In both humans and laboratory animals, females and males can respond differently to cocaine. Women can progress more rapidly from initial cocaineuse to addiction. Studies in laboratory rodents have also demonstrated that females can be more vulnerable to the reinforcing and incentive motivational effects of cocaine. Most preclinical studies characterizing the effects of cocaineusein females and males have been conducted using continuous-access self-administration procedures [e.g., (6 h sessions), Long Access or ShA (1-2 h sessions), Short Access]. These procedures achieve produces high brain concentrations of drug. However, human addicts take cocaine intermittently during a bout of intoxication, and this would produce spiking brain cocaine levels. A recent intermittent-access (IntA) cocaine self-administration procedure models this inrats. Compared to LgA, IntA self-administration is uniquely effective in producing the neurobiological, psychological and behavioral changes that underlie the transition to cocaine addiction. Here, we compared cocaineusein female and male rats that self- administered the drug (0.25 mg/kg/ infusion, i.v.) during 10 daily, 6-h LgA or IntA sessions. LgA rats took more cocaine than IntA rats, and only female LgA rats escalated their intake. However, only IntA rats (both sexes) developed psychomotor sensitization and sensitization was greatest in the females. Five and 25 days after the last self- administration session, we quantified incentive motivation for cocaine by measuring breakpoints for the drug under progressive ratio under progressive ratio schedule. There were no significant sexdifferences on this measure in LgA rats. However, under IntA, females reached higher breakpoints for cocaine than males. Thus, LgA might be best suited to study sexdifferencesincocaine intake, while IntA might be best suited to study sexdifferencesin incentive motivational processes incocaine addiction.
Toxicological Evaluation of Perinatal Exposure to BPA. Intestinal paracellular permeability is high at birth and mucosal immune mechanisms are immature in neonates. A high-permeability state in early infancy reﬂects the development of thegutmucosal barrier (38) that precedes a period of epithelial TJ sealing to provide an effective barrier against foreign agents (19, 20). BPA ingested by mothers is present in fetal tissues (39) and human milk (40), and leakage of BPA from polycarbonate baby bottles under real use conditions has been demonstrated (6). As ERβ is the only ER detectable in human fetal colon (41), we speculated that a BPA-mediated alteration of gut permeability in the perinatal period of life may have restricting effects on the developing immune system in the colon. Indeed, the maturing gut is highly permeable to sugars and proteins from human milk to promote infant growth (19, 20), as well as to nucleotides that improve infant immune status (21). Mice prenatally exposed to BPA show immune system impairment, particularly regarding the devel- oping T cell population (42). We showed that rats exposed in utero and during lactation to BPA NOAEL ingested by their dams ex- hibited intestinal barrier dysfunctions in adulthood, with sex-related differencesin gut susceptibility to BPA effects. First, we observed decreased colonic paracellular permeability in the female offspring, as reported herein in OVX rats fed with oral BPA at NOAEL or TDI, but not in male offspring. Furthermore, we showed that neu- trophil inﬁltration in the colon faced with inﬂammation was dra- matically enhanced in female rats only. The latter correlated with an increase in the tissue levels of MIF, a stimulating factor of proin- ﬂammatory cytokines in acute colitis (28). It is noteworthy that, in contrast with the antiinﬂammatory effect of a direct NOAEL ex- posure in OVX rats, the same dose administered to pregnant and lactating mothers resulted in opposite effects in female offspring in adulthood. It is also remarkable that this deleterious impact was observed during estrus, i.e., at a time of the reproductive cycle when the colon of adult females is more resistant to injury, and produces less proinﬂammatory cytokines when challenged by experimental inﬂammation, as a result of a protective inﬂuence of plasma estrogen dominance (22, 28). Our results clearly show that the endocrine disruptor effects of BPA vary depending on age of the gut, and that a perinatal exposure has a long-term effect on mucosal immune de- fenses that predisposes female offspring to enhanced proin- ﬂammatory responses in the colon. Interestingly, male offspring perinatally exposed to BPA showed decreased ERβ expression inthe Fig. 6. (A) Intestinal permeability and (B) relative expression levels of ER α
Figure 3: Novelty preference, but not locomotor reactivity to novelty, predicts the development of cocaine addiction-like behaviour.
