Neurobiology of Stress 12 (2020) 100208 Contents lists available at ScienceDirect Neurobiology of Stress journal homepage: www.elsevier.com/locate/ynstr Early postnatal allopregnanolone levels alteration and adult behavioral T disruption in rats: Implication for drug abuse Iris Bartolomé, Anna Llidó, Sònia Darbra, Marc Pallarès∗ Institut de Neurociències, Departament de Psicobiologia I Metodologia en Ciències de La Salut, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain A R T I C LE I N FO A B S T R A C T Keywords: Several studies have highlighted the role that early postnatal levels of allopregnanolone play in the development Emotional behavior of the CNS and adult behavior. Changes in allopregnanolone levels related to stress have been observed during Neurodevelopment early postnatal periods, and perinatal stress has been linked to neuropsychiatric disorders. The alteration of early Alcohol abuse postnatal allopregnanolone levels in the first weeks of life has been proven to affect adult behaviors, such as Allopregnanolone anxiety-related behaviors and the processing of sensory inputs. This review focuses on the first studies about the Neurosteroids Neuropsychiatric disorders vulnerability possible relationship between the early postnatal allopregnanolone levels and the vulnerability to abuse of drugs such as alcohol in adulthood, given that (1) changes in neonatal allopregnanolone levels affect novelty ex- ploration and novelty seeking has been linked to vulnerability to drug abuse; (2) early postnatal administration of progesterone, the main allopregnanolone precursor, affects the maturation of dopaminergic meso-striatal systems, which have been related to novelty seeking and drug abuse; and (3) alcohol consumption increases plasma and brain allopregnanolone levels in animals and humans. Manipulating neonatal allopregnanolone by administering finasteride, an inhibitor of the 5α-reductase enzyme that participates in allopregnanolone synthesis, increases alcohol consumption and decreases the locomotor stimulant effects of low alcohol doses. At a molecular level, finasteride decreases dopamine and serotonin in ventral striatum and dopamine release in nucleus accumbens. Preliminary results suggest that serotonin 5HT3 receptors could also be affected. Although an in-depth study is necessary, evidence suggests that there is a relation between early postnatal allopregna- nolone and vulnerability to drug use/abuse. 1. Introduction excitability, revealed that they are extremely important, given the huge array of brain activities in which they may be involved. Thus, in recent In decades, it has been shown that steroids are important in many years, NS have been reported to participate in several brain processes brain processes. In 1981, Baulieu and colleagues studied the steroid and changes in their physiological levels have been related to a variety dehydroepiandrosterone sulfate, and concluded that the brain was a of neurological/psychiatric disorders and pathologic behaviors. It has steroidogenic organ (Corpéchot et al., 1981). This led to the term also been shown that administering NS is beneficial in a number of ‘neurosteroid’, which is used to describe the subclass of steroids that are experimental models of certain disorders (see Dubrovsky, 2005; synthesized in the central nervous system (Baulieu et al., 1981). A few Melcangi and Panzica, 2014; Porcu et al., 2016 for reviews). years later, in 1986, Majewska and cols. found that the neurosteroid NS levels in the brain are not static but rather naturally fluctuate (NS) allopregnanolone (AlloP) acted as a positive allosteric modulator over the life-time in response to different physiological and environ- of γ-aminobutyric acid type A (GABAA) receptors (Majewska et al., mental conditions. For example, the concentration of AlloP oscillates 1986). This last discovery led to the term ‘neuroactive steroids’ (NS), during development (Grobin and Morrow, 2001), the menstrual cycle which is used to describe all those steroids that, whether they are (Monteleone et al., 2000), pregnancy and post-partum (Concas et al., synthesized in the brain, the adrenal gland or gonads, are capable of 1998), as well as faster changes occur in response to adverse events, modifying neuronal activity via membrane receptors modulation such as stress (Purdy et al., 1991; Serra et al., 2000), or drugs, such as (Dubrovsky, 2005; Paul and Purdy, 1992). The discovery of steroids the systemic administration of alcohol (Barbaccia et al., 1999; Cook that not only act as endocrine messengers but also in a paracrine or et al., 2014a, 2014b). Surprisingly, although the actions of NS in the autocrine manner, and their capacity to rapidly modulate neural adult brain have been studied extensively, their role in the developing ∗ Corresponding author. E-mail address: Marc.pallares@uab.cat (M. Pallarès). https://doi.org/10.1016/j.ynstr.2019.100208 Received 2 August 2019; Received in revised form 14 November 2019; Accepted 23 December 2019 Available online 27 December 2019 2352-2895/ © 2019 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 brain and their long-term effects are still poorly understood. The en- Changes in AlloP levels related to stress have also been observed zymes responsible for steroidogenesis are present in the brains of rats during early postnatal stages (Frye et al., 2006; Kehoe et al., 2000). from early fetal stages (Compagnone et al., 1995; Lauber and Given that adequate inhibitory GABAergic control is necessary for stress Lichtensteiger, 1996; Lephart et al., 1990) and the ability of the fetal regulation, variations in physiological AlloP levels may imply changes brain to synthesize NS has been proven (Pomata et al., 2000). Fur- in stress responses, and thus different outcomes of neonatal stress. thermore, pre and postnatal fluctuations of AlloP in the frontal cortex of Previous works studying the effects of administering AlloP on neonatal rats are time-related to significant changes in brain development stress have shown that administering AlloP prior to a brief EMS at (Grobin and Morrow, 2001). Thus, studying the involvement of NS, postnatal day 7 decreased the ultrasonic vocalizations of rats such as AlloP, in brain development and the possible effects of changes (Zimmerberg et al., 1994; Zimmerberg and Kajunski, 2004). Ultrasonic in their levels on vulnerability to neurological/psychiatric disorders is vocalizations are emitted by neonatal rat pups in the range of of great interest. 