Brain Iron Accumulation and the Formation of Calcifications After Developmental Zika Virus Infection.

Intracranial calcifications (ICC) are the most common neuropathological finding in the brains of children exposed in utero to the Zika virus (ZIKV). Using a mouse model of developmental ZIKV infection, we reported widespread calcifications in the brains of susceptible mice that correlated in multiple ways with the behavioral deficits observed. Here, we examined the time course of ICC development and the role of iron deposition in this process, in 3 genetically distinct inbred strains of mice. Brain iron deposits were evident by Perls' staining at 2 weeks post infection, becoming increasingly dense and coinciding with calcium buildup and the formation of ICCs. A regional analysis of the brains of susceptible mice (C57BL/6J and 129S1/SvImJ strains) revealed the presence of iron initially in regions containing many ZIKV-immunoreactive cells, but then spreading to regions containing few infected cells, most notably the thalamus and the fasciculus retroflexus. Microglial activation was widespread initially and later delineated the sites of ICC formation. Behavioral tests conducted at 5-6 weeks of age revealed greater deficits in mice with the most extensive iron deposition and calcification of subcortical regions, such as thalamus. These findings point to iron deposition as a key factor in the development of ICCs after developmental ZIKV infection.

Mouse Strain and Sex-Dependent Differences in Long-term Behavioral Abnormalities and Neuropathologies after Developmental Zika Infection.

Exposure of the developing fetus to Zika virus (ZIKV) results in a set of brain abnormalities described as the congenital Zika syndrome. Although microcephaly is the most obvious outcome, neuropathologies, such as intracranial calcifications and polymicrogyria, can occur in the absence of microcephaly. Moreover, the full impact of exposure on motor, social, and cognitive skills during development remains uncharacterized. We examined the long-term neurobehavioral consequences of neonatal ZIKV exposure in four genetically divergent inbred mouse strains (C57BL/6J, 129S1/SvImJ, FVB/NJ, and DBA/2J). Male and female mice were infected on postnatal day 1, considered comparable with exposure late in the second trimester of humans. We demonstrate strain differences in early susceptibility to the virus and the time course of glial reaction in the brain. These changes were associated with strain- and sex-dependent differences in long-term behavioral abnormalities that include hyperactivity, impulsiveness, and motor incoordination. In addition, the adult brains of susceptible mice exhibited widespread calcifications that may underlie the behavioral deficits observed. Characterization of the neuropathological sequelae of developmental exposure to the Zika virus in different immunocompetent mouse strains provides a foundation for identifying genetic and immune factors that contribute to long-term neurobehavioral consequences in susceptible individuals.SIGNIFICANCE STATEMENT Developmental Zika virus (ZIKV) infection is now known to cause brain abnormalities in infants that do not display microcephaly at birth, and the full impact of these more subtle neuropathologies has yet to be determined. We demonstrate in a mouse model that long-lasting behavioral aberrations occur after developmental ZIKV exposure. We compare four divergent mouse strains and find that the effects of Zika infection differ greatly between strains, in terms of behavioral changes, sex differences, and the intracranial calcifications that develop in the brains of susceptible mice. These findings provide a foundation for identifying susceptibility factors that lead to the development of abnormal behaviors secondary to ZIKV infection early in life.

Staufen2 deficiency leads to impaired response to novelty in mice.

Staufen2 (Stau2) is a double-stranded RNA-binding protein (RBP) involved in posttranscriptional gene expression control in neurons. In flies, staufen contributes to learning and long-term memory formation. To study the impact of mammalian Stau2 on behavior, we generated a novel gene-trap mouse model that yields significant constitutive downregulation of Stau2 (Stau2GT). In order to investigate the effect of Stau2 downregulation on hippocampus-dependent behavior, we performed a battery of behavioral assays, i.e. open field, novel object recognition/location (NOR/L) and Barnes maze. Stau2GT mice displayed reduced locomotor activity in the open field and altered novelty preference in the NOR and NOL paradigms. Adult Stau2GT male mice failed to discriminate between familiar and newly introduced objects but showed enhanced spatial novelty detection. Additionally, we observed deficits in discriminating different spatial contexts in a Barnes maze assay. Together, our data suggest that Stau2 contributes to novelty preference and explorative behavior that is a driver for proper spatial learning in mice.

