elicited. Receptivity in ovx, E₂-primed hamsters requires P to both the VMH and the ventral tegmental area (VTA) [5], [44]. Because of their extreme reliance on P, hamsters have been utilized extensively to examine the mechanisms of P’s actions in the VTA. Studies utilizing P conjugated to the macromolecule bovine serum albumin (P:BSA), which cannot permeate cell membranes [45], reveal that P:BSA quickly induces lordosis when applied to the VTA following free P to the VMH [3], [4], [9], [13], [15]. The rapid and membrane-relegated action of P:BSA suggests that P’s enhancing effects in the VTA may be membrane mediated.

Traditionally, P’s actions are understood as occurring through E₂-induced intracellular progestin receptors (PRs) [6]. While P in the VMH has actions via PRs in hamsters and rats, P can have membrane effects in the VTA. To date, analysis of P’s mechanism in the VTA has been examined most fully in hamsters [3], [4] because of their extreme reliance on P for the manifestation of sexual receptivity. The nature of P’s effects in the VTA of hamsters $\text{and}$ rats [13] is the topic of this investigation.

Neuroanatomical studies using autoradiography, immunocytochemistry, and behavioral assays have found a sparse number of E₂-induced PRs in the rodent VTA [22], [39], [48]. In attempts to understand how P facilitates receptivity by actions in the VTA in the absence of E₂-induced intracellular PRs, our laboratory and others have examined γ-aminobutyric acid (GABA)_A/benzodiazepine receptor complexes (GBRs) as a site of action.

Progesterone’s membrane effects in the VTA of rats and localization of GABAergic interneurons in the VTA [1] suggest GBRs in the VTA may be an important substrate for P’s actions. For example, muscimol, a GBR agonist, infused into the VTA of E₂- and subthreshold P-primed hamsters facilitates P’s behavioral effects [3], [4], [8]. Furthermore, the GBR antagonist, bicuculline, inhibits P-induced receptivity when infused directly into the VTA of E₂- and suprathreshold P-primed hamsters [16]. In rats, the same effects were seen, but GABAergic manipulations’ alteration of sexual behavior [14] were less dependent on P. These findings suggest that GBRs in the VTA may be a substrate for P’s facilitation of receptivity in rats and hamsters.

The present study investigates the importance of PRs and GABAergic neurons in the VMH and VTA of rats for the mediation of feminine sexual receptivity. The importance of PRs or glutamic acid decarboxylase (GAD) in mediating lordosis was examined following infusion of anti-sense oligonucleotides into the VMH or VTA of hamsters and rats. The use of anti-sense oligonucleotides (oligos), synthetic DNA, has proven to be an effective tool for the blockade of targeted behaviors [41]. In vivo, the anti-sense method can directly demonstrate the role of molecular and cellular processes in the regulation of behavior [28], [32], [35], [38], [41]. Because oligos’ effects are reversible and do not damage brain sites, researchers have directly administered PR-specific oligos into the lateral ventricle or VMH and observed reductions in P-induced lordosis. Unmodified oligos, specific for PRs, effectively block E₂-induced upregulation of PRs necessary for facilitation of receptivity when administered 12, but not 24 h, after E₂ [40]. Similar reductions in lordosis were seen when a PR-specific phosphorothioated oligo (s-oligo) was infused into the lateral ventricle concurrent with E₂ and 24 h later [25]. Thus, PR blockade in the VMH is achievable if a PR oligo is administered before synthesis of E₂-induced PRs [41].

The few studies examining GABA’s effects on receptivity using oligos were targeted at the enzyme, GAD, which is responsible for the conversion of l-glutamic acid to GABA and is expressed in most major classes of GABA neurons [35]. Two separate forms of GAD, GAD₆₅ and GAD₆₇, exist; they differ in terms of their molecular weight and type of activation. Anti-sense oligonucleotide infusions also have demonstrated that the time frame for GAD₆₅ and GAD₆₇ to block lordosis is different [35]. Infusions of GAD₆₅ oligo to the VMH and central gray were effective at blocking lordosis 48 h after infusion, whereas similar GAD₆₇ oligo infusions were effective at reducing lordosis 24 h after infusion [35].

Regardless of an oligo’s sequence, the type used is important for successful results. McCarthy et al. [35] used an s-oligo for their combined GAD studies. While s-oligos are commended for guarding against degradation, their questionable toxicity and specificity can be problematic [26], [28], [49]. Unmodified oligos may also produce major problems due to degradation and stability [2], [50]. Attempts to resolve these concerns have involved combining s-oligos and unmodified oligos to aid in the reduction of toxic side effects associated with the sulfur in s-oligos, while improving intracellular stability and decreasing degradation associated with unmodified oligos. The present studies utilized the chimeric, combination s- and unmodified oligos, to test the hypothesis that receiving an oligo specific for PRs to the VMH, or to the VMH $\text{and}$ VTA, will reduce receptivity in E₂- and P-primed rodents compared to animals receiving the same oligo to the VTA-only. Furthermore, it is hypothesized that infusions of GAD oligos will significantly reduce lordosis of E₂- and P-primed rodents when applied to the VTA or to both the VMH $\text{and}$ VTA compared to reductions seen following VMH only infusions.