20–50 kHz and are thought to contribute to the formation of the ma- ternal-infant bond as infant mammals vocalize when separated from 2. Neonatal allopregnanolone levels and stress their mothers to elicit protection, nourishment and warmth (Zimmerberg and Kajunski, 2004). Therefore, the analysis of ultrasonic GABAergic inhibitory control plays an important role in regulation vocalizations is used to measure the pup's affective state (Zimmerberg hypothalamic-pituitary-adrenal (HPA) axis activation (see Gunn et al., et al., 2003). Other authors have shown that when the neurosteroid 2015 for a review). Several studies have shown that the GABAergic THDOC, which is a GABAA-positive modulator, is administered to- transmission rapidly decreases (Biggio et al., 1990; Concas et al., 1998) gether with EMS, it can attenuate the adult behavioral and neu- while AlloP brain and plasma levels increase (Barbaccia et al., 1996; roendocrine consequences of this repeated stress during early life (i.e. Purdy et al., 1991) in adult rats after acute stress, such as the exposure increased anxiety, enhanced HPA axis responses to stress, such as an to CO2 or swimming. Moreover, this increase in AlloP levels correlates exaggerated adrenocortical secretion, and impaired glucocorticoid with the restoration of the GABAergic transmission (Barbaccia et al., feedback). (Patchev et al., 1997). Similarly, a single administration of 1998). Therefore, it has been suggested that endogenous AlloP re- 2 mg/kg of AlloP injected just prior to 12h of EMS on postnatal day 5 presents a homeostatic mechanism in the context of adaptation to stress counteracted the increased HPA axis response to subsequent stressors in by limiting the extent and duration of the reduction in GABAergic in- both infants and adult rats (Mitev et al., 2003). Thus, administering NS, hibitory transmission and activation of the HPA axis (Girdler and such as AlloP, which acts as a positive modulator of GABAA receptors, Klatzkin, 2007). In this line, it has been observed in humans that a might prevent or reverse some of the negative neuroendocrine and pretreatment with 50 mg of progesterone prior to psychosocial stress behavioral effects of neonatal stress. In fact, it has been suggested that a diminished stress-induced cortisol increase (Childs et al., 2010), transient increase in NS biosynthesis may contribute to the stress hypo- showing the possible action of progesterone-derived metabolites, such responsive period (SHRP) (Mitev et al., 2003). The early postnatal as AlloP, on the HPA axis. However, it has been reported that chronic period between postnatal days 2 and 14 is known as SHRP and is a stress due to social isolation induces large reductions in AlloP cere- phase of high plasticity within the CNS with neuronal migration, sy- brocortical and plasma concentrations (Serra et al., 2000), as well as naptogenesis and apoptosis (Gunn et al., 2015). During this period, alterations in brain NS levels, such as AlloP or tetra- animals have low corticosterone (CORT) and adrenocorticotropic hor- hydrodeoxycorticosterone (THDOC), in response to acute stressors. mone (ACTH) levels in response to stress (Sapolsky and Meaney, 1986; Thus, it has been proposed that a disruption in this homeostatic me- Sapolsky et al., 2000; Rosenfeld et al., 1992), which reflects the activity chanism may play a pathogenic role in some psychiatric disorders re- of the HPA axis that is still developing (Ellenbroek and Cools, 2002). lated to chronic stress, for instance, depressive disorders (Girdler and The hypoactivity of this axis in response to stress is key during early life Klatzkin, 2007). Therefore, the administration of exogenous AlloP ei- since it protects the developmental nervous system of stressful events ther during or following a period of chronic stress, such as social iso- (Brunton et al., 2014; deKloet et al., 1988; Ellenbroek and Cools, 2002). lation, can prevent or normalize HPA axis dysfunction, precluding the If a stressor is able to raise the CORT and ACTH levels during SHRP it establishment of depressive/anxiety-like behaviors in the rat (Evans can cause multiple alterations in neurodevelopment (Bock et al., 2014; et al., 2012). On the other hand, it seems that there are some in- Chen and Baram, 2016), such as, a reduction in the density of GA- dividuals more sensitive to these NS levels fluctuations. In this sense, it BAergic interneurons in the medial prefrontal cortex and the hippo- has been observed that NS level changes are relevant to the induction of campus (Fride et al., 1986; Barros et al., 2006), which have been linked the altered affective state in women with premenstrual dysphoric dis- to several neuropsychiatric disorders, such as schizophrenia, autism, order (for a review see Wei et al., 2018), which are more vulnerable to and anxiety (Fine et al., 2014). increased anxiety (Kim et al., 2004) and alcohol abuse (Stout et al., Besides the possible protective role of AlloP for neonatal stress, it 1986; Caan et al., 1993; Chuong and Burgos, 1995).The possibility that has to be considered that since alterations in neonatal AlloP levels have these women have suffered NS levels alterations during neurodeve- been related to several developmental disruptions, some of the detri- lopment that could have made them more behaviorally sensitive to mental effects of neonatal stress may be in part elicited by an alteration these fluctuations in adulthood is a question that opens new lines of in neonatal NS levels. Zimmerberg and Kajunski (2004) showed that research. both pups submitted to 6h per day of social isolation between postnatal During the perinatal period the brain is highly sensitive to en- days 2 and 6, and pups that were not socially isolated and that were vironmental factors, and thus early exposure to adverse experiences like administered AlloP (5 mg/kg) daily emitted fewer ultrasonic vocaliza- stress have been related to several developmental alterations, such as tions than control rats during EMS at postnatal day 7. These authors impoverished dendritic trees and reduced hippocampal