Not only did HNP rats show higher addiction score than LNP rats (A) but they scored higher on each of the three addiction-like criteria, namely motivation for cocaine (measured by the breakpoint during a progressive ratio session) (B) and inability to stop seeking cocaine when it was not available and signalled as so (non reinforced active nose-pokes) (C) and resistance to punishment (maintenance of drug use and drug seeking despite contingent electric foot-shocks) (D), Compared to LNP rats, HNP rats even displayed a progressive development of this inability to refrain from drug seeking over time that has been previously described for 3crit addicted rats (E). These behavioural differences between HNP and LNP rats could not be attributable to differential cocaine intake since the two groups have been exposed to the same amount of cocaine throughout the experiment (F). When compared to LR rats, HR rats showed no difference in the addiction-like behavioural measures, thereby illustrating that locomotor reactivity to novelty, as opposed to novelty preference, does not predict addiction-like behaviour for cocaine.
causes. First, sex-specific exposure to pollutants could result from sexdifferencesin spatio-
temporal use of urban areas: in Basel (Switzerland), male feral pigeons spent less time outside
the loft and flew shorter distances than females (maybe due to sex-specific constraints, such
as territory defence in males vs. food search in females; Rose et al. 2006), potentially
characteristics can play a major role in explaining different findings.
Overall, we found that 89.9% of the participants already knew what PrEP is. This is a large proportion compared to earlier studies (Bil et al., 2015 ; Frankis et al., 2016 ; Grov et al., 2016 ), suggesting that knowledge of PrEP is increasing over the years. Despite the high level of PrEP knowledge, we found low actual use of PrEP (6.6%). This is comparable to findings of other studies in Europe (Bourne et al., 2019 ), and globally (Kamitani et al., 2018 ), and likely reflects the early stages of PrEP implementation. Nevertheless, about half of the participants (45.3%) were interested in taking PrEP, and a similar number of participants (46.9%) had the intention to use PrEP when available. This is in line with the major- ity of earlier studies, reporting a willingness to use PrEP among about 50% of participants, as described in a review by Young and McDaid ( 2014 ). Covariates of interest in PrEP were being single, having correct prior PrEP knowledge, not having used a condom for last sex, having ever used drugs in a sexual context, and not being participant of the Amster- damPinkPanel. The same covariates were found for intention to use PrEP, except for the covariate having correct prior PrEP knowledge. In contrast with the notion that there is a clear distinction between willingness to take PrEP and inten- tion to take PrEP (Rendina et al., 2017 ), we did not encounter this distinction in our results. Because PrEP had only limited availability in the Netherlands at the time of our study, PrEP use was fairly distant for most MSM and, therefore, it may not have been possible to find this fine distinction between interest in PrEP and intention to use PrEP, as was found in the U.S.-based study of Rendina et al. ( 2017 ).
This differential response to sex steroids develops during ontogeny under the inﬂuence of estrogenic ovarian secretions in females during a critical period that ends on day 12 of incubation ( Adkins, 1975 ; Adkins-Regan et al., 1982 ; Balthazart and Adkins- Regan, 2002 ). This process can be pharmacologically manipulated in both sexes to obtain in adult birds a male or female behavioral phenotype irrespective of the genetic sex of the subjects. Speciﬁ- cally, injections of estradiol benzoate into male embryos demas- culinizes (i.e., completely suppresses) adult copulatory behavior, whereas injections of an aromatase inhibitor in female embryos block demasculinization and lead to adult females that show the full range of male sexual behavior if exposed to exogenous T ( Balt- hazart and Adkins-Regan, 2002 ; Balthazart et al., 2009a ). During embryonic life, ovarian estrogens thus block the subsequent abil- ity of female quail to perform masculine sexual behavior whereas the male phenotype seems to develop in the absence of hormonal inﬂuences. This pattern of sexual differentiation is based on simi- lar principles but is opposite of the mammalian pattern, in which steroids secreted by the male, not the female, play the critical role in the sexual differentiation of reproductive behaviors. Male rats are masculinized and defeminized by T secreted by the embryonic testes, even if T exerts most of its effects on brain and behavior after being aromatized into an estrogen ( Goy and McEwen, 1980 ;
unusual longevity (more than 80 years old) have revealed lower testosterone in the survivors, itself related to lipid and lipoprotein metabolism (metabolism of fat). Thinner people have significantly longer life expectancy. The life-preserving effects of post-menopausal estrogen include resistance to osteoporosis and subsequent hip injuries, and resistance to coronary heart disease. Though there are life-threatening effects of estrogen replacement therapy, including endometrial and breast cancer, these risk factors seem to be calculable from a woman's genealogy, and can be weighted into the decision, such that overall, we are looking ahead to an even greater chasm between the longevities of the two sexes, especially if we assume that smoking in women will reach a plateau soon. A recent multi-center large scale study found that post-menopausal estrogen replacement therapy prevented mortality by 17.4% in women without heart diseases at outset, and by a whopping 52.55 % in women with heart disease at outset ! Since only about 20% of North American women are now opting for estrogen replacement therapy, imagine the effect full conversion would have on life expectancy ! There seem to exist metabolic sexdifferences, in mammals, due to gonadal hormone action, which cause diffential risk for hypertension. For example, one study determined the effects of gonadectomy on the pathogenesis of hypertension. Male and female rats were gonadectomized at age three weeks. In intact rats, hypertension developed more rapidly and to a higher level in males than in females. By contrast, in gonadectomized rats, there was no sex difference in blood pressure because the development of hypertension was attenuated in males and exacerbated in females. None of these differences could be attributed to differencesin either saline consumption or vasopressin release since no differences were found among the groups for either variable. Although the underlying mechanisms remain uncertain, the results of this investigation clearly show that the gonadal hormones affect the development of hypertension in the rat.