Neurodegeneration by activated microglia across a nanofiltration membrane.

Microglia have been implicated in the pathogenesis of several neurodegenerative diseases, but their precise role remains elusive. Although neuron loss in the presence of lipopolysaccharide-stimulated microglia has been well documented, a novel coculture paradigm was developed as a new approach to assess the diffusible, soluble mediators of neurodegeneration. Isolated microglia were plated on membrane inserts that were coated with a layer of cellulose acetate. The cellulose acetate-coated membranes have nanofiltration properties, in that only molecules with masses less than 350 Da can pass through. Products released from activated microglia that were separated from primary ventral mesencephalon cells beneath the nanofiltering membrane were able to kill the dopamine neurons. Microglial cytokines cannot diffuse through this separating membrane. Addition of a nitric oxide synthase inhibitor prevented the loss of the dopamine neurons. These data describe a novel coculture system for studying diffusible factors and further support nitric oxide production as an important mediator in microglia-induced neuron death.

Nicotinamide improves motor deficits and upregulates PGC-1α and BDNF gene expression in a mouse model of Huntington's disease.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an expansion of the polyglutamine (polyQ) repeat in exon-1 in the Huntingtin gene (HTT). This results in misfolding and accumulation of the huntingtin (htt) protein, forming nuclear and cytoplasmic inclusions. HD is associated with dysregulation of gene expression as well as mitochondrial dysfunction. We hypothesized that by improving transcriptional regulation of genes necessary for energy metabolism, the HD motor phenotype would also improve. We therefore examined the protective effects of nicotinamide (NAM), a well-characterized water-soluble B vitamin that is an inhibitor of sirtuin1/class III NAD(+)-dependent histone deacetylase (HDAC). In this study, both mini-osmotic pumps and drinking water deliveries were tested at 250 mg NAM/kg/day, using the B6.HDR6/1 transgenic mouse model. Results were similar for both modes of delivery, and there was no evidence of toxicity. We found that NAM treatment increased mRNA levels of brain-derived neurotrophic factor (BDNF), and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis. Protein levels of BDNF were also significantly increased. In addition, NAM treatment increased PGC-1α activation in HD mice, pointing to a possible mode of action as a therapeutic. Critically, NAM treatment was able to improve motor deficits associated with the HD phenotype, tested as time courses of open field, rotarod, and balance beam activities. These improvements were substantial, despite the fact that NAM did not appear to reduce htt aggregation, or to prevent late-stage weight loss. Our study therefore concludes that NAM or similar drugs may be beneficial in clinical treatment of the motor dysfunctions of HD, while additional therapeutic approaches must be added to combat the aggregation phenotype and overall physiological decline.

Early or late-stage anti-N-terminal Huntingtin intrabody gene therapy reduces pathological features in B6.HDR6/1 mice.

Huntington disease (HD) is a progressive neurodegenerative disease caused by an expansion of a polyglutamine sequence in mutant huntingtin (mhtt) that produces abnormal folding and aggregation that results in the formation of nuclear and cytoplasmic neuronal inclusion bodies. Although the precise role of mhtt aggregates in the pathogenesis is unclear, attempts to reduce accumulated mhtt protein have ameliorated the phenotype in multiple cellular and in vivo HD models. Here, we provide critical results on intracranial delivery of a single-chain Fv intrabody, C4, which targets the first 17 amino acids of the htt protein, a region of httExon1 that is increasingly being recognized as pivotal. To assess long-term efficacy and safety issues, we used adenoassociated viral vectors (AAV2/1) to deliver intrabody genes to the striatum of inbred B6.HDR6/1 mice. Treatment initiation at various stages of the disease showed that early treatment preserved the largest number of cells without nuclear aggregates and that the accumulation of aggregated material could be delayed by several months. Even when intrabody treatment was not initiated until the clinical disease stage, significant, albeit smaller, effects were seen. These data indicate that neuronal intrabodies against critical N-terminal epitopes can be safely and effectively delivered using AAV2/1 to delay the aggregation phenotype during a sustained period in this HD model, even when delivery is initiated after disease onset.