volume, and proposed that the effects of social isolation on ultrasonic vocalizations adult disorders, such as long-lasting memory deficits (see Bale et al., could be mediated by an endogenous increase in AlloP; and therefore, 2010; Bock et al., 2014; Chen and Baram, 2016 for a review). In neo- administering it exogenously leads to the same effect (Zimmerberg and natal animals, early stress arises mainly from disruptions in maternal Kajunski, 2004). Interestingly, when rats submitted to social isolation care. For this reason, several models of neonatal stress in rodents con- received a previous injection of AlloP, the rate of ultrasonic vocaliza- sist in manipulating maternal-pup interactions. One of the main tions was unaffected. This may again indicate a protective effect of methods used to cause stress for the pups which has consequences for AlloP against stress due to EMS when AlloP is administered prior to their cognitive and emotional networks and functional outcomes, is stress, as well as the endogenous AlloP increase that occurs as an early maternal separation (EMS) (Chen and Baram, 2016), in its many adaptive response. In a set of experiments carried out in our laboratory procedure variants (see Fumagalli et al., 2007 for a review). we observed that early postnatal AlloP administration (from postnatal 2 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 days 5–9) played a protective role against posterior effects of neonatal in animal models that this change in GABAergic polarity would occur stress (maternal deprivation for 24 h at postnatal day 10). For instance, from the second postnatal week in several brain areas, such as the and consistent with previous works in adolescent (Marco et al., 2007) striatum and the spinal cord (Dehorter et al. 2011, 2012) or the hip- and adult rats (Rentesi et al., 2013), EMS increased locomotion and pocampus (Sipilä et al., 2006), and would be able to temporarily co- decreased exploration of holes in a new environment in adolescent age incide with the increase in AlloP levels during postnatal day 10. In a (postnatal day 40), and these effects were prevented by previous neo- previous work, we reported that the alteration of physiological AlloP natal AlloP administration (Llidó et al., 2013). These data suggest that levels by means of sub-chronic administration (from postnatal day 5–9) the previous increase in AlloP levels produced by administering it of AlloP or finasteride, which acts as an inhibitor of the 5α-reductase exogenously could alter the function of GABAA receptors, which in turn enzyme and decreases plasmatic AlloP levels in adult rats (Azzolina could interfere with the physiological response produced by EMS. et al., 1997; Mukai et al., 2008), alters the developmental expression of However, the recorded anxiolytic effect of EMS measured in the ele- KCC2 in the hippocampus (Modol et al., 2014). These results may in- vated plus-maze test at adult age, which has also been reported by other dicate that AlloP participates in the GABA hyperpolarizing shift. authors who studied rats (Burke et al., 2013; Llorente-Berzal et al., As discussed in the previous sections, NS can play an important role 2011) and mice (Fabricius et al., 2008), was not prevented or neu- in regulating early neurodevelopment. Thus, when the endogenous NS tralized by administering AlloP previously to EMS (Llidó et al., 2013). levels are altered, for example, through the neonatal administration of Thus, it seems that the kind of behavior and the developmental period AlloP, then the number and location of GABAergic interneurons in the are key factors for obtaining the protective effects of AlloP on neonatal adult prefrontal cortex are modified (Grobin et al., 2003), the total stress. number of neurons in the dorsomedial nucleus of the thalamus de- creases (Gizerian et al., 2004) and the basal levels of striatal dopamine 3. The effects of changes in neonatal allopregnanolone levels on decrease (Muneoka et al., 2009). The neonatal administration of pro- gesterone, the main precursor of AlloP, also induces a decrease in basal 3.1. Brain maturation dopamine levels, together with a decrease in serotonin levels in the striatum (Muneoka et al., 2010). Furthermore, it has been documented As mentioned above, the brain in the fetal stage already acts as a that neonatal treatment with estradiol, which induces a persistent de- steroidogenic organ (Pomata et al., 2000) and has NS levels similar to crease in cerebral and plasma concentrations of progesterone and AlloP those found in the adult brain (Kellogg et al., 2006). AlloP levels fluc- (McCarthy, 2008; Berretti et al., 2014; Calza et al., 2010), increases tuate during neurodevelopment, and are higher in fetal nervous tissue production of extracellular dopamine in the medial prefrontal cortex, during late gestation (Kellogg and Frye, 1999; Grobin and Morrow, an effect that can be reversed through subsequent treatment with 2001). According to Hirst and his collaborators (Hirst et al., 2016), the progesterone (Porcu et al., 2017). However, the neonatal administra- high fetal levels of AlloP during late gestation would play a key role in tion of the 5α-reductase inhibitor finasteride, which has been observed regulating the activity of the fetal brain. In addition, stressful events to tend to increase the AlloP levels in the neonatal hippocampus (Mòdol during late pregnancy stimulate the release of AlloP; thus, this NS may et al., 2013; Darbra et al., 2014), causes an increase in the expression of play a neuroprotective role during pregnancy (Brunton et al., 2014; the α4δ subunits of the GABAA receptor in this same structure (Modol Hirst et al., 2014). et al., 2014). This data set highlights the importance of basal levels of Grobin and Morrow (2001) characterized AlloP pre and postnatal NS during early neurodevelopment because manipulating it alters the fluctuations in the frontal cortex of rats. These authors reported that maturation of diverse brain structures. fetuses have elevated AlloP levels during the last gestational days, which has been related to the mentioned protective role played by 3.2. Adolescent and adult behavior AlloP against gestational stress (see Brunton et al., 2014 for review). These high levels decrease progressively during the first week of life, At