Altered localization of choline transporter sites in the mouse hippocampus after prenatal heroin exposure

Brain Research Bulletin 63 (2004) 25–32

Altered localization of choline transporter sites in the mouse hippocampus after prenatal heroin exposure Ori Vatury a , Jacob Barg a , Theodore A. Slotkin b , Joseph Yanai a,b,∗ a

b

The Ross Laboratory for Studies in Neural Birth Defects, Department of Anatomy and Cell Biology, The Hebrew University-Hadassah Medical School, Box 12272, 91120 Jerusalem, Israel Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA Received 7 September 2003; received in revised form 15 November 2003; accepted 30 November 2003

Abstract Prenatal heroin exposure disrupts hippocampal cholinergic synaptic function and related behaviors. Biochemical studies indicate an increase in the number of presynaptic high-affinity choline transporter (HACT) sites, as assessed by [3 H]hemicholinium-3 (HC-3) binding. The present study was designed to assess whether this effect involves global upregulation of the transporter, or whether disruption occurs with a specific tempero-spatial distribution. Pregnant mice were given 10 mg/kg per day of heroin subcutaneously on gestational days (GD) 9–18. Autoradiographic distribution of HC-3 binding sites was evaluated in the hippocampus of the offspring at postnatal days 15, 25, and 53. These results, suggestive of hippocampal “miswiring,” are likely to explain the net impairment of cholinergic synaptic function after prenatal heroin exposure, despite the simultaneous upregulation of both presynaptic cholinergic activity and postsynaptic receptors. Understanding the subregional selectivity of hippocampal defects can lead to the development of strategies that may potentially enable therapeutic interventions to offset or reverse the neurobehavioral defects. © 2004 Elsevier Inc. All rights reserved. Keywords: Acetylcholine; Autoradiography; Choline transporter; Hemicholinium-3; Heroin; Hippocampus

1. Introduction Heroin is a major drug of abuse [3,31] and is one of the drugs of choice of pregnant women in the drug addicted population [55]. Several studies have demonstrated teratogenic [56,57] and neuroteratogenic effects of heroin in humans [22,32,33,42,50,51]. Studies on animals exposed prenatally to opioids have shown alterations in multiple brain regions and innervations, resulting in numerous behavioral deficits [52,53,62]. In our model of heroin neurobehavioral teratogenicity, mice prenatally exposed to heroin exhibited deficits in hippocampus related behaviors [44,46,59] associated with a specific and unusual alteration in septohippocampal cholinergic function. Presynaptic terminals were hyperactive, as indicated by increased acetylcholine (ACh) release [1] and by an increase in the number of choline transporter sites, assessed by [3 H]hemicholinium-3 (HC-3) [44–46]. Unexpectedly, the presynaptic hyperactivity, which ordinarily would

∗

Corresponding author. Tel.: +972-2-675-8439; fax: +972-2-675-8443. E-mail address: [email protected] (J. Yanai).

0361-9230/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2003.11.004

evoke postsynaptic desensitization [30,58] was instead accompanied by postsynaptic hyperactivity [44,45], suggesting a deficiency in the ability of presynaptic neurons to evoke postsynaptic cell signals. This was confirmed by the specific loss of protein kinase C (PKC) responses to cholinergic receptor stimulation in the heroin-exposed offspring [45,46]. There is a causal relationship between cholinergic synaptic alterations evoked by prenatal heroin exposure and the resultant behavioral deficits, as both are restored to normal by subsequent grafting of septal cholinergic cells into the hippocampus in adulthood [44]. The fact that the neurochemical and functional alterations are reversed when new cholinergic connections are made suggests that at least some of the heroin-induced impairment of synaptic communication reflects architectural “errors” in the distribution of cholinergic terminals within the hippocampus. Indeed, such an effect would account for simultaneous presynaptic and postsynaptic hyperactivity in the face of impaired net synaptic function. In the present study, we evaluated quantitative localization of the presynaptic choline transporter using [3 H]HC-3 autoradiography [15] on postnatal (PN) days 15 and 25, periods that represent critical milestones

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of hippocampal synaptic development [7,8,15,17,29,34,38, 43,61], as well as in adulthood (PN53), when behavioral manifestations are fully evident [60]. In adulthood, sex-dependence of the effects were evaluated, since previous studies indicate sex-selectivity of the effects of prenatal exposure to morphine on other neurotransmitter systems [54].

