Brain Struct Funct DOI 10.1007/s00429-015-1014-y

ORIGINAL ARTICLE

Impact of prenatal nicotine on the structure of midbrain dopamine regions in the rat Natalia Omelchenko • Priya Roy • Judith Joyce BalcitaPedicino • Samuel Poloyac • Susan R. Sesack

Received: 2 July 2014 / Accepted: 15 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract In utero exposure of rats to nicotine (NIC) provides a useful animal model for studying the impact of smoking during pregnancy on human offspring. Certain sequelae of prenatal NIC exposure suggest an impact on the development of the midbrain dopamine (DA) system, which receives a robust cholinergic innervation from the mesopontine tegmentum. We therefore investigated whether prenatal NIC induced structural changes in cells and synapses within the midbrain that persisted into adulthood. Osmotic minipumps delivering either sodium bitartrate (vehicle; VEH) or NIC bitartrate at 2 mg/kg/day were implanted into nine timed-pregnant dams at E4. At birth, rat pups were culled to litters of six males each, and the litters were cross-fostered. Plasma levels of NIC and cotinine from killed pups provided evidence of NIC exposure in utero. Pups separated from dams at weaning showed a trend toward reduced locomotor activity at this time point but not when tested again in adulthood. Adult rats were killed for anatomical studies. Estimates of brain size and

volume did not vary with NIC treatment. Midbrain sections stained for Nissl or by immunoperoxidase for tyrosine hydroxylase and analyzed using unbiased stereology revealed no changes in volume or cell number in the substantia nigra compacta or ventral tegmental area as a result of NIC exposure. Within the ventral tegmental area, electron microscopic physical disector analysis showed no significant differences in the number of axon terminals or the number of asymmetric (putative excitatory) or symmetric (putative inhibitory) synapses. Although too infrequent to estimate by unbiased stereology, no obvious difference in the proportion of cholinergic axons was noted in NIC- versus VEH-treated animals. These data suggest that activation of nicotinic receptors during prenatal development induces no significant modifications in the structure of cells in the ventral midbrain when assessed in adulthood.

N. Omelchenko  P. Roy  J. J. Balcita-Pedicino  S. R. Sesack (&) Department of Neuroscience, Langley Hall, Room 210, University of Pittsburgh, Pittsburgh, PA 15260, USA e-mail: [email protected]

Introduction

N. Omelchenko Department of Natural Sciences and Mathematics, West Liberty University, West Liberty, WV 26074, USA S. Poloyac Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA S. R. Sesack Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15260, USA

Keywords

Glutamate  Ultrastructure

Maternal smoking is associated with spontaneous abortions, preterm births, low birth weight, and a 150 % increase in overall perinatal mortality (Bardy et al. 1993; DiFranza and Lew 1995; Andres and Day 2000). Smoking during pregnancy also can result in learning disabilities and behavioral problems in children (Eskenazi and Castorina 1999; Ernst et al. 2001) as well as a greater likelihood of acquiring the smoking habit (Kandel et al. 1994; Cornelius et al. 2000; Niaura et al. 2001; Buka et al. 2003; Shenassa et al. 2003). Although tobacco contains many harmful substances, nicotine (NIC) underlies the addictiveness of tobacco consumption (Lichtensteiger et al. 1988) and is

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also reportedly both neurotoxic and neuroteratogenic (Joschko et al. 1991; Roy and Sabherwal 1994; Berger et al. 1998; Roy and Sabherwal 1998; Roy et al. 2002). To quit smoking, pregnant women may resort to NIC patches, electronic cigarettes or other replacement therapies that are considered less dangerous (Jorenby 1998; Scalera and Koren 1998; King et al. 2013) but still introduce NIC to the subjects. In animal models, prenatal exposure to NIC alone provokes similar behavioral deficits as observed in humans consuming tobacco, including a hyperlocomotor state that resembles human hyperactivity (Fung 1988; Tizabi et al. 1997; Ajarem and Ahmad 1998; Newman et al. 1999; Thomas et al. 2000; Pauly et al. 2004; Vaglenova et al. 2004). In addition, developmental exposure to NIC in rats leads to a higher rate of NIC selfadministration in adulthood (Adriani et al. 2002, 2003). The developing brain becomes vulnerable to NIC exposure at the end of neurulation when mRNA for neuronal nicotinic receptor subunits and NIC receptor binding sites are detected (Zoli et al. 1995; Tribollet et al. 2004) and become operational (Atluri et al. 2001; Schneider et al. 2002). After migration from the mitotic zone, future neurons express regionally specific patterns of mRNA for different nicotinic receptor subunits (Naeff et al. 1992; Zhang et al. 1998). In the rat substantia nigra compacta (SNc) and ventral tegmental area (VTA), mRNA expression levels for a3, a4, and b2 subunits are maximal before parturition (Zoli et al. 1995), and a4, a7, and b2 mRNA levels are maintained in the brainstem at adult levels throughout the postnatal period (Zhang et al. 1998). This complexity of ontogenetic events suggests that there is a precise regulation of cholinergic actions at different developmental stages and multiple ways for NIC to provoke structural damage to the brain. In its actions on nicotinic receptors, acetylcholine at early developmental stages acts not as a neurotransmitter but in a morphogenetic capacity by controlling and coordinating proper assembly of the brain (Seidler et al. 1994; Smith 1994; Buznikov et al. 1996; Nguyen et al. 2001; Hohmann 2003). Therefore, cholinergic ligands like NIC have the ability to act as neuroteratogens (Joschko et al. 1991; Chen et al. 1999) by disrupting the timing and intensity of acetylcholine-mediated commands, most likely via alterations in gene expression (Greenberg et al. 1986; Slotkin et al. 1997; Trauth et al. 1999). Such neuroteratogenic effects have been revealed as structural disturbances in the cortex and hippocampus (Roy and Sabherwal 1994, 1998; Roy et al. 2002; Onal et al. 2004; Muhammad et al. 2012). Morphological changes in brainstem development following prenatal or adolescent NIC treatment also have been suggested on the basis of indirect measures (Trauth et al. 2000; Abreu-Villaca et al. 2003, 2004a) but never examined directly. At later stages of development, NIC has