in control women for either the right hand [22, 23] or for both hands [24, 25], although a “feminine” digit ratio in CAH was reported in yet another study . Individ- uals with Klinefelter syndrome, who have 1 Y- and 2 or more X-chromosomes and might have reduced fetal an- drogen exposure, showed a higher, i.e., “feminized”, digit ratio than control men . A study in 16 women with CAIS showed that their digit ratios were feminized, which, along with the majority of findings in DSDs, points to a link between fetal androgen exposure and the 2D:4D ratio . However, some important caveats have been discussed by the authors of this study  and in a commentary on this paper . First, the effect size of the difference between women with CAIS and control men was only moderate, and second, the vari- ability in digit ratios was not smaller in women with CAIS than in either the male or female control subjects. If fetal androgen exposure alone determines 2D:4D ra- tios, then, one would expect less variability in women with CAIS because they are completely insensitive to any androgen actions. This has led to the conclusion that 2D:4D digit ratio is partially determined by fetal an- drogen exposure but that the relationship is too small to use digit ratio as a reliable marker for fetal androgen ex- posure in individual subjects (as cited in ). Regard- less of this conclusion and other important critical arguments against AR-gene-related effects (summarized by ), numerous investigators continue to use the digit ratio as an index of fetal androgen action. The study by Berenbaum et al.  included a sample of only 16 women with CAIS, which is a reasonable sample con- sidering the rareness of the syndrome. However, the stat- istical power of the comparisons made with control subjects was quite low. For this reason, we sought to replicate this study using a larger sample of CAIS women as well as control men and women. Thus, this is the first study analyzing both CEOAE and 2D:4D in women with CAIS.
Chapter 1 Introduction
1.1 Imaging Modalities
Aging is associated with an increasing incidence of cerebrovascular, cardiovascular and neurodegenerative diseases (Niccoli and Partridge 2012; Harman 1990; Najjar et al. 2005). Recent advances in neuroimaging techniques have now begun to improve our understanding of the effects of aging on the brain, and how this is linked to the pathogenesis of diseases of aging. Age-related cerebrovascular alterations have been reported with the use of arterial spin labeling (ASL), a magnetic resonance imaging based-technique used for quantifying local perfusion in the brain (Leoni et al. 2017; Badji et al. 2019; Gauthier et al. 2013; Grade et al. 2015). Noninvasive ASL techniques directly estimates cerebral blood flow (CBF), the rate of delivery of arterial blood to the capillary bed in brain tissue, by applying a magnetic tag to inflowing arterial blood at the carotid level and measuring the delivery of tagged blood into each imaging voxel (Zhang et al. 2017). The magnetic tag is applied by inverting the magnetization of blood water using radiofrequency (RF) pulses that are similar to those used in the MR imaging process (Leoni et al. 2017; Williams; Intzandt et al. 2019; Grade et al. 2015). In addition, blood oxygenation level dependent (BOLD) MR imaging has been used in the aging literature as a common means of observing changes in the functional state of the brain (D'Esposito et al. 2003). This technique is based on the T2*‐weighted signal, to indirectly measure flow changes by observing changes in the local concentration of oxygenated and deoxygenated hemoglobin in the brain (Golestani et al. 2016; Williams). The BOLD signal is however ambiguous in nature, as it simultaneously reflects changes in oxidative metabolism, blood flow and blood volume (Gauthier et al. 2013; Gauthier and Fan 2019). This ambiguity can be harnessed however to obtain measures of vascular health. Since the BOLD contrast is highly dependent on the local blood volume and flow, it can be used in combination with a vasoactive challenge such as breathholding or inhalation of carbon dioxide (CO 2 ) to map quantitative cerebrovascular