Opioids activate brain analgesic circuits through cytochrome P450/epoxygenase signaling.

To assess the importance of brain cytochrome P450 (P450) activity in mu opioid analgesic action, we generated a mutant mouse with brain neuron-specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.

Prenatal inflammatory effects on nigrostriatal development in organotypic cultures.

Maternal intrauterine infection, and the accompanying inflammation in the fetal brain, represent a significant risk to the developing fetus. Dopamine (DA) neurons have been shown to be particularly vulnerable to inflammation induced by injection of the bacterial endotoxin lipopolysaccharide (LPS). In order to further examine the nature of this vulnerability, we used a combination of in vivo prenatal LPS exposure, and in vitro analysis of nigrostriatal development in organotypic cultures prepared from LPS-exposed rat fetuses. Control co-cultures prepared from unexposed E14 substantia nigra (SN/VTA) and E21 striatum exhibited numerous DA neurons in the nigral piece and robust ingrowth into the striatal piece. When E14 SN/VTA was obtained from fetuses exposed to LPS (0.1 mg/kg) on E10, initial DA cell numbers and striatal innervation in co-cultures were normal, but at longer durations in vitro, a reduction in DA neurons was observed. When striatal tissue from fetuses exposed to LPS on E14 or E18 was used in combination with non-exposed SN/VTA, DA neurons initially exhibited a normal pattern of ingrowth into LPS-exposed striatum. However, with longer durations in vitro, DA neurons were lost more rapidly when co-cultured with LPS-exposed striatum. Despite the loss of DA neurons, striatal DA innervation was only reduced in cultures prepared with striatum exposed to LPS at E18, at the longest time period examined. Experiments in which unexposed SN/VTA was given the choice to grow toward control striatum or toward LPS-exposed striatum supported the idea that the tropic qualities of the striatum were not altered by LPS-induced inflammation. Thus, the inflammation induced by LPS not only affects the SN/VTA DA neurons, but also alters the neurotrophic--although not the neurotropic--characteristics of the striatum. Such alterations in nigrostriatal development may demonstrate how adverse perinatal events predispose the developing brain toward the later development of Parkinson's disease.

Afferent influences on striatal development in organotypic cocultures.

Organotypic cocultures of striatum, cortex, and ventral mesencephalon were used to study the anatomical and physiological development of striatal neurons in the presence or absence of cortical and nigral (SN/VTA) inputs. Striatum and cortex were dissected from prenatal (E18-E22) or early postnatal (P0-P2) rats, and SN/VTA was dissected from E14-15 fetuses; pieces were maintained up to 3 weeks in static slice culture. Triple cocultures containing SN/VTA exhibited rapid and robust dopamine (DA) innervation of the striatum in a patchy pattern, and homogeneous distribution within the cortical piece, regardless of the orientations of the three pieces. DA fibers within the striatal piece overlapped striatal patch neurons, marked by DARPP-32 immunoreactivity, in striatal cultures prepared from all age rats, but development most analogous to that seen in vivo was observed with the use of late prenatal (E20-E22) striatum. The patch/matrix organization was maintained in cultures prepared from late prenatal striatum in the presence of cortical and nigrostriatal DA afferents. In addition, a more complete transition to a patchy organization was observed in E18/19 striatal cultures in the presence of cortical and DA innervation. Electrophysiological recording demonstrated the presence of both spontaneous and cortically evoked activity in striatal medium spiny neurons; this activity was greatly influenced by the presence of DA innervation. These findings demonstrate the importance of afferent innervation in the maturation of striatal neurons in organotypic cultures.

Polychlorinated biphenyl-induced neurotoxicity in organotypic cocultures of developing rat ventral mesencephalon and striatum.