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potential neuroprotective actions by promoting cell survival during the period of naturally occurring cell death, approximately E15-P8, depending on the brain region (Messi et al. 1997; Fucile et al. 2004). Although developmental NIC exposure may cause structural changes in many brain regions, the behavioral impairments reported following early NIC exposure are consistent with malfunctions of the midbrain dopamine (DA) system and its afferent regulation. DA neurons modulate cognition, affect, and motor behaviors related to reward through projections to forebrain structures including the prefrontal cortex and nucleus accumbens (Koob 1996; Redgrave et al. 1999; Sesack and Grace 2010). Early NIC exposure may contribute to learning disabilities and cognitive disturbances through direct cholinergic actions on these forebrain regions, for which there is considerable evidence (Schneider et al. 2011; Muhammad et al. 2012; Mychasiuk et al. 2013a, b). It is also possible that prenatal NIC might cause behavioral alterations via indirect effects mediated through the midbrain DA system. In this study, we utilized unbiased stereological methods to estimate persistent changes in regional volume and cell number in the SNc and VTA, as well as axon and synapse number in the VTA of adult rats following prenatal NIC exposure. The VTA was chosen for ultrastructural analysis because of studies showing morphological changes in the forebrain targets of this DA projection system (Muhammad et al. 2012; Mychasiuk et al. 2013a, b). We also examined locomotor activity to determine whether structural changes in the midbrain DA system might correlate with altered behavior that persists into adulthood.

Materials and methods Subjects, surgeries and handling All the proposed experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and as approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Multiparous, timed-pregnant dams (i.e. with pups at first embryonic day, E1) were purchased from Breeding Laboratories (Hilltop Lab Animals Inc). All animals were housed in a large temperature controlled room with lights on between 07:00 a.m. and 07:00 p.m. daily. Rats had free access to food and water. To minimize discomfort following the surgery and to ensure effective maternal care for litters, solid bottom caging and individual housing were supplied for dams prior to parturition and for 21 days afterwards. Post-weaning animals were contained in wire bottom cages.

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Dams were treated and their pups were handled in three cohorts. Cohort 1 originated from three dams (1 NIC and 2 vehicle) and contributed 5 rats (2 NIC and 3 vehicle) for behavioral testing and the same 5 rats (2 NIC and 3 vehicle) for anatomical analysis. Cohort 2 originated from two dams (one NIC and one vehicle) and contributed 12 rats (6 NIC and 6 vehicle) for behavioral testing and 8 rats (4 NIC and 4 vehicle) chosen at random for anatomical analysis. Cohort 3 matched cohort 2 in all respects. The unused animals from each litter were euthanized at various stages as described below. Specific details regarding behavioral and anatomical studies also appear below. We exposed pups to NIC in utero following a wellestablished experimental procedure (Benowitz 1996b; Crooks and Dwoskin 1997; Slotkin 1998; Audesirk and Cabell 1999; Dwoskin et al. 1999; Pichini et al. 2000; Hohmann 2003; Abreu-Villaca et al. 2004a, b; Godding et al. 2004). Dams in three separate cohorts were implanted with osmotic minipumps (type 2ML4; Alzet) on E4, before implantation of the embryo in the uterine wall. The minipump was loaded under sterile conditions with NIC bitartrate (Sigma-Aldrich) dissolved in bacteriostatic water to deliver a daily dose of 2 mg/kg (calculated as free base) or sodium bitartrate as a vehicle (VEH) control. This dose was chosen to match previous experiments using rodents to model human exposure (Newman et al. 1999; Roy et al. 2002) and so as to produce approximately 30 ng/ml plasma drug levels, which corresponds to regular smoking in humans (Pichini et al. 2000; Godding et al. 2004). To insert osmotic minipumps in the subcutaneous dorsum, rats were anesthetized by inhalation of isoflurane (2–5 % for induction, 0.25–4 % for maintenance). A 4-cm2 area on the back was shaved, sterilized and incised, and a minipump warmed to 36 °C was inserted. After minipump insertion, the incision was closed with wound clips and treated with a topical antibiotic/pain reliever agent (Neosporin). The animals were further treated with Penicillin G benzathine and Penicillin G procaine (Pen BP-48) at 2 ml/kg. For the next 14–18 days, the tissue around the minipumps was gently massaged to avoid immobilization of the devices that could have interfered with effective drug delivery. The pump supplied drug for 18 days up to the point when it was removed after parturition at E22/P0 using the same procedure described above. The morning after birth, the number and weight of pups was documented, and the litter was culled to six pups. The other pups, including at least one male, were killed and used to measure NIC and cotinine concentrations as an estimate of the in utero NIC exposure (see ‘‘Nicotine and cotinine measurements’’). The remaining pups were weighed every 3 days to assess their physical development. At each weighing, all the pups in each litter were cross-fostered to the other dam within the same cohort. All pups were separated from the dams at the