the behavioral level, it has been observed that the manipulation when rats have low levels similar to those found in the adult brain. of neonatal levels of NS such as AlloP, can modify adolescent and adult Then, during the second week of life there is another rise in AlloP le- behavior. It has been shown that administrating AlloP during the early vels, and maximum values are reached between postnatal day 10 and postnatal stage in rats causes a decrease in responses related to anxiety 14, decreasing again to similar adult levels on postnatal day 15 and in the elevated plus-maze (EPM) in adult age, because animals spent remaining low until puberty. This second peak in AlloP is time-related more time and entered the open arms of the apparatus more often to the functional GABA shift from depolarization to hyperpolarization without showing differences in locomotor activity (Zimmerberg and in both the neocortex and hippocampus (reviewed in Dehorter et al., Kajunski, 2004; Darbra and Pallarès, 2012). It has also been shown that 2012). Unlike the adult brain, during early neurodevelopment, GABA the early postnatal administration of AlloP causes a decrease in the has a depolarizing profile caused by the activation of GABAA to trigger anxiolytic effects of lorazepam in the EPM in adult animals, suggesting Cl- (Ben-Ari et al. 2007, 2012). The depolarization induced by GABA that an increase in neonatal levels of this NS could decrease sensitivity eliminates the voltage-dependent blockade exerted by magnesium in to the effects of this benzodiazepine (Darbra and Pallarès, 2009). the NMDA-channel receptors, which generates a large influx of calcium However, it has been observed that the neonatal administration of fi- and activates various neurotrophic mechanisms that mediate neuronal nasteride causes a decrease in the time and number of entries in the proliferation, migration and differentiation processes (see Owens and open arms of the EPM in adult animals that have undergone stereotactic Kriegstein, 2002 for a review). The depolarizing profile of GABA is surgery (Martín-García et al., 2008). Concerning the behavior induced promoted by the higher concentration of intracellular Cl-contained in by novelty, in our laboratory we have observed that neonatal treatment immature neurons, since the sodium-potassium-chloride cotransporter with finasteride can induce a decrease in hole exploration in the Bois- type 1 (NKCC1), which transports Cl-within the cell, is expressed much sier test during adolescence (Darbra and Pallarès, 2010). In contrast, a more than the KCC2 potassium-chloride cotransporter (which extracts single administration of AlloP in the early postnatal stage can cause an the Cl-out of the cell). This causes the intracellular accumulation of this increase in the exploratory behavior induced by a new environment in anion. In contrast, an increase in KCC2 expression is observed to the an open field (OF) in adulthood (Darbra and Pallarès, 2009). Regarding detriment of NKCC1 in the adult brain, providing the hyperpolarizing locomotor activity, other studies have shown that the neonatal ad- profile of the GABAergic system (Ben-Ari et al. 2007, 2012). Even so, it ministration of AlloP causes an increase in the activity induced by has to be kept in mind that this change in the response of the GABAA amphetamine during adulthood (Gizerian et al., 2006). This suggests receptor depends on the age and sex of the subject, in addition to the that this manipulation during the early postnatal period would increase area of the brain (Ben-Ari et al., 2007). However, it has been observed the sensitivity to the stimulant effects of this drug. Our group has also 3 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 Table 1 Effects of neonatal neuroactive steroid levels alteration on adult and adolescent behavior (Accioly and Guedes, 2019; Bayless et al., 2013; Bonansco et al., 2018; Dib et al., 2018; Jorge et al., 2005; Komine et al., 2017; Li et al., 2019; Locci et al., 2017; Muneoka et al., 2002; Shiga et al., 2016; Velásquez et al., 2019). Abbreviations: PN, postnatal day; PN0, day of birth; s.c., subcutaneous injection; i.p., intraperitoneal injection; EMS, early maternal stress; PREGS, pregnenolone sulfate; THDOC, tetrahydrodeoxycorticosterone; EPM, elevated plus maze; OF, open field; PPI, prepulse inhibition; ORT, object recognition test; CPP, conditioned place preference; NOP, novel object preference. (continued on next page) 4 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 Table 1 (continued) (continued on next page) 5 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 Table 1 (continued) conducted studies on prepulse inhibition (PPI). This is a pre-attentional both experiments could have been due to the stages of development task consisting of a weak stimulus (prepulse) preceding a stronger sti- (postnatal day 30 vs. postnatal day 90) in which progesterone was mulus (pulse) that generates a startle reaction. Under normal conditions administered as well as the behavioral test was carried out. In this the prepulse produces an attenuation of the startle response reflex sense, we had already observed an effect of age on the behavioral caused by the pulse. The neonatal administration of AlloP both injected evaluation, regardless of neonatal treatment, in our laboratory. Speci- acutely (Gizerian et al., 2006), or during several consecutive days fically, the hole exploration levels in the Boissier test were higher in (Darbra and Pallarès, 2010; Llidó et al., 2013), has been observed to postnatal day 40 than in postnatal day 60 (Darbra and Pallarès, 2010). decrease PPI in adults. Altering the PPI has been linked to affecting the These results show that the specific behavioral effects observed during a dopaminergic system and mesocortical and prefrontal cortex GA- period of development do not necessarily have to manifest in other BAergic systems (Swerdlow et al., 2001; Gizerian et al., 2006) in ad- maturation stages (Doremus-Fitzwater et al., 2012; Barbayannis et al., dition to the dorsal and ventral hippocampus (Zhang et al., 2002). Thus, 2017; Blume et al., 2018). However and as explained in section 2, an- the behavioral results obtained in the PPI test are consistent with some imals subjected to EMS stress had low exploration scores