Anxiolytic and anti-stress effects of brain prolactin: improved efficacy of antisense targeting of the prolactin receptor by molecular modeling

Abstract

We provide the first evidence that prolactin is a neuromodulator of behavioral and neuroendocrine stress coping in the rat. In virgin female and male rats, intracerebral infusion of ovine prolactin (oPRL) into the lateral cerebral ventricle (intracerebroventricular) exerted an anxiolytic effect on the elevated plus-maze in a dose-dependent manner (0.1 and 1.0 microg/5 microl; p < 0.01). In contrast, downregulation of the expression of the long form of brain prolactin receptors by chronic intracerebroventricular infusion of an antisense oligodeoxynucleotide (ODN) (osmotic minipump, 0.5 microg. 0.5 microl(-1). hr(-1); 5 d) increased anxiety-related behavior on the plus-maze compared with mixed bases-treated and vehicle-treated rats (p < 0.01), again demonstrating an anxiolytic effect of PRL acting at brain level. Furthermore, in jugular vein-catheterized female rats, the stress-induced increase of corticotropin secretion was decreased after chronic intracerebroventricular infusion of oPRL (osmotic minipump, 1.0 microg. 0.5 microl(-1). hr(-1); p < 0.05) and, in contrast, was further elevated by antisense targeting of the brain prolactin receptors (p < 0.01). This provides evidence for a receptor-mediated attenuation of the responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis by prolactin. The antisense ODN sequence was selected on the basis of secondary structure molecular modeling of the target mRNA to improve antisense ODN-mRNA hybridization. Receptor autoradiography confirmed the expected improvement in the efficacy of downregulation of prolactin receptor expression [empirically designed antisense, 30%; p > 0.05, not significant; adjustment of target position after mRNA modeling, 72%; p < 0.05). Taken together, prolactin acting at brain level has to be considered as a novel regulator of both emotionality and HPA axis reactivity.

Fig. 1.

Secondary structure of mRNA coding for the rat PRL-R as predicted by sequential energy minimization. Noncanonical base pairs are allowed. The regions selected for targeting are magnified.A, Two antisense ODNs selected empirically, used also in comparative autoradiographic experiments. B, Antisense ODNs selected on the basis of molecular modeling, used in behavioral and neuroendocrine experiments. The beginning of the sequence is marked by two concentric circles. The simulation strategy is based on a homogeneous Markovian jump process that considers each transcription step. The state space of interest is the set of structures constructible on the part of the mRNA already transcribed. A formula permitting the computation of the structures' probabilities at the end of the mRNA transcription is derived and successively approximated to reduce the size of the state space. This permits the design of a prediction algorithm (Breton et al., 1997).

Fig. 2.

Behavioral parameters on the EPM of female and male rats treated with intracerebroventricular or intravenous oPRL.A, B, Acute intracerebroventricular oPRL administered 10 min before EPM exposure decreased the anxiety-related behavior in a dose-dependent manner in virgin female (A) or male (B) rats. For comparison, an anxiolytic effect could also be revealed 30 min after injection of diazepam (1 mg/kg, i.p.) in male but not female rats (insets). C, Acute intravenous oPRL administered 20 min before EPM also decreased anxiety-related behavior in female rats. The percentage of entries into (entries) and the time spent on (time) the open arms to overall arm entries and time, respectively, and the number of full entries into the open arms of the EPM are given as indicators of anxiety-related behavior. The number of entries into the closed arms (entries closed) is given as an indicator of locomotor activity. Data are the mean ± SEM. *p < 0.05, **p < 0.01 versus vehicle.

Fig. 3.

Anxiety-related behavior on the EPM after downregulation of brain PRL-R by antisense targeting. Anxiety-related behavior was increased after 5 d of chronic intracerebroventricular infusion of PRL-R antisense (PRL-R-AS) ODN compared with vehicle (Veh)-treated or mixed bases ODN (MB)-treated virgin female rats, as shown by a decrease in the percentage of entries into, the percentage of time in, and the number of full entries into the open arms of the EPM. Data are the mean ± SEM. *p < 0.05, **p < 0.01.

Fig. 4.

Plasma ACTH concentrations after chronic intracerebroventricular infusion with either oPRL (A) or antisense ODN against the PRL-R (B). Blood samples were collected under basal conditions and 5, 15, and 60 min after exposure to the EPM (arrows). A, Chronic intracerebroventricular oPRL (5 d; osmotic minipump; 1.0 μg · 0.5 μl⁻¹ · hr⁻¹)-attenuated stress-induced secretion of ACTH. B, Chronic intracerebroventricular PRL-R antisense ODN (PRL-R-AS) (5 d; osmotic minipump; 0.5 μg · 0.5 μl⁻¹ · hr⁻¹) further elevated the stress-induced ACTH response to EPM exposure compared with mixed bases (MB) and vehicle treatment.Insets show the stress-induced rise (percentage of basal) in ACTH secretion. Data are the mean ± SEM. *p < 0.05, **p < 0.01 versus vehicle/MB; # p < 0.05, ## p< 0.01 versus respective basal values.

Fig. 5.

PRL-R binding at the choroid plexus after chronic treatment with PRL-R antisense ODN. A, Treatment with empirically designed antisense ODN (AS1) directed against the PRL-R mRNA resulted in a downregulation of PRL-R binding of ∼30% (n.s.) compared with vehicle (Veh) and mixed bases (MB). B, Treatment with an AS selected on the basis of mRNA secondary structure prediction (AS2) (Fig. 1) resulted in a downregulation of PRL-R binding of ∼72% (*p < 0.05 vsVeh and MB). Shown are relative label density values in arbitrary units, obtained from optical density values measured at the choroid plexus of the right cerebral ventricle by appropriate logarithmic transformation. C, Distribution of binding sites in the choroid plexus of Veh-,MB-, and AS2-treated virgin female rats. Autoradiograms illustrate total binding (TB) and nonspecific binding (NSB).