2. Methods 2.1. Animal treatment All experiments were carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Heterogeneous stock (HS/Ibg) mice [27] were housed in mating cages maintained at 24 ◦ C with a 12 h-light:12 h-dark cycle where each cage housed four females and one male. The females were checked daily for vaginal plugs at 08:00 h and those that had mated were then housed with other pregnant females until gestational day (GD) 18, at which point the mice were housed individually. Heroin was administered on GD 9–18, where the vaginal plug date was considered as GD 1 and the day of birth was considered postnatal day (PN) 1. To isolate potential drug effects on maternal caretaking, some of the control and heroin pups were fostered by control dams within 24 h after birth. Since, as in previous studies, the results obtained from fostered and non-fostered offspring did not differ [60], the data of the two groups were pooled for presentation. Pups were weaned on PN25, segregated by sex and housed in groups of five until used in the experiment. Each experiment utilized not more than two pups from each litter. 2.2. Drug administration The drug administration paradigm was based on our previous work showing neurobehavioral deficits after heroin administration without compromising fetal growth and viability [40,44–46]. Pregnant mice received daily subcutaneous injections of 10 mg/kg of heroin in saline on GD 9–18, whereas controls received the vehicle on the same schedule. The termination of heroin on GD 18 is important since heroin causes acute withdrawal effects [6] that may affect the ability to nurse and interfere with the cues needed to elicit maternal care [4,9,25,35]. Consequently, terminating drug treatment prior to parturition in the present study prevented withdrawal symptoms in the dam and avoided the potential confound of withdrawal-induced mortality and growth retardation in the offspring [25]. The dose used in the present study represents the maximum tolerated exposure commensurate with continuation of pregnancy, lack of fetal resorption, and postnatal survival, as established in our earlier work [40,44–46,59]. Variables related to physiology and maternal

behavior as well as offspring development were monitored in an extensive study that has already been published [37]. 2.3. Tissue preparation Offspring were decapitated on PN15, 25, or 53 and the brains were rapidly removed, frozen in −20 ◦ C isopentane and stored at −70 ◦ C. Tissues were mounted on cryostat chucks with OCT imbedding medium (Tissue-Tek by Sakura, Torrance, CA, USA), frozen on dry ice and cut coronally (16 ␮m). Sections containing the hippocampus were identified during sectioning by periodically staining a sample section with Toluidine Blue O (Sigma Chemical Co., St. Louis, MO, USA) and were subjected to microscopic examination. The sections containing the prominent pyramidal and granular layers of the hippocampus were then thaw-mounted onto pre-prepared Super Frost Plus slides (Menzel-Glaser, Braunschweig, Germany) and were stored at −70 ◦ C. 2.4. Autoradiography [3 H]HC-3 autoradiography was conducted essentially as described earlier [15]. Slide-mounted hippocampal tissue sections were thawed for 14 min. They were then incubated for 25 min at 25 ◦ C by layering onto each slide 400 ␮l of assay medium containing 50 mM glycylglycine buffer, pH 7.8, 200 mM NaCl, and 33 nM [3 H]HC-3 (127.8 Ci/mmol; NEN, Boston, MA, USA), a saturating concentration approximately three times the Kd of HC-3 for the high-affinity choline transporter [15]. Previous studies showed that the Kd values of HC-3 binding did not differ between the adult and neonatal brains [15]. Non-specific binding was determined using an additional 10 ␮M unlabeled HC-3. Following the incubation, slides were washed in two changes of ice-cold 50 mM glycylglycine buffer, pH 7.8, with 200 mM NaCl, for 2 min each, followed by two 15 s rinses in ice-cold distilled water to remove salts [15]. Slides were then dried and placed in autoradiography cassettes (Amersham, Arlington Heights, IL, USA) apposed to tritium sensitive film (Hyperfilm 3 H; Amersham). PN53 slides were exposed to film for 2 months, whereas PN15 and 25 were exposed for 3 months. [3 H]MicroscalesTM (Amersham) were co-exposed with the tissue sections, one for each cassette. Films were developed in Kodak (Rochester, NY, USA) D76 developer for 5 min, Kodak Indicator Stop Bath for 30 s and Kodak Rapid Fixer for 2 min at 18 ◦ C. The developed films were scanned using a standard white backlight and a high resolution CCD camera (M-852; Sony, Japan) placed 45 cm from the films. Images were acquired using ImageProTM software (MediaCybernetics, Silver Spring, MD, USA) and data were analyzed using Scion Image Beta 4.02 software (Scion Corporation, Frederick, MD, USA). For each brain section the background was determined by measuring the mean density of the area outside the brain section. The scores of non-specific binding were added to