time of weaning (P21), and the dams and female pups that had been used in some groups to maintain constant litter size were euthanized. Some gender-related differences in the outcomes of NIC treatment have been reported. For example, biochemical evidence for altered DA transmission was reported to be more profound in male than female offspring of dams treated with NIC (Fung and Lau 1989; Ribary and Lichtensteiger 1989; Romero and Chen 2004). Hence, male animals were chosen for the present anatomical studies. Because stress can affect the development of the DA system (Saal et al. 2003; Choy et al. 2009; Kunzler et al. 2013; Buchmann et al. 2014), every effort was made to minimize any distress to the pups that could potentially obscure the effects of NIC exposure. Prior to weaning, manipulations were restricted to non-painful marking (to trace pups during cross fostering) and infrequent weighing. From weaning to killing at adulthood (P75), handling was limited to behavioral testing and routine animal habitat maintenance. Moreover, both NIC and VEH-treated groups were subjected to the same manipulations. Behavioral testing for NIC-induced changes in locomotor activity was conducted over 3 days, first at P22–P26 and again at P69–P73 (slight variations in the days depended on the cohort, see ‘‘Behavioral testing of locomotor activity’’ below). The early time point was chosen based on published studies reporting hyperlocomotion in the postweaning period (Tizabi et al. 1997; Ajarem and Ahmad 1998; Newman et al. 1999). Behavior in adulthood was also examined to determine the persistence of prenatal nicotine’s effect. Nicotine and cotinine measurements Animals assigned for plasma measures were anesthetized with pentobarbital (100 mg/kg i.p.), and 1–2 ml of blood was collected by cardiac puncture exsanguination. The animals were then decapitated. The blood of all the pups within each cohort was then combined, so that plasma concentrations represent an average across these animals. Plasma NIC and cotinine concentrations were measured using HPLC with single quadrupole MS detection (ThermoFinnigan MSQ, San Jose, CA, USA) (Jacob et al. 1981; Ghosheh et al. 1999). The limit of quantitation of this method was 10 ng/ml for cotinine and 0.5 ng/ml for NIC. Plasma samples (900 ll) were alkalinized by 50 ll of 10 M NaOH and double extracted with 4 ml per extraction of methylene chloride. The bottom organic layer was collected and acidified to pH 3 with HCl. Samples then were evaporated to dryness under nitrogen gas, and the dried residue was reconstituted with 150 ll of methanol. Samples were injected onto a Surveyor HPLC with an MSQ single quadrupole detector (ThermoFinnigan). NIC and

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cotinine were separated via HPLC using a Betasil Diol 100 9 50 9 2.1 analytical column (ThermoHypersil Keystone, Bellefonte, PA, USA). The mobile phase consisted of 95 % methanol and 5 % ammonium acetate 50 mM at an isocratic flow rate of 200 ll per min. Mass detection was completed by electrospray ionization under selective ion monitoring mode. Probe temperature was 100 °C with a cone voltage of 65 V and a 0.5 s dwell time. Mass to charge ranges monitored included NIC m/z 163.1, d3-nicotine m/z 166.1, cotinine m/z 177.1, d3-cotinine m/z 180.1. NIC and cotinine were quantified based on the ratio of each analyte to its deuterated internal standard. Sample concentrations were then determined from linear standard curve ratios. Weighted linear regression was used to determine the best fit of the standard curve ratios using Xcaliber software (ThermoFinnigan). Behavioral testing of locomotor activity Individual behavioral testing was conducted between 06:00 and 09:00 a.m. near the end of the animals’ active period. Tests were conducted for 1 h in a soundproof room at 30 LUX of light intensity using an open field activity system (17.000 L 9 17.000 W 9 12.000 H) from Med Associates, Inc. (St. Albans, VT, USA). Prior to each trial, the internal surface of the chamber was wiped with Nolvasan dissolved 1:10 in water to eliminate olfactory cues. The subjects were then placed in the same corner of the chamber, and horizontal activity (total distance traveled), vertical activity (rearing), and rest time were automatically recorded by a personal computer. The same procedures were conducted over a 3-day period (see above) to allow animals to acclimate to the environment, and statistical analyses were conducted on the third day to assess differences between NIC and VEH-treated animals. Perfusion and sectioning for anatomical studies Rats were anesthetized with pentobarbital (100 mg/kg i.p. with supplemental doses if needed), and the vasculature was perfused with 50 ml heparin saline (Elkins-Sinn, NJ, USA; 1000 U/ml) followed by 3.75 % acrolein and 2 % paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB) and then 400 ml of 2 % paraformaldehyde in 0.1 M PB. The brains were removed and weighed. The volume of the brain was determined according to Archimedes’ principle following the detailed description by Dorph-Petersen et al. (2005). Coronal blocks 4–5 mm thick were post-fixed in 2 % paraformaldehyde for 0.5–1 h. Adjacent sections through the VTA/SNc were stained for Nissl, by immunoperoxidase labeling for tyrosine hydroxylase (TH) as a selective marker for DA cells (Bayer and Pickel 1990), or by immunocytochemistry for the vesicular acetylcholine