in the hole- of the structural alterations observed after the manipulation of neonatal board exploration test (Boissier test) during adolescence, which was NS levels (see section 3.1). neutralized or prevented by the neonatal administration of AlloP prior Other studies have focused on observing behavioral effects in ad- to separation (Llidó et al., 2013). In summary, quite a lot of evidence verse situations, such as stress, after manipulating neonatal NS levels. shows that manipulating neonatal levels of NS, such as AlloP, modifies As previously mentioned, acute stress increases the cerebral and plasma the maturation of the nervous system at a structural and functional concentrations of AlloP in rodents (Purdy et al., 1991; Barbaccia et al. level, which is reflected at the behavioral level. A systematic review of 1996, 1997; Cozzoli et al., 2014). This kind of response has also been the main studies modifying neonatal NS levels and the corresponding observed in experimental studies in humans, since some types of stress, behavioral effects is presented in the Table 1. such as psychosocial stress, can increase circulating levels in healthy humans which do not present a lower basal AlloP concentrations 4. Why to study the alteration of neonatal allopregnanolone and (Girdler et al., 2001; Droogleever Fortuyn et al., 2004; Crowley and its possible effect on alcohol abuse vulnerability? Girdler, 2014). In this way, it has to be kept in mind that the nature of the stressor may influence the magnitude of AlloP response in humans, First of all, it is important to highlight that if applying early post- as well as the moment when AlloP is sampled and because of inter- natal stress can alter plasmatic and cerebral AlloP levels (Frye et al., individual characteristics. Moreover, periods of neurosteroid fluctua- 2006; Kehoe et al., 2000) (see section 3.1), and neonatal stress has been tion, such as during the ovarian cycle, can influence that reactivity of considered to be a key factor that increases the vulnerability to devel- AlloP in acute stress situations and it may be linked to alterations in the opmental alterations and adult neuropsychiatric disorders (see Bale sensitivity of GABAA receptors to NS, resulting in mood instability in et al., 2010; Bock et al., 2014; Chen and Baram, 2016 for a review), it is vulnerable women, such as those with premenstrual dysphoric disorder reasonable to consider that early postnatal alterations in AlloP levels (Crowley and Girdler, 2014). Interestingly, in our laboratory, we have are an important factor to be considered for studying the development observed that male wistar rats subjected to forced swimming during of behavioral disorders. adulthood show an increase in plasma levels of AlloP, which are not observed if finasteride is previously administered (Pallarès et al., 2015). 4.1. What has previously been observed at a behavioral level? One way to mimic the increase in AlloP levels observed after acute stress is by administering its precursor, progesterone (Moran and Smith, In relation to drug abuse, as mentioned above (see section 3.2), 1998; Pisu et al., 2016). In this way, it has been previously reported that altering early postnatal AlloP levels can affect novelty exploration and the administration of progesterone during early adolescence induces an anxiety. Interestingly, an increase in novelty exploration has been re- anxiolytic-like behavior in the EPM regardless of previous neonatal NS lated to a novelty or sensation seeking pattern, which has been linked to levels manipulations (Bartolomé et al., 2017). Nevertheless, our group vulnerability to drug use and/or abuse (Belin and Deroche-Gamonet, has documented that administering progesterone during adulthood 2012; Deroche-Gamonet and Piazza, 2014; Manzo et al., 2014). It has causes an anxiogenic profile in EPM in those adult animals neonatally been proposed that novelty seeking (tendency to explore new en- treated with finasteride (Modol et al., 2014). Therefore, altering neo- vironments) and sensation seeking (tendency to search intense emo- natal levels of NS by inhibiting the enzyme 5α-reductase may modify tional experiences) are two related (Arnett, 1994; Belin and Deroche- sensitivity to the anxiolytic effects caused by administering this AlloP Gamonet, 2012; Belin et al., 2016) but dissociable traits (Cain et al., precursor during adulthood. These differences between the results of 2005; Belin et al., 2001; Flagel et al., 2014). Sensation seeking is usually 6 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 inferred by measuring locomotor activity in a new environment (Dellu adulthood (Holdstock et al., 2006; Pierucci-Lagha et al., 2006). Thus, et al., 1996; Piazza et al., 1989), whereas novelty seeking is studied by making a comparative study between different ages and alcohol con- determining the preference for new versus familiar environments using sumption levels would be interesting. the conditioned place preference or the new object preference tests In parallel, the systemic administration of AlloP leads to an increase (Belin et al., 2016; Ennaceur et al., 2010; Parkitna et al., 2013; in alcohol intake in rats that had low levels of basal consumption, and Montagud-Romero et al., 2014; delaPeña et al., 2015). These two be- decreases alcohol ingestion in rats with high levels of alcohol con- havioral traits have been related to different aspects of drug abuse. It sumption (Janak et al., 1998; Morrow et al., 2001), even when AlloP seems that a sensation seeking pattern could be related to the acquisi- was injected directly into the hippocampus (Martín-García et al., 2007). tion of drug intake (Piazza et al., 1989; Klebaur et al., 2001; Belin et al., In other brain structures, such as the ventral tegmental area, the over- 2008), whereas novelty seeking has proven to be important for drug expression of the steroidogenic enzyme cytochrome P450 side chain abuse in addition to the severity of addiction behavior (Belin et al., cleavage increased AlloP and reduced long-term operant ethanol self- 2011; Belin et al., 2016; Flagel et al., 2014; Foulds et al., 2017). A administration in rats (Cook et al., 2014b). In mice, although differ- possible link between sensation/novelty seeking and drug use/abuse ences in the two sexes have