O. Vatury et al. / Brain Research Bulletin 63 (2004) 25–32

the background and were then subtracted from the measured density (Fig. 1). The results were corrected for the amount of protein per measured area [5], assessed in several 16 ␮m sections from each age group. It should be noted that quenching is known to be similar in the hippocampus in all ages studied [14]. 2.5. Data analysis The data were analyzed using multivariate ANOVA incorporating all factors (treatment, age, region, sex), with lower order ANOVAs to evaluate effects in specific regions. Data were log transformed whenever variance was heterogeneous. Significance was assumed at P < 0.05 for main effects; however, for interactions at P < 0.1, we also examined whether lower order main effects were detectable after subdivision of the interactive variables [41]. Because prior, biochemical work indicated that HC-3 binding increases with age and with prenatal heroin treatment [45] a one-tailed criterion was used.

3. Results 3.1. Regional development of HC-3 binding in control mice Overall, HC-3 binding increased significantly with age but the pattern was not identical in all of the hippocampal regions (Fig. 2). Values increased monotonically in CA1 and CA3 but the increase occurred somewhat earlier in CA3. In contrast to these two regions, binding in the dentate gyrus was already high by PN15 and remained relatively unchanged into adulthood. Accordingly, the relative distribution of HC-3 binding sites changed substantially across development. In CA1, there were parallel increases in molecular and pyramidal cell layers. In CA3, the molecular layer showed a greater proportional increase between PN15 and PN53 than did the pyramidal layer, so that the distribution of sites shifted in favor of the molecular layer. In the dentate gyrus, although the proportion of binding sites fell in both the molecular and pyramidal layers, the decrement was much larger for the molecular layer: only 3% of the binding sites were found in the molecular layer of the dentate gyrus by PN53 (see Fig. 2). 3.2. Effects of heroin on HC-3 binding sites In light of the radical, age-dependent shifts in the distribution of HC-3 binding sites, we assessed the effects of heroin both as the overall effect (across all regions and ages) as well as within each hippocampal region. Prenatal heroin exposure evoked a significant overall increase in HC-3 binding (main effect, P < 0.05) as well as a shift in the distribution of sites (treatment × age interaction, P < 0.08). However, we did not find sex-dependent effects (no interaction of treatment × sex, or treatment × sex × other variables), so

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the results were compiled for males and females combined. Sex effects may have been present but were not detected here because of the relatively small sample size. In the CA1 region, heroin treatment evoked a significant overall increase (main treatment effect, P < 0.01) that was also statistically significant in each of the two subregions, the molecular and pyramidal layers (Fig. 3). The increase occurred predominantly over the developmental phase in which HC-3 binding sites were increasing toward their adult values. In contrast to CA1, the overall effects of prenatal heroin on CA3 were highly selective (treatment × layer, P < 0.02): significant increases were seen in the molecular layer but not the pyramidal layer (Fig. 4), and if anything, the latter values were decreased in adulthood. Accordingly, the effect of heroin exposure in this hippocampal region entailed both changes in the number of transporter sites as well as in their relative distribution. As already noted, the developmental pattern in the dentate gyrus differed from the other two regions, characterized by a relatively constant total number, but with an ontogenetic decrease in the proportion of sites found in the molecular layer. Prenatal heroin exposure again evoked an overall increase in the total number of sites (main treatment effect, P < 0.05) but the pattern was entirely different in the two different areas (Fig. 5). The heroin group showed an initial, robust increase in HC-3 binding in the molecular layer and the effect decayed with age, so that by PN53, values were indistinguishable from those of controls. Alterations in the granule cell layer exhibited a different time course, with the increase centered primarily around PN25.