Over the last two decades, misuse of prescription drugs (e.g. opioids, tranquilizers, stimulants, antipsychotics, etc.) has become a major public health concern in various parts of the world due to the increasing number of people who abuse them (SAMHSA, 2010a) and the associated harmful health and social consequences (Huang, Dawson, Stinson, Hansin & Ruan, 2006; SAMHSA, 2004). Prescription drug misuse, particularly that of opioids and tranquilizers, has been associated with numerous risk behaviours and harms such as dependence, mental health problems, fatal and non-fatal overdose, initiation into injection drug use, syringe sharing and unsafe sex (Hayashi, Suwannawong, Ti, Kaplan, Wood, & Kerr, 2012; Lake & Kennedy, 2016; Kecojevic, Silva, Sell & Lankenau, 2014; Lankenau et al., 2007; Lankenau, Teti, Silva, Bloom, Harocopos, & Treese, 2012a; SAMHSA, 2004, 2010b; Tucker et al., 2016). Public health programs have been particularly challenged by people who usecocaine (PWUC) whose complex drug use patterns often involve polydrug consumption that includes non-medical use of prescription drugs (Fischer et al., 2010; Guindalini, Vallada, Breen, & Laranjeira, 2006; Latkin, Knowlton, & Sherman, 2001; Prinzleve et al., 2004; Roy et al., 2012; Shaw, Shah, Jolly, & Wylie, 2008). A significant area of concern is the frequent misuse by this population of psychotropic medications 1 (PM) such as tranquilizers, sedatives, stimulants and antipsychotics; a problem compounded by high rates of mental health problems among these individuals (Conway, Compton, Stinson, & Grant, 2006; Roy et al., 2015). In the North American context where cocaine misuse plays a major role in the HIV and hepatitis C virus (HCV) epidemics (Edlin et al., 1994; Nelson, Galai, Safaeian, Strathdee, Celentano & Vlahov, 2002; Patrick et al.,
The shunt apparatus consisted of silicone tubing (0.040 ” ID × 0.085” OD Silclear tubing, Degania Sili- cone, VWR International, Benelux) mounted on a Plexi- glas stand with secure mounts for the T-connections (Harvard Apparatus Standard tube T-connector #72- 9275 1.5 mm ID, Harvard University, Cambridge, MA, USA) and a secure, adjustable, mounting point for a microprobe, as illustrated in Figure 1. Plexiglas was cho- sen for the stand as it could readily be cleaned and would not corrode after contact with saline. The T-con- nections and microprobe are clamped in position with custom machined Plexiglas clamps, such that the appa- ratus can readily be dismantled for cleaning or replace- ment of parts but with precise reassembly. The clamp for the microprobe clamps at the solid base of the probe and is height adjustable via two machined slots to account for differencesin probe lengths. The shunt was prefilled with 250 U/ml heparinized saline (volume approximately 400 μl). The probe connection was situ- ated such that arterial blood first passed over the probe tip for counting, followed by the peristaltic pump, to prevent back-flow when infusing via the second T-con- nection. Finally, the blood was pumped back into the animal via the femoral vein. A probe was fixed with its tip in the blood flow of the shunt for the measurement of IF. In this position, the probe has a measured sensi- tivity of 0.045 cps/kBq/ml (4.5%). The sensitivity of the probe is a function of crystal size at the measuring tip and detection volume. The detection volume is depen- dent on the energy of the radioisotope used and is approximately spherical around the probe tip . In the shunt apparatus, the detection volume is partially filled with blood. Utilization of the full detection volume would require larger T-connections and therefore sub- stantially increase the dead volume.