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transporter (VAChT), which labels cholinergic fibers (Omelchenko and Sesack 2006). The boundaries and subdivisions of the VTA were based on the descriptions by Swanson (1982) and Phillipson (1979). All subdivisions were included for volume and cell number assessments, whereas the parabrachial and paranigral regions were the focus of ultrastructural analysis for synapse counting (see ‘‘Stereological data analysis’’). Immunocytochemistry and tissue preparation Four sets of vibratome sections cut at 50 lm were collected in PB and treated in 1 % sodium borohydride in PB for 30 min to reduce free aldehydes and improve immunolabeling. Three sets of sections through the VTA/SNc were stained for Nissl or immunolabeled for VAChT or TH. Sections were chosen by systematic random sampling so that all portions of the region had an equal probability of being sampled. To facilitate antibody penetration for light microscopic analysis, sections were cryoprotected in 8 % glycerol and 20 % sucrose in 0.04 M PB before being subjected to freeze–thaw at -80 °C for 20 min. This treatment, in combination with 0.4 % Triton X-100 in antibody solutions, resulted in penetration of antibodies throughout the section thickness. To enhance antibody penetration but preserve membrane integrity for ultrastructural analyses, 0.04 % Triton X-100 was added to antibody solutions as described previously (Omelchenko and Sesack 2006, 2007). Immunoperoxidase labeling procedures were performed on free-floating sections with constant agitation at room temperature; all immunoreagent incubations were followed by extensive rinses in buffer. After rinsing in 0.1 M trisbuffered saline (TBS; pH 7.6), sections were placed for 30 min in TBS blocking solution containing 1 % bovine serum albumin, 3 % donkey serum, and 0.4 or 0.04 % Triton X-100 to improve antibody penetration. Sections then were incubated for 12–48 h in primary antibody in blocking solution: goat anti-VAChT (1:20,000; ImmunoStar) or mouse anti-TH (1:8000; Chemicon). The primary antibodies have been used to label brain sections in several prior publications from our laboratory as well as others (Sesack et al. 1995; Carr and Sesack 2000; Omelchenko and Sesack 2005, 2006, 2007) and have been shown to be specific by Western blot analysis, in vitro cell transfection, labeling of primary cultures, and preadsorption with the immunizing peptide. Following incubation in primary antibodies, sections were then incubated for 30 min in the blocking solution containing biotinylated secondary antibodies: donkey anti-mouse or anti-goat IgG (1:400; Jackson ImmunoResearch Labs). After that, sections were incubated for 30 min in 1:100 avidin–biotin peroxidase complex (Vectastain Elite; Vector Labs). The

Brain Struct Funct

peroxidase reaction was run using 0.022 % diaminobenzidine and 0.003 % hydrogen peroxide in TBS for 3–5 min; the reaction was stopped by TBS rinses. Sections for light microscopy were mounted onto glass slides; those stained for Nissl were dehydrated, defatted, rehydrated and stained with thionin. All tissue sets were then dehydrated and coverslipped with DPX. The average section thickness following this procedure was specifically measured using StereoInvestigator and determined to be 15–17 lm. Alternate sections for electron microscopy were post-fixed in osmium tetroxide, dehydrated through graded ethanol and propylene oxide solutions and embedded in epoxy resin according to standard methods. Ultrathin sections cut from the surface of these sections were collected in serial order on formvar-coated slot grids. Section thickness (*60 nm) was determined according to the small fold method (De Groot 1988). Stereological data analysis Quantitative data were collected by one operator blinded to the treatment conditions using unbiased stereological principles (German et al. 1999; Leranth et al. 2000; Day et al. 2006) and StereoInvestigator software (MicroBrightField) for light microscopy or the physical disector method for electron microscopy (Mayhew 1992; Geinisman et al. 1996). The volume of the VTA/SNc was assessed by the Cavalieri estimator (i.e. point counting) (Gundersen et al. 1988) using a 2.59 objective on THimmunolabeled sections. Region of interest (ROI) boundaries were traced on live video images of the brain. A lattice of points with spacing selected to produce roughly 400 points per ROI per animal was superimposed at a random rotation angle. Cell counts for both DA (TH-immunolabeled) and total neurons (Nissl staining) in the VTA/SNc were determined using the optical fractionator method (West 1993). In each section, the ROI was outlined using a 2.59 objective, and cells were counted using a 609 objective. Grid spacing was set to produce 150 probes within the ROI across all sections per animal and the volume of the counting frame was set to include 2–3 neurons (total of 300–400 neurons per ROI). In each case, there were no differences between left and right hemispheres, and so the two were added together for volume estimates and cell counts. The region of VTA sampled for electron microscopy was chosen by dropping a 0.5 cm2 square from 15 cm above the corresponding section of the rat atlas (Paxinos and Watson 1998), as described previously (Ingham et al. 1998). Regions were rejected if they fell outside the VTA or included only white matter. In some cases, a second random region for analysis was chosen from the same section but from the side contralateral to the first. With the