been detected, a dose-response relation has can be related to dopamine release in the nucleus accumbens. It has been proposed in several alcohol intake paradigms: low AlloP doses been documented that the animals who showed more locomotion in (3.2 mg/kg i.p.; 50 ng i.c.v.) increased EtOH ingestion, and high doses response to novelty also showed higher levels of mesolimbic dopamine (17 or 24 mg/kg i.p.; 400 ng i.c.v.) decreased it (Ford et al., 2005, 2007, release (Flagel et al., 2010; Hooks et al., 1991; Marinelli and White, 2008, 2015). These results suggest that changes in central AlloP pro- 2000), and similar results have been obtained in Roman High Avoid- duction as a consequence of alcohol ingestion may be involved in some ance rats (Tournier et al., 2013). aspects of the alcohol abuse spectrum, such as the development of tolerance. Therefore, it has also been proposed that the anxiolytic ef- 4.2. What has previously been observed at a neurochemical level? fects of alcohol are, at least in part, mediated by the increase in central AlloP levels induced by the drug (Hirani et al., 2005). It is important to note that early-postnatal manipulations of neu- In conclusion, taking these results together, it seems conceivable roactive steroid levels by administering progesterone, the main pre- that alterations in AlloP levels during development can affect drug in- cursor of AlloP, affect the normal development and maturation of the take or drug seeking behaviors, and alcohol abuse is a reasonable study dopaminergic meso-striatal systems (Muneoka and Takigawa, 2002; subject. Muneoka et al., 2010). These results are similar to those previously reported in neonatally AlloP-treated rats (Muneoka et al., 2009b). It has 5. The effects of altering neonatal allopregnanolone levels on been observed that the administration of progesterone increases cor- alcohol abuse tical and plasmatic AlloP levels in animals (Pisu et al., 2016) as well as in the brain of progesterone-treated neonates (Palliser et al., 2015). The The developing brain shows a variety of neuroplasticity responses developing brains can synthesize AlloP from progesterone (Pomata that are not normally shown in adult age. Several mechanisms con- et al., 2000) and, furthermore, the action of progesterone on many tribute to the equilibrium between neuronal plasticity and homeostasis neuronal receptors, such as GABAA, seems to be mediated by its re- in the developing brain, which allow synaptic stability and flexibility duced metabolite AlloP (Melcangi et al., 2014; Schumacher et al., (Johnston, 2004). Adverse events or pathological states can alter the 2014). Thus, it is conceivable that at least part of the dopaminergic normal homeostasis in the developing brain and induce an aberrant alterations observed in those progesterone-treated animals were in- neuroplastic effect that can contribute to a neurophysiologic or beha- duced by AlloP. Thus, if neonatal AlloP manipulation affects the ma- vioral abnormal phenotype (Ismail et al., 2017). Recent works carried turation of dopamine systems, then it is conceivable that these animals out in our laboratory have shown that neonatal manipulation of AlloP have alterations in drug seeking behavior. levels by administering finasteride increased the consumption of a On the other hand, GABAA receptors participate in regulating ex- sweetened alcohol solution (10% EtOH + 3% glucose) in non-selected citability of the dopaminergic neurons in the ventral tegmental area adult male rats (Bartolomé et al., 2018; Llidó et al., 2016a, 2016b). This (Xiao et al., 2007). Regarding this receptor, previous experiments car- increase in alcohol intake was reported in the initial period of con- ried out in our laboratory showed that early postnatal AlloP manip- sumption, specifically in a two-week procedure of EtOH access. Thus, it ulation produced an over-expression of hippocampal GABAA receptors seems that early postnatal AlloP levels could be important for de- containing α4/δ subunits, and these changes were maintained in adult termining the initial sensitivity to alcohol seeking behavior in adult- age (Modol et al., 2014). This result is relevant for drug abuse vulner- hood. Furthermore, when monoamine levels are analyzed in ventral ability because this kind of GABAA receptor has been related to alcohol striatum by means of HPLC, we observed that the neonatal finasteride use. Several studies have shown that a low expression of α4/δ GABAA group consumed a higher amount of alcohol and had lower levels of receptors in the mesolimbic system seems to be related to low alcohol dopamine and serotonin in this brain structure compared to control consumption (Mihalek et al., 2001; Nie et al., 2011; Rewal et al., 2009, groups when they were sacrificed at the end of the two-week con- 2012). sumption period (Llidó et al., 2016a). Moreover, the levels of the main metabolites of dopamine (3,4-dihydroxyphenylacetic and homovanillic 4.3. The interaction between allopregnanolone levels and alcohol acid) and serotonin (5-hydroxyindoleacetic acid) were not affected. This indicates that animals neonatally treated with finasteride had an Another important factor to take into consideration is that alcohol increased turnover ratio of dopamine and serotonin, which possibly consumption or its administration (i.p.) increases brain cortical and indicates a decrease in dopaminergic and serotoninergic activities hippocampal AlloP levels in adult animals (Barbaccia et al., 1999; (Llidó et al., 2016a). Interestingly, the effects of neonatal finasteride VanDoren et al., 2000; Morrow et al. 2001, 2006). Moreover, the de- administration (postnatal days 5–9) on ethanol intake and ventros- velopment of tolerance to depressing alcohol effects has been linked to triatal dopamine and serotonin levels described above were neutralized a reduction in AlloP production (Janis et al., 1998; Morrow et al., by later EMS (24 h of maternal separation at postnatal day 10) (Llidó 2001). Regarding human studies, although AlloP levels increase in the et al., 2016a). This last result suggests that in the same way that the plasma of adolescent men and women after alcohol intoxication (Torres effects of neonatal stress can be neutralized or prevented by the pre- and Ortega, 2003, 2004), it has been observed