Sexdifferencesin the eCB system may be involved in the aforementioned effects. Cortical CB1R expression and function are higher in juvenile male rats (PND 28–35) as compared to adolescent sex-matched subjects (PND 40), and CB1R levels decrease thereafter towards young adult levels (PND 70; Heng et al., 2011 ). Compared to females, male rats have a higher density of CB1R. However, a higher G-protein activation after CB1R stimulation is observed in adolescent females in several brain areas ( Rubino et al., 2008; Burston et al., 2010 ). Additional molecular mechanisms may help explain the observed sexdifferences. Sexual differencesin the eCB system appear early in development in rodents ( Craft et al., 2013 ). Sexually dimorphic regulation of synaptic plasticity or intrinsic neuronal activity in the amygdala ( Fendt et al., 2013; Chen et al., 2014; Bender et al., 2017 ), hippocampus ( Huang and Woolley, 2012; Inoue et al., 2014; Harte-Hargrove et al., 2015; Qi et al., 2016 ) and PFC ( Nakajima et al., 2014; Li et al., 2016 ) has been described. Female rats exhibit greater concentrations of the metabolic enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) as early as PND 4 compared to males ( Krebs-Kraft et al., 2010 ). Moreover, CB1R expression reaches its peak earlier in females (PND 30) than in males (PND 40; Romero et al., 1997 ), whereas at adulthood, CB1R density is lower in the PFC and amygdala of cycling females ( Castelli et al., 2014 ). Finally, it was recently shown that the CB1 agonist CP 55, 940 has widespread effects on the brain lipidome in adolescent female mice ( Leishman et al., 2018 ). Thus, females’ eCB systems appear more sensitive to the deleterious influence of exogenous cannabinoids.
studies finding a positive correlation tended to be conducted earlier. These studies however,
concern Scandinavian populations with generally higher T-C levels than other ethnic white
population and use of logistic regression and lack of adjustment for vascular factors could be
problematic. LDL-C was only examined in the Northern Manhattan study which also found no
These sex dependent effects of progesterone in presence of caffeine may be related to sex-dependent-modulation of GABAa receptor function by chronic caffeine administration. In line with this suggestion is that bicucculine, a specific GABAa receptor antagonist, induces similar effects on breathing or on apnea frequency and apnea time (Figure 7) in both control and caffeine treated female rats. While in male rats bicucculine enhanced breathing and decreased apnea inrats treated with caffeine (Uppari et al., 2016). On the other hand, several studies showed that chronic caffeine administration modulates other inhibitory or excitatory transmitter function in the central nervous system such as dopamine and glutamate (Ferre, 2010; Nehlig et al., 1992). In the respiratory areas, simply blocking the inhibitory effects of adenosine receptors induced sex-specific effects in adult rodent (Montandon et al., 2008a; Montandon et al., 2008b; Tchekalarova et al., 2014). Finally, the progressive establishment of the inhibitory effects of GABA on hippocampal neuronal activity is almost completed in male rats by postnatal day 7 but in female that occurs around P14 (Nunez and McCarthy, 2007). It is difficult to draw a firm explanation from our observations on that sex-differences of progesterone in caffeine treated rats but at least we may suggest that P12 female rats are less sensitive to the side effects of chronic caffeine administration on GABAergic functions than males.
Subsequently, we demonstrated that the NKB-ir volume in the INF is sexu- ally dimorphic in adulthood (women ⬎ men). Surprisingly, no such significant sex difference was found in the total number of NKB- positive cells in the INF, suggesting a sexually dimorphic NKB innervation rather than a sex difference in the number of NKB-expressing cells. A female-dominant number of NKB neurons has been identified in the rat (8) and sheep (9, 10) ARC. It is interesting to note that although a sex difference was observed in the number of kisspeptin-immunoreactive neurons in humans, the most prominent sex difference was observed in the den- sity of kisspeptin-immunoreactive fibers (6). The sta- tistical discrepancy observed between our two measures of NKB expression could be partly explained by limi- tations related to the use of postmortem brain tissue, whose conditions at death cannot be tightly controlled [i.e. a long postmortem delay may cause fast axonal transport of the neuropeptide between death and fixa- tion (23)]. It should also be noted that NKB gene ex- pression varies with the rat estrous cycle (19), which could partly explain the lack of significant differencesin NKB-positive cells between adult men and women, given that the stage of the menstrual cycle at death was not reported in the patient’s medical folder.