exception of two cases in which single regions were analyzed, multiple regions (2–5) were typically examined per rat. Ultrathin sections through the VTA regions were sampled close to the tissue surface where immunoreagent penetration was optimal and surface damage was minimal. At low magnification (*30009), 15 disector fields per section were chosen from the gray matter. Disector heights were chosen to maximize efficiency in particle counting but minimize exclusion of the smallest particles. For synapse counting, this required a disector height of 60 nm (i.e., adjacent sections). For counting of axon terminals, a disector height of 360 nm (i.e., sections 1 and 6) was chosen. Hence, for each disector field, serial photomicrographs were taken at 18,0009 from sections 1 to 6 in succession. An unbiased counting frame (18 lm2) was superimposed on the same field within each serial micrograph, using dendrites and myelinated axons as reference points. Axon varicosities containing at least three vesicles were counted when they first appeared in the reference plane (section 1) but not the look-up plane (section 6). To increase efficiency, the reference and look-up planes were reversed, and additional axons were counted when they first appeared (Calverley et al. 1988; Mayhew 1992; Woolley and McEwen 1992; Francis et al. 2006). Each varicosity was marked so that it was never counted a second time when the reference and look-up planes were reversed. The total number of axons counted per animal ranged from 29 to 84, with a mean of 62. The total density of axons per disector volume (Nv) was then expressed as the average of the two counts. Axons were further characterized as those immunoreactive for VAChT and those that were immunonegative; the latter formed the bulk of the counts. Synapses were counted when they first appeared in the reference plane (section 1) but not the look-up plane (section 2). The reference and look-up planes were then reversed, and additional synapses were counted when they first appeared. Each synapse was marked so that it was never counted a second time. Synapse counting then continued, comparing sections 2 and 3, 3 and 4, 4 and 5, and 5 and 6, taking care to avoid any double counting. The total number of synapses counted per animal ranged from 32 to 127, with a mean of 38. The total density of synapses per disector volume (Nv) was expressed as the average of the ten counts. Synapses met established morphological criteria and were further characterized as those with symmetric or asymmetric character (Peters et al. 1991). The thickened post-synaptic density associated with asymmetric synapses, in addition to accumulated pre-synaptic vesicles, allowed synapse identification despite occasional oblique planes of section that obscured the synaptic cleft (Calverley et al. 1988; Mayhew 1992). Symmetric synapses characterized

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Brain Struct Funct

by little or no post-synaptic density, however, required observation of the synaptic cleft for identification. This difference in criteria could contribute to an underestimation of symmetric versus asymmetric synapses, but such an underestimation would occur equally in all sections that were analyzed blinded to treatment groups. Finally, either synapse type was also characterized as to whether they were formed by axons immunopositive or immunonegative for VAChT. Again, the latter formed the bulk of the counts. Finally, the total number of axon varicosities or synapses was determined as the product of Nv 9 Vref, where Vref is the volume of the VTA as determined by the Cavalieri estimator described above.

no drug-dependent difference in distance traveled on the third successive day after acclimation to the chamber (Fig. 1b; NIC 4648 cm ± 989, N = 13 versus VEH 4776 ± 1131, N = 14; p = 0.8, t test, two-tailed; note that each group contained one less animal than tested postweaning due to the unexplained death of two rats). At the same time, the average velocity of the animals’ movement in the activity chamber was not different between NIC- and VEH-treated groups regardless of the age of testing (data not shown). As animals from both groups were moving at the same speed when roaming around the chamber, the trend for a decrease in the total distance traveled displayed by the NIC-treated rats post-weaning is suggestive of increased periods without movement.

Results

Anatomical measures

Litters

The observed decrease in total body weight due to in utero NIC exposure might suggest a corresponding decline in overall brain growth or development of specific brain regions. At the time of killing in adulthood, however, prenatal NIC exposure was associated with no overall change in brain weight or brain volume indicating limited effect of prenatal NIC on gross anatomy of the central neural system of adult animals (Table 2). Despite early reports suggesting injury to specific brain regions resulting from prenatal exposure to NIC (Navarro et al. 1988; Fung and Lau 1989; Ribary and Lichtensteiger 1989; Richardson and Tizabi 1994; Muneoka et al. 1997, 1999; Kane et al. 2004), significant changes in the DA-rich midbrain were not observed in this study (Fig. 2; Tables 2, 3). Light microscopic examination did not reveal any gross abnormalities in the structure of the SNc or VTA from NIC- versus VEH-treated animals. The results of stereological study were consistent with this qualitative assessment. Neither the volume of the SNc nor the VTA, as estimated using the Cavalieri method, showed any significant difference between NIC-exposed and VEH-treated rats (Table 2). Similarly, the counts of DA cells from THimmunolabeling or of total neurons based on Nissl staining (Fig. 3) showed no drug treatment-related differences (Table 3). The electron microscopic analysis using the unbiased physical disector stereological method indicated that prenatal NIC exposure had also little impact on the ultrastructure of the VTA of adult animals (Fig. 4; Table 4). Figure 4 shows a representative set of images from adjacent ultrathin sections that constituted the disector set used for quantitative analysis. No significant differences were observed in the total number of axon terminals counted in the VTA of NIC- or VEH-treated rats. The random approach used to select regions for stereological assessment resulted in too few axon terminals immunoreactive for

Nicotine-treated dams delivered 16.0 ± 1.0 (mean ± stdev) pups in each litter; the average number of pups delivered by VEH-treated animals was similar (15.3 ± 0.6). The average NIC and cotinine plasma levels for pups killed for individual cohorts are reported in Table 1 and verify NIC exposure in utero. Despite comparable litter sizes, the birth weight of pups treated with NIC prenatally (5.75 g ± 0.21; mean ± stdev) was slightly but significantly lower than the weight of VEH-exposed pups (6.24 g ± 0.30; p = 0.04, t test). Any potential variations due to litter size or maternal care were addressed by culling the litters to equal size and by regular cross-fostering (see Materials and methods). After weaning at P21, no difference in the weight of animals in the NIC- or VEH-treated groups was observed (NIC 57.03 g ± 5.03 versus VEH 58.44 g ± 3.15). Similarly, at the time of killing (P75– P82), no difference in weight was observed between groups (NIC 430.8 g ± 35.6 versus VEH 440.6 g ± 25.3). Behavior The effect of prenatal NIC on animal behavior also appeared to be transient. Rat pups tested just after weaning showed a trend toward less locomotion in an open field, as measured by the total distance traveled (cm), if they were exposed in utero to NIC versus VEH. The total distance calculated on the third successive day of testing was 16 % lower for NIC-treated animals (1637 cm ± 265, mean ± stdev, N = 14) than for VEH-treated rats (1949 cm ± 537, N = 15; p = 0.06, t test, two-tailed). Figure 1a illustrates the distance traversed by each rat on the third successive day of testing when animals were relatively habituated to the environment. Conversely, the same rats tested as adults a few days before killing showed