4. Discussion Previous work on the development of the hippocampus indicates a dichotomy between the major biochemical markers of cholinergic nerve terminals [38,61]. Whereas choline acetyltransferase, the enzyme that synthesizes acetylcholine, shows monotonic increases with development, high affinity choline uptake or HC-3 binding to the choline transporter show distinct developmental “spikes.” ChAT is a constitutive marker for presynaptic terminals and thus does not change with neuronal impulse activity; on the other hand, the transporter is rate-limiting in acetylcholine biosynthesis and is correspondingly regulated by synaptic activity [16,21,36,39]; accordingly, the distinct ontogenetic profiles of ChAT and HC-3 binding connote the fact that neuronal activity undergoes major changes that are superimposed on the ingrowth of cholinergic terminals. In the current study, we used autoradiographic techniques to examine the subregional distribution of transporter sites in the developing hippocampus and found disparities among the CA1, CA3, and dentate gyrus areas. The developmental spikes seen for biochemical evaluations of HC-3 binding and/or choline transporter activity in the whole hippocampus, thus actually represent

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Fig. 1. (a) A coronal section of a non-specific binding of HC-3 (10 ␮M of unlabeled HC-3 with 33 nM of [3 H]HC-3) in a 53-day-old mouse. (b) Coronal section of a representative [3H]hemicholinium labeling autoradiogram of a 53-day-old mouse. (c) An enlargement of the hippocampal region with the areas CA1, CA3, and Dg (dentate gyrus) marked. Note the extensive labeling of the hippocampal layers.

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Fig. 2. Development of HC-3 binding sites in hippocampal regions of control mice; values were compiled as the sum of the two layers examined for each region. Data represent means and standard errors obtained from three to seven animals at each age. The upper panel shows the developmental increase in absolute terms; note the developmental increases in CA1 and CA3, but not in dentate gyrus. The bottom panel shows the relative distribution within the various layers. Note the parallel increases in the proportion of HC-3 binding found in the molecular and pyramidal layers of CA1 and CA3. Consequently, the proportion in dentate gyrus falls with age, with disproportionately larger decrements in the molecular layer.

shifts in either the distribution of cholinergic terminals, or more likely in neuronal activity that is specific to each hippocampal subregion. Similarly, when HC-3 binding is perturbed by prenatal drug exposure, the global effects on transporter sites assessed biochemically may obscure more focal aspects, that actually dictate an adverse behavioral outcome. In our previous work, prenatal heroin exposure evoked elevations in HC-3 binding measured biochemically in the whole hippocampus [45,46]. It is thus of critical importance that, in the current study, we found that the increase actually represents disparate patterns of effects on various hippocampal subregions. The most consistent increases were seen in CA1 but even there, the largest effects occurred in the preweaning period, during the phase of most rapid synaptogenesis. In contrast, effects in CA3 and the dentate gyrus were far more focal and limited in duration. CA3 showed a transient increase limited to the molecular layer,

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Fig. 3. Effects of prenatal heroin treatment on HC-3 binding in subregions of hippocampal CA1. Data represent means and standard errors obtained from five to nine animals in each group, except on PN15, where the heroin group consisted of two animals. ANOVA for each layer appears at the top of the panels; subtesting of individual ages was not carried out because of the absence of treatment × age interactions.

but the effect was so large in magnitude that it virtually reversed the ontogenetic pattern; that is, activity in this part of CA3 actually decreased over age in the heroin group, instead of increasing as was the case for normal animals. The dentate gyrus also showed transient increases of large magnitude, with the earliest effect in the molecular layer and a later effect in the granular layer. This pattern is consistent with the fact that granule cells are among the last to mature in the hippocampus [2]. Our results are thus consistent with the idea that the heroin-induced overall upregulation of hippocampal HC-3 binding actually represents a family of changes in the various hippocampal areas, each with a different time frame and site of alteration. In effect, heroin leads to miswiring of the hippocampus and/or disruption of synaptic input/output relationships. This conclusion is likely to explain the apparently anomalous observations of a failure of effective cholinergic synaptic signaling, despite simultaneous hyperactivity of cholinergic inputs and upregulation of receptors [45]: the activation of cholinergic pathways may not correspond spatially or temporally to the proper location of impaired synaptic output. Accordingly, establishing new, properly wired connections, such as by grafts of immature cholinergic neurons into the damaged hip-

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Fig. 4. Effects of prenatal heroin treatment on HC-3 binding in subregions of hippocampal CA3. ANOVA for each layer appears at the top of the panels; subtesting of individual ages was not carried out because of the absence of treatment × age interactions.