that they don't increase vious application of AlloP (see section 2), the effects of an alteration in or even decrease when the doses of alcohol consumption are moderate AlloP levels produced by finasteride administration can also be modu- (Holdstock et al., 2006) and/or when the subjects have reached lated by the posterior application of postnatal stress and the consequent 7 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 increase in endogenous AlloP levels induced by it. order to better define a profile of vulnerability (searching for sensations In a parallel experiment, we measured dopamine release in the and/or novelty) together with evidence of drug use and abuse. nucleus accumbens during alcohol drinking in animals submitted to However, as we also observed decreased ventro-striatal levels of early postnatal manipulation of AlloP levels. Microdialysis results re- serotonin in the early postnatal finasteride treated rats, and serotonin vealed that dopamine release in the nucleus accumbens (expressed as a together with dopamine has been proposed to modulate the motiva- baseline percentage), which was increased in controls after alcohol tional properties of alcohol (Deehan et al., 2016) and participate in solution presentation, was not affected in neonatal finasteride treated alcohol abuse vulnerability (Barker et al., 2014; Yaakob et al., 2011). In animals (Llidó et al., 2016b). Accordingly, the overall increase in do- addition, 5HT3 receptors could regulate the brain function and network pamine release with respect to the baseline in response to solutions formation during early development (Engel et al., 2013). Thus, we in- (ethanol and glucose) and food presentation was lower in finasteride itiated a set of experiments focusing on the serotoninergic system. subjects than in control subjects (Llidó et al., 2016b). Taken together, Taking into account that AlloP, like the rest of neuroactive steroids, the ventral striatum HPLC and nucleus accumbens microdialysis results binds with high affinity to ionotropic receptors (for a review see seem to indicate a hypo-dopaminergic activity in early postnatal fi- Rupprecht, 2003), we decided to study possible alterations in the 5HT3 nasteride treated rats, which could be related to the increase in alcohol receptor function, the unique ionotropic subtype of serotonin receptors. drinking reported in these animals. Previous experiments have shown that AlloP at high doses negatively In this way, it has been described that a hypo-dopaminergic activity, modulates 5HT3 receptors (Wetzel et al., 1998), and it has also been induced either by a decrease in dopamine D2 receptors or by a de- described that mesolimbic dopamine release can be promoted by acti- creased presynaptic dopamine release in the striatum, leads to an ex- vating central 5HT3 receptors (for a review see Barnes and Sharp, cessive craving and seeking for substances known to cause dopamine 1999). In a first experiment, we analyzed the effects of ondansetron on release in nucleus accumbens (Blum et al., 1996, 2014; Trifilieff and alcohol consumption (in the previously mentioned two-week free access Martínez, 2014). Several studies in rats have shown that lines selec- procedure) in animals submitted to the manipulation of AlloP levels in tively bred for high ethanol preference had a decreased dopamine D2 the early postnatal period. Ondansetron is a 5HT3 antagonist that has receptor density in the nucleus accumbens (McBride at al. 1993), and been shown to effectively reduce alcohol intake in rats in free choice lower dopaminergic innervation (Casu et al., 2002; Zhou et al., 1995), conditions, i.e., when the animals can choose between drinking a with lower dopamine content in the nucleus accumbens (McBride et al., sweetened alcohol solution or drinking a sweetened control solution 1995; Murphy et al., 1982). Similarly, alcohol addiction in humans has (Heiling and Egli, 2006; McKinzie et al., 1998; Sari et al., 2011). Al- been related to significant reductions in D2 receptor availability as well though its efficacy varies depending on drinking conditions after sta- as reduced dopamine release in the striatum (see Volkow et al., 2009 for bilization of ethanol intake, no studies have focused on their possible a review). Therefore, our data could indicate that the higher ethanol effects in the initial period of ethanol intake (for instance the first two consumption in neonatal finasteride treated rats might be related to a weeks of consumption), which is related to the initial alcohol seeking blunted dopamine response in ventral striatum and nucleus accumbens. behavior. In our experiment, the highest dose of ondansetron (0.1 mg/ Precisely, early postnatal finasteride administration decreased the kg) tested in male wistar rats decreased the consumption of alcohol, but locomotor activity induced by a low ethanol dose (0.5 g/kg), thus re- only in the animals that were administered finasteride postnatally, ducing the sensitivity to the stimulant motor effects of alcohol which were the ones that consumed the highest amount of alcohol (Bartolomé et al., 2017). Reduced sensitivity to ethanol stimulating (Bartolomé et al., 2018). In control animals, the initial alcohol intake effects has been related to dopamine inhibition (see Brabant et al., 2014 was not affected by ondansetron administration. As previously pro- for a review). In accordance whit this, our results indicate that neo- posed, it seems that 5HT3 antagonists could be effective or ineffective natally finasteride treated rats: (1) have lower dopamine levels in the depending on several variables such as the dose, the ethanol drinking ventral striatum and release in nucleus accumbens; (2) exhibit lower conditions (initial versus stabilized), the duration of the treatment, or sensitivity to the motor stimulant effects of a low alcohol dose; and (3) the sex or strain of the animals (McKinzie et al., 1998; Knapp et al., show higher alcohol consumption in the two-week access procedure. 