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Brain Struct Funct Table 1 Nicotine and cotinine levels Cohort

Birth

Sacrifice

Plasma Nicotine levels in killed pups (ng/ml)

Plasma Cotinine levels in killed pups (ng/ml)

9.3

223

No. of adult male rats for anatomical studies P75

P82

1

1

2

1

Nicotine

2

2

Vehicle

2

2

Nicotine

2

2

Vehicle

2

2

Cohort 1 Nicotine Vehicle Cohort 2

Cohort 3

3.5

5.4

85

220

Fig. 1 Scatter plots illustrating the total distance (in cm) traversed in an open field by rats exposed prenatally to nicotine (gold circles) or vehicle (blue circles). Horizontal lines represent the mean value

within each group. Locomotor activity data is shown for the last of three consecutive days of testing (i.e. after acclimation) either postweaning (a) or in adulthood just prior to killing (b)

VAChT (Fig. 5) for quantitative measurements. Nevertheless, it was noted that the proportion of overall axon profiles that were cholinergic was similar between groups (3.8 versus 3.7 %, respectively, for NIC- or VEH-treated animals). Significant differences in the total number of asymmetric (putative excitatory) or symmetric (putative inhibitory) synapses formed by unlabeled axon terminals also were not detected, nor were differences observed in specific cholinergic synapses in NIC- versus VEH-treated rats.

developmental organization of the SNc and VTA. The findings of this study are inconsistent with earlier reports using indirect measures that suggested compromised organization in these regions (Fung 1989; Fung and Lau 1989; Ribary and Lichtensteiger 1989; Slotkin et al. 1987; Navarro et al. 1988). Hence, our light and electron microscopic studies in rats suggest that changes in human brain and behavior that may occur as a result of NIC exposure during pregnancy are more likely to be expressed at physiological levels within the midbrain DA complex than at the level of cellular and circuit structure.

Discussion

Methodological considerations

The goal of this study was to evaluate the impact of prenatal NIC exposure on the structural characteristics of the DA-rich midbrain in adult rats. The data indicate that NIC exposure had minimal structural impact on the

Given the largely negative results of this study, it is prudent to examine evidence that pups were exposed to NIC in utero. First, osmotic minipumps are currently the most common mode of drug delivery for studies of NIC effects

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Brain Struct Funct Table 2 Brain size and regional volumes No. of rats

Avg weight (g)

Table 3 Cell counts SD

p value

No. of rats

Avg cell count

SD

p value

CE

0.44

0.09

NissI Brain weight

SNc

Nicotine

10

2.00

0.13

Vehicle

10

1.99

0.08

No. of rats

Avg volume (cm3)

SD

0.42

10

1.95

0.14

Vehicle

10

1.96

0.11

8

26,472

4465

Vehicle

9

20,555

3454

Nicotine

8

33,524

9660

Vehicle

9

34,025

6651

0.08 0.08

SNc

No. of rats

Avg volume (mm3)

SD

p value

CE

10

1.19

0.32

0.17

0.03

Nicotine

0.03

Vehicle

SNc volume Vehicle

0.38

TH

0.44

Nicotine

Nicotine

0.10

VTA

p value

Brain volume Nicotine

Nicotine

11

1.40

0.33

No. of rats

Avg volume (mm3)

SD

p value

CE

2.48 2.61

0.57 0.43

0.59

0.02 0.02

Vehicle VTA

7

20,555

5761

10

19,356

5272

7

34,025

10,957

10

31,286

7276

0.33

0.11 0.10

0.27

0.09 0.09

In a few animals, full tissue sets were not available for some regions

VTA volume Nicotine Vehicle

10 11

Brain weight and volume were not available for one vehicle animal

on development in rodents (Tizabi et al. 1997; Newman et al. 1999; Roy et al. 2002; Abreu-Villaca et al. 2004a), most likely because of their reliable delivery of consistent doses over long periods of time that lead to relatively stable blood levels. Minipumps also avoid the stress and potential negative consequences associated with daily systemic NIC injections (Slotkin et al. 1987; Navarro et al. 1988).