pocampus, repairs the biochemical and behavioral damage [46]. Our finding of subregional selectivity of hippocampal effects of prenatal heroin exposure also has implications for underlying mechanisms and functional consequences. The specific relationship of HC-3 binding to neuronal activity implies that the affected pathways undergo bursts of hyperactivity during the period of cholinergic synaptogenesis. Opioidergic innervation generally inhibits cholinergic activity, through receptors which are located on the presynaptic cholinergic terminal and that reduce acetylcholine release in an age-dependent fashion [10,19]. Prenatal exposure to opiates is known to elicit subsequent decreases in opioid receptor expression [13,20,47–49], which would lead to disinhibition of cholinergic presynaptic activity. The results obtained here are thus consistent with the fact that hippocampal opiate receptors are not uniformly distributed [28]. Regardless of the underlying mechanism, in terms of functional consequences, the targeting of specific hippocampal subregions or layers is likely to be critical to behavioral outcomes. For the purpose of this discussion, it is worthwhile to focus on CA1, the region in which hyperactivity was most consistent. CA1 is implicated in spatial memory and electrical hyperactivity in this area impairs this behavior [11,12,23,24]. Moreover, grafting of embryonic CA1 tissue into the adult CA1 area, but not into the

Fig. 5. Effects of prenatal heroin treatment on HC-3 binding in subregions of hippocampal dentate gyrus. ANOVA for each layer appears at the top of the panels; subtesting of individual ages was not carried out because of the absence of treatment × age interactions.

CA3 area, corrects spatial performance deficits caused by ischemic damage to the hippocampus [18]. In addition to our findings for prenatal heroin exposure, CA1 appears to be vulnerable prenatal cocaine [26]. Accordingly, specific targeting of this subregion is likely to account for at least some of the actual neurobehavioral deficits caused by heroin or other neuroteratogens. Our findings of disparate patterns of cholinergic disruption in specific layers of CA3 and dentate gyrus indicate the need to pursue similar approaches to understand the potential role of alterations in these areas in the subsequent emergence of behavioral anomalies. The use of autoradiography thus gives a fuller picture of the effects of prenatal heroin exposure on hippocampal cholinergic function than does the biochemical approach. However, there are a number of limitations in applying this methodology to all situations. Obviously, autoradiography is far more cumbersome and variable than biochemical determinations and cannot easily assess overall quantitative differences. Biochemical determinations of HC-3 binding indicate a heroin-induced shift in both the number of transporter sites and in the binding affinity [45]; whereas autoradiography can assess the number of sites, the evaluation of binding affinity is extremely difficult, given the corresponding need to evaluate binding at multiple concentrations of radioligand. An additional problem is that, whereas autora-

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diography pinpoints effects in specific areas, the biochemical approach enables the assessment to be made over the entire tissue, giving appropriate weight to the contribution of each region. This can lead to a dichotomy in results because the effects are integrated over different masses of tissue with the two techniques. As just one example, radioligand binding performed on membranes from the whole hippocampus indicate a clear-cut increase in the heroin group in adulthood [45], whereas this effect was not discernible with autoradiography in the current study. Either the biochemical increases involve contributions from areas of the hippocampus not assessed here, or alternatively, the higher variability inherent in autoradiographic techniques confounds the ability to detect these changes unless much higher numbers of animals are evaluated. In the current study, for example, we were unable to detect sex effects in adulthood and thus combined results from males and females, likely increasing the overall variability of the results. On the other hand, biochemical approaches are deficient in that they cannot detect even large changes that may involve just a small subregion, one that would not contribute significantly to overall measurements involving the entire tissue. Accordingly, an actual understanding of the effects of prenatal heroin on hippocampal cholinergic function requires the combination of biochemical and autoradiographic techniques. In conclusion, prenatal heroin evokes an increase in the number of choline transporter sites with distinct ontogenetic profiles for each hippocampal subregion, and even for specific layers within those subregions. The temporal and spatial disparities of the effects in different areas of the hippocampus, indicative of miswiring of synaptic connections, are likely to explain the net impairment of cholinergic synaptic function, despite the simultaneous upregulation of both presynaptic cholinergic activity and postsynaptic receptors. Understanding the subregional selectivity of heroin-induced biochemical defects can lead to the development of strategies that may potentially enable therapeutic interventions to offset or reverse the neurobehavioral defects.

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