Up until 20 years ago, evidence-based treatments for adolescents were practically unknown; nevertheless, antidepressants drugs developed for adults were administered to adolescents, even though their use may possibly interfere with brain maturation. For example, mice and rats that were treated with fluoxetine during the postna- tal period (from postnatal days 4–21) showed increased anxiety- and depression-related behaviour when tested drug-free in adult- hood. However, similar effects did not occur as a result of fluoxe- tine-treatment during adolescence (13). In a study conducted by Norcross et al. , two different mouse strains were treated with clinically relevant doses of fluoxetine during the mouse ‘adoles- cence’ period (between 3 and 7 weeks of age). When tested drug- free in adulthood, these mice did not express any behavioural abnormalities and displayed normal fear-, anxiety- and stress- related phenotypes (14). Regrettably, sex was not taken into account in these studies. By contrast, Hodes et al. (15) revealed that fluoxetine treatment inrats around puberty (between weeks 4 and 6 of age) did not affect cell proliferation in either males or females. However, increased hippocampal cell proliferation was observed in adult males as a result of fluoxetine treatment, whereas such effects did not occur in female rats at any age or stage of the oestrous cycle (16). It still remains to be determined, however, whether such sexdifferencesin treatment responses are also pres- ent in humans.
II receptor type-1 receptors (AGTR1). 63 Using the elastase perfusion model, Ailawadi et al. similarly found higher incidence and an increase in abdom- inal aortic diameter in male compared with that in female rat abdominal aortas. This apparent female protection was mediated by inhibitory effect of estradiol on the aortic wall macrophage infiltration and secretion of MMP9. 64 They also noticed that estrogen-related resistant phenotype was lost after transplantation of the female aorta into male rats. Moreover, the male and female aortas had nearly identical aortic structure before any intervention, suggesting a postinjury action of estrogen. Sinha et al. 65 focused on the first week after elastase perfu- sion, and the protective effects of female rats were associated with a decrease of macrophage and neutrophil infiltration and lower levels of multiple members of bone morphogenetic protein, C-C che- mokine ligand, C-C chemokine receptor, inter- leukin (IL), transforming growth factor (TGF), tumor necrosis factor (TNF), and vascular endothe- lial growth factor (VEGF) families in the early stages of AAA formation. Furthermore, Cho et al. 66 inves- tigated sex-related changes of ECM proteins and found that sex disparities were associated with lower levels of types I and III collagen, Tgfb1, and higher leukocyte infiltration and MMP13 levels in male rats.
tones, the envelope following responses (EFRs), thought to reflect the strength of phase locking of the stimulus in the auditory nerve and possibly brainstem to the envelope, was also shown to differ between male and female cowbirds, Molothrus ater [ 57 ].
In addition, an abundant literature demonstrates that in mammals PNN limit plasticity in the visual system and develop following exposure to light. Following monocular light deprivation, plasticity is maintained in the “blind” hemisphere and PNN become established in much smaller amounts [ 46 , 47 ]. There is to our knowledge no direct evidence demonstrating sexdifferencesin the activity of visual pathways in birds but such evidence exists in mammals [ 58 ]. Furthermore aromatase has been identified in fibers and perikarya of the visual system in quail [ 59 ]. Aroma- tase-immunoreactive fibers are also present throughout the visual system of neonate rats (optic tract, suprachiasmatic nucleus, lateral hypothalamus, geniculate nuclei, lateral posterior thalamic nucleus and all layers of the superior colliculus). Immunoreactive cell bodies were also seen in the superior colliculus, suprachiasmatic nucleus and intergeniculate leaflet [ 60 , 61 ]. Estrogen re- ceptors have finally been identified in the developing rat cortex [ 62 – 64 ]. Furthermore sex differ- ences were detected in the total dendritic length for three populations of cortical neurons within the visual cortex of the rat brain [ 65 ]. These observations therefore support the possibility of a differential control of visual activity by peripheral or centrally-synthesized estrogens.
Once animals performed this task efficiently, sound stimuli were introduced in the next session. At the start of each trial, a repeating pure tone ‘reference’ (200 ms duration, 200 ms inter-tone inter- val, 60 dB SPL) was presented to indicate that the central spout could be activated. Nose poking at the central spout resulted in the presentation of a repeating complex tone ‘target’ (200 ms duration, 200 ms inter-tone interval, 70 dB SPL) after a 100 ms delay. The animal was again required to remain at the centre for 300 ms, and early releases now resulted in the presentation of an ‘error’ broadband noise burst (200 ms duration, and 60 dB SPL) and a 3 s timeout before a new trial began. The target tone could take one of two possible F0 values, which corresponded to rewards at one of the two peripheral spouts (right rewards for high F0 targets, and left for low F0s). For all training and testing stages, the target tones contained harmonics within the same frequency range, so that animals could not use spectral cut-offs to classify the sounds. The target tone continued to play until the animal responded at the correct peripheral spout, resulting in a water reward. Once the animals could per- form this final pretraining task with >70% accuracy across trials, they advanced to pitch classification testing.