Fig. 2 Coronal sections through the left hemisphere of the rat midbrain stained by immunoperoxidase for TH in DA cells and processes. Sections are shown every 300 lm from rostral to caudal and illustrate the boundaries of the SNc and VTA that were used to

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Second, despite frequent monitoring of dams that were close to delivery, blood sampling of the pups may have occurred as late as 5 h after parturition, when NIC plasma levels were reduced compared to the in utero condition. To address this concern, we based our estimate of NIC exposure on the levels of cotinine, an obligatory product of NIC metabolism that is commonly used to evaluate exposure levels (Benowitz 1996a; Lambers and Clark 1996; Dempsey et al. 2000). Cotinine is readily distributed into the brain and has its own effects on developmental processes (Crooks and Dwoskin 1997; Audesirk and Cabell 1999; Dwoskin et al. 1999). The maximum 5 h lag time to blood sampling is close to the half-life of cotinine in the brain

determine regional volumes. cp cerebral peduncle, fr fasciculus retroflexus, IPN interpeduncular nucleus, ml medial lemniscus, MB mammillary body. Scale bar corresponds to 500 lm

Brain Struct Funct Table 4 Total axon and synapse numbers No. of rats

Mean total number

SD

p value

0.31

Axon terminals Nicotine

10

6.14E?08

1.67E?08

Vehicle

11

7.00E?08

2.09E?08

Asymmetric synapses Nicotine

10

5.00E?08

1.89E?08

Vehicle

11

6.16E?08

1.89E?08

0.18

Symmetric synapses

Fig. 3 High magnification images of the rat VTA showing neurons labeled by immunoperoxidase for TH or for Nissl substance (asterisks show representative cells in panels a and b, respectively). In b, glial cells (g) are also evident. Scale bar corresponds to 20 lm

Nicotine

10

4.27E?08

1.93E?08

Vehicle

11

4.95E?08

1.92E?08

0.42

(Ghosheh et al. 1999), indicating that our measures were adequate for confirming exposure of brain tissue to NIC and cotinine in utero. The observation of a significant reduction in birth weight further substantiates the exposure of rat pups to NIC

prenatally, given the similarity of our findings to numerous prior investigations (Tizabi et al. 2000; Vaglenova et al. 2004; Santiago and Huffman 2012). Although birth weight was lower in NIC-treated rats, the equivalent body weight, brain weight and brain volume of drug and VEH-treated animals at the time of killing suggests that the loss of body weight caused by NIC did not persist into adulthood,

Fig. 4 Serial electron micrographic images through the rat VTA illustrating the physical disector method used for axon terminal and synapse counting. Each field represents the full counting frame, with the left and bottom edges being exclusion lines and the right and top edges being inclusion lines. Axon terminals (a) and asymmetric

(black arrows), and symmetric (white arrows) synapses were counted when they first appeared moving forward in the series (red font). Additional elements were then counted when they first appeared moving backward in the series (blue font). The total count for each disector series was then averaged. Scale bar represents 0.5 lm

123

Brain Struct Funct

suggesting that our experimental animals stopped receiving NIC before the equivalent end of prenatal exposure in humans. Furthermore, neurogenesis in rats and humans proceeds at roughly equivalent rates until P0 in the rat, after which the trajectories are divergent (Bayer and Altman 2007). Hence, future studies of the rat brainstem will need to examine the structural impact of NIC exposure during the early postnatal period. Light microscopic observations

Fig. 5 Electron micrograph from the rat VTA illustrating an axon varicosity labeled by immunoperoxidase for VAChT (VAChT-a) forming a symmetric synapse (white arrow) onto an unlabeled dendrite (ud). Scale bar represents 0.5 lm

consistent with prior studies (Santiago and Huffman 2012), and did not cause a lasting effect on brain size. Finally, evidence for exposure to NIC in utero is provided by the trend toward reduced total distance traveled in a locomotor activity chamber compared to VEH-treated rats, at least in the post-weaning period. It is not immediately clear why the drug-treated animals showed a reduction in locomotion versus more commonly reported increases. Nevertheless, according to a recent review, several other laboratories investigating this same measure have indicated reduced activity or no change in locomotion with prenatal NIC exposure (LeSage et al. 2006). Taken together, our findings strongly suggest that rats were exposed to amounts of NIC in utero sufficient to cause demonstrable, albeit transient, alterations in body weight and behavior. The anatomical analyses were conducted in young adult animals killed at P75 to focus on changes in brain structure that were persistent following early NIC exposure. This choice reflected the notion that morphological alterations that could not be fully restored are likely to have the most practical importance for understanding the clinical sequelae of prenatal NIC. Nevertheless, we acknowledge that investigation of animals killed at earlier stages might reveal evidence for transient structural alterations and ought to be examined in future studies. It remains possible that NIC exposure during the postnatal phase of development might induce structural changes in the ventral midbrain as reported in other brain regions (Chen et al. 2003). The brain development of a newborn baby corresponds approximately to a rat of 12–13 days old (Romijn et al. 1991; Bayer et al. 1993),

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This study represents the first application of unbiased stereological assessments of ventral midbrain volume, cell number, and synapse number in adult rats exposed prenatally to NIC. Contrary to expectation, we observed no significant changes in these parameters in NICversus VEH-exposed animals. The expectation of alterations in regional volume and cell number was based on evidence that NIC can negatively impact the proliferation of progenitors by provoking cellular destruction through neurotoxicity and apoptosis (Joschko et al. 1991; Roy and Sabherwal 1998). Furthermore, prenatal NIC exposure is associated with a loss of fetal brain weight (Onal et al. 2004) as well as a reduction of DNA synthesis, indirect measures of cell production (McFarland et al. 1991; Qiao et al. 2003). Third, morphological studies of the rat hippocampus and somatosensory cortex following prenatal NIC report decreases in cell density in rats tested at E21 or P30 (Roy et al. 2002; Onal et al. 2004). The latter studies did not, however, conduct unbiased estimates of total cell number, nor did they examine the brains of adult rats treated prenatally with NIC. Unbiased estimation of neuron number (both DA and total) in the SNc and VTA of drug- and VEH-treated animals did not show an effect of prenatal NIC as measured in adulthood. The findings are inconsistent with biochemical analyses of DNA and protein measurements in midbrain homogenates that were thought suggestive of cell loss at P75 (Abreu-Villaca et al. 2004a). Our observation of normal cell number could suggest that NIC did not induce neurotoxic effects in midbrain DA areas or, perhaps more likely, that prenatal NIC damage was repaired prior to adulthood. Indeed, prenatal NIC has been shown to have neurotoxic effects that impact neuron number in other regions, in particular, cortical pyramidal cells and cerebellar Purkinje neurons, at least when tested at P31–60 (Roy et al. 2002; Abdel-Rahman et al. 2005), but see (Chen and Edwards 2003). Potential regional or cellular selectivity in the neurotoxic effects of prenatal NIC might reflect differences in the expression of nicotinic receptor subunits (Lv et al. 2008).