1992; Moore et al., 2014; Pei et al., 1993). Nevertheless, the fact that Thus, following the hypo-dopaminergic hypothesis (Blum et al., 1996, ondansetron was effective in our finasteride animals but not in controls 2000; Febo et al., 2017), it is reasonable to think that finasteride ani- suggests that 5HT3 receptors work differently in these animals as a mals consumed greater amounts of ethanol in order to compensate the consequence of the neonatal treatment, making them more sensitive to deficiency of mesolimbic dopamine; however, more experiments are the effects of ondansetron. Thus, alterations in early postnatal ser- needed to confirm this (see section 6). otoninergic pathways including 5HT3 receptors, together with those Moreover, in order to characterize the effects of early postnatal previously described that affect dopaminergic systems (Muneoka and administration of finasteride on drug use/abuse behavior, we evaluated Takigawa, 2002; Muneoka et al., 2009), could be related to the increase the novelty object preference, since the trait of novelty seeking has been in ethanol intake in initial periods and the corresponding higher alcohol related to both consumption and substance abuse (Deroche-Gamonet seeking behavior observed in the neonatal finasteride treated animals. and Piazza, 2014, Manzo et al., 2014; Belin et al., 2016) (see section 4). The results of the ondansetron experiment suggesting that 5HT3 The results indicated that animals neonatally treated with finasteride receptors could be more sensitive to antagonists in neonatal finasteride had a higher preference index for exploring the new object versus the animals, together with the possible role of these receptors in brain familiar one (Bartolomé et al., 2018). Consequently, this increase in the maturation, led us to study the expression of 5HT3 receptors in the preference for novelty could indicate that these subjects are more vul- early postnatal period. Therefore, we analyzed the density of 5HT3 nerable to consuming addictive substances, which would be related to receptors by means of Western Blot analyses in the midbrain of rats the increase in EtOH consumption exhibited by these animals. How- submitted to neonatal manipulation of AlloP. Preliminary results of our ever, as we have already mentioned, in previous studies we have ob- group (Bartolomé, 2018) indicate that early postnatal administration of served that animals neonatally treated with finasteride have low levels either AlloP or finasteride alters the pattern of 5HT3 expression in male of both exploratory behavior and locomotor activity with respect to the Wistar rat offspring. Specifically, neonatal AlloP administration an- control group (Darbra and Pallarés, 2010; Mòdol et al., 2013). This ticipated the normal peak of 5HT3 expression (shown in controls) from suggests that the neonatal administration of finasteride can produce a postnatal day 12 to postnatal day 9, whereas the 5HT3 receptor den- novelty but not sensation seeking profile. This result would be in ac- sities in the finasteride treated group did not show statistical differences cordance with the hypothesis that proposes that these two traits are between the different days compared to control subjects. These results dissociable (Cain et al., 2005; Belin et al., 2012; Flagel et al., 2014). can be seen in Fig. 1. However, it would be interesting to carry out other behavioral tests in Therefore, these preliminary results seem to highlight the possible 8 I. Bartolomé, et al. Neurobiology of Stress 12 (2020) 100208 Fig. 1. Percentage of 5HT3 protein receptor expression. A) 5-HT3 expression is different between PN6 and PN12 in control vehicle group (t = −2.73; p < 0.05; the differ- ence is indicated by the symbol: *). Instead, 5-HT3 expression is different between PN6 and PN9 in Allopregnanolone group (t = −2.3; p < 0.05; the difference is in- dicated by the symbol: #). On the other hand, there were no statistically significant differences between the different days of samples extraction in Finasteride group. B) Western Blot images. role played by serotonin systems containing 5HT3 receptors in the ef- life has been proven to affect the adult response to novel environmental fects of early postnatal manipulation of AlloP levels on adult alcohol stimuli, anxiety-like behaviors and the processing of sensory inputs. intake in initial periods of consumption, and thus on initial ethanol Moreover, recent studies indicate that early postnatal AlloP could play seeking behavior. an important role in vulnerability to alcohol consumption. As evidence However, to our knowledge, there is very little information in the of this, it has been observed that inhibiting the enzyme 5α-reductase, literature about the role of early postnatal manipulation of AlloP levels which participates in AlloP synthesis, by administering finasteride in on the consumption or effects of the abuse of drugs other than alcohol. the early postnatal period: (1) increases initial alcohol consumption and The only exception is a study that showed that AlloP administration at the preference for novelty in adult age; (2) decreases the locomotor postnatal day 2 or 5 increased locomotor response to amphetamine stimulant effects of low alcohol doses in adults; (3) promotes a hypo- (28% and 20% respectively) in adulthood (postnatal day 80) (Gizerian dopaminergic profile in adulthood that is characterized by a decrease in et al., 2006). This suggests that possible variations in neonatal AlloP dopamine levels in the ventral striatum and in dopamine release in the levels can be important for determining individual differences in the nucleus accumbens in response to alcohol and/or food presentation; response to psycho-stimulant drugs, such as amphetamines, and the and (4) increases the 5HT3 receptors’ sensitivity to their antagonist subsequent vulnerability to their abuse or addiction. ondansetron for reducing alcohol consumption, probably by altering early postnatal 5HT3 receptor expression. Thus, taken together, these results highlight the important role played by fluctuations in AlloP le- 6. Perspectives vels during the first two weeks of life for modulating adequate neuro- developmental processes to prevent behavioral alterations related to Based on the above evidence it is clear that a set of new experiments neuropsychiatric and neurologic diseases. are needed to determine the role of early postnatal neurosteroids, particularly AlloP, on drug seeking behavior and vulnerability to drug Acknowledgements use or abuse in adult ages. 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