Brain Struct Funct

Electron microscopic observations Despite the observation of normal neuron number in the SNc/VTA, it remains possible that prenatal NIC induced alterations in the synaptic input to these cells. Evidence suggestive of altered synaptic regulation comes from reports of reduced monoamine metabolite levels in the midbrain and cortex of young adult rats treated prenatally with NIC (Navarro et al. 1988; Muneoka et al. 1997, 1999). Axonal proliferation within the midbrain would be consistent with reports of elevated membrane/total protein and decreased protein/DNA ratios in midbrain homogenates at P75 (Abreu-Villaca et al. 2004a) and a more specific finding of increased choline acetyltransferase activity in P75 homogenates following prenatal NIC exposure (Abreu-Villaca et al. 2004b). In cortical structures, prenatal NIC has been shown to alter laminar thickness, dendritic branching, dendritic length, and spine density, consistent with changes in synaptic inputs (Roy and Sabherwal 1994, 1998; Muhammad et al. 2012; Mychasiuk et al. 2013a); some of these structural alterations persist into adulthood (Mychasiuk et al. 2013b). Of interest regarding cortical morphology, children whose mothers smoked during pregnancy exhibit reduced cortical gray matter thickness at the age of 6–8 years (El Marroun et al. El Marroun et al. 2014). Using unbiased estimates of axon and synapse numbers, we observed no significant alterations in these structural measurements in the VTA of adult animals that were exposed prenatally to NIC. The findings suggest that the overall density of excitatory and inhibitory synaptic inputs to VTA neurons is relatively normal despite prenatal NIC exposure. Although we intended to also estimate cholinergic axon terminal number, the requirements of the unbiased stereological method made accomplishing this goal prohibitive. Specifically, fields for analysis had to be selected randomly and also had to be sufficiently deep from the plastic interface so as to contain well-preserved tissue across six adjacent sections. These requirements resulted in the observation of too few cholinergic axons to allow estimation of total number. Qualitative observations revealed no obvious change in cholinergic axon terminals resulting from prenatal NIC administration. It is important to note that the VTA neurons receiving synaptic input were not further differentiated by phenotype and therefore represented a combination of both DA and non-DA cell populations (Nair-Roberts et al. 2008). It is possible that a differential change in synapse number onto these two major populations might have resulted in offsetting effects that were not detected. It is also possible that within these two major populations, prenatal NIC may have caused differential alterations in synaptic input to cells with different forebrain targets (Carr and Sesack 2000; Omelchenko et al. 2009). A further possibility is that alterations

in individual afferents to the VTA induced by prenatal NIC were compensated by opposing changes in other afferents (Omelchenko and Sesack 2007). Hence, it remains to be investigated whether prenatal NIC exposure alters the density of synaptic inputs from specific sources to individual cell populations in the VTA (Carr and Sesack 2000; Omelchenko et al. 2009). The present study indicates only that there is no significant structural change in the overall excitatory and inhibitory afferent innervation to VTA neurons. Functional considerations In certain respects, it is reassuring that prenatal NIC exposure did not cause gross structural alterations of ventral midbrain neurons or their synaptic innervation. A considerable number of women smoke during pregnancy without overt deleterious consequences to their offspring. Nevertheless, lack of evidence for morphological changes does not mean that physiological function is normal. Prenatal NIC exposure disrupts normal DA transmission in the midbrain and forebrain as measured in pre-adolescence or adolescence, although studies do not agree on whether DA and/or DA metabolites are elevated or reduced (Slotkin et al. 1987; Navarro et al. 1988; Fung and Lau 1989; Ribary and Lichtensteiger 1989; Richardson and Tizabi 1994; Muneoka et al. 1997; Kane et al. 2004). Studies in adult animals report that neurochemical measures of DA are decreased, suggesting that the most persistent effect of prenatal NIC is a suppression of DA transmission (Ribary and Lichtensteiger 1989; Muneoka et al. 1999). Several mechanisms may lead to such outcomes (Navarro et al. 1988) and underlie behavioral abnormalities including attention deficit/hyperactivity disorder and the heightened susceptibility for NIC dependence later in life. In addition to alterations in DA physiology, studies in human children and animals following prenatal NIC exposure have consistently reported structural changes in cortical regions. Hence, despite the largely negative outcomes of the present study, there remain multiple important reasons for abstaining from cigarette smoking or NIC replacement therapies during pregnancy. Acknowledgments

Grant support: USPHS grant DA021276.

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Impact of prenatal nicotine on the structure of midbrain dopamine regions in the rat.

In utero exposure of rats to nicotine (NIC) provides a useful animal model for studying the impact of smoking during pregnancy on human offspring. Cer...
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