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that are highly toxic to the developing nervous system, particularly via their disruption of dopamine (DA) function. In order to characterize the effects of PCBs on the developing basal ganglia DA system, we utilized an organotypic coculture system of developing rat striatum and ventral mesencephalon (VM). Exposure of the cocultures to an environmentally relevant mixture of PCBs for 1, 3, 7, or 14 days reduced tissue DA concentrations and increased medium levels of DA, homovanillic acid, and 3,4-dihydroxyphenylacetic acid. PCB exposure also increased neuronal cell death in both the VM and the striatum and reduced the number of DA neurons in the VM. Decreases in both tyrosine hydroxylase and DA transporter protein expression were shown by Western blot analysis in PCB-exposed cocultures. There was also an increase in neuronal cell death, identified by Fluoro Jade B, prior to a reduction in the number of VM DA neurons; we hypothesize this increase to be partly due to a loss of gamma-aminobutyric acid (GABA) neurons. Indeed, Western blot analysis revealed up to a 50% reduction in both VM and striatal glutamic acid decarboxylase 65/67. Analysis of tissue PCB levels revealed that concentrations were at or below 10 ppm following all exposure paradigms. This coculture system provides an excellent model to examine the chronology of PCB-induced neurotoxic events in the developing basal ganglia. Our results suggest that PCB-induced neurotoxicity in the developing basal ganglia involves GABAergic neuronal dysfunction, in addition to PCBs' better-recognized effects on DA function. These findings have important implications for disease states such as Parkinson's disease and for developmental deficits associated with exposure to PCBs and toxicologically similar environmental contaminants.

Dopaminergic development of prenatal ventral mesencephalon and striatum in organotypic co-cultures.

Using organotypic co-cultures of rat embryonic day 14 (E14) ventral mesencephalon (VM) and E21 striatum, we have described the developmental changes in (i) dopamine (DA) neurochemistry; (ii) numbers of DA neurons; and (iii) protein expression of tyrosine hydroxylase (TH), DA transporter (DAT), and glutamic acid decarboxylase (GAD 65/67), over 17 days in vitro (DIV). Co-cultures demonstrated changes in DA development similar to those observed in vivo. The numbers of VM DA neurons remained relatively constant, while levels of VM DA progressively increased through 10 DIV. After 3 DIV, the levels of striatal DA increased substantially, through 10 DIV. Tissue levels of DA metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) reflected changes in tissue DA concentrations, indicating that release and metabolism of DA are similar to these characteristics observed in vivo. Western blot analysis of TH protein expression revealed large increases in VM TH after only 3 DIV, followed by a decline in levels through 17 DIV; levels of striatal TH, in contrast, increased through this period. Additionally, DAT and GAD 65/67 expression increased, in both the VM and striatum, over 17 DIV. By 17 DIV, many measures of DA function had decreased from those assessed at 10 DIV, thus providing an approximate limit to the effective duration of use of this co-culture model. Our results provide a much-needed description of the neurochemical changes that occur during the maturation of VM and striatum in organotypic co-cultures. Additionally, these results provide a foundation for future studies to assess toxic challenges of the developing nigrostriatal DA system, in vitro.

Dopaminergic modulation of striatal plateau depolarizations in corticostriatal organotypic cocultures.

RATIONALE: It has been proposed that dopamine (DA) sustains up states in striatal medium spiny neurons (MSN). Testing this hypothesis requires an in vitro preparation, but up states are typically only observed in vivo. OBJECTIVES: In this study, we used corticostriatal organotypic cocultures, a preparation in which up states have been previously observed, to test the DA control of cortically-driven plateau depolarizations. RESULTS: After 7-21 days in vitro in serum-free conditions, plateau depolarizations resembling up states were only observed in cultures with a critical extent of striatal DA innervation. These plateaus were completely blocked by the non-NMDA antagonist CNQX and significantly shortened by the NMDA antagonist APV or the D(1) antagonist SCH23390. Intracellular interruption of Ca(++) or protein-kinase A (PKA) signaling also eliminated the plateaus. The D(2) antagonist eticlopride failed to disrupt the plateaus, but significantly increased MSN excitability. CONCLUSIONS: These results suggest that coincident activation of corticostriatal glutamatergic and mesostriatal DA transmission may set ensembles of MSN into prolonged depolarizations through a D(1) enhancement of striatal NMDA function in a Ca(++) and PKA-dependent manner.

Regional expression of constitutive and inducible transcription factors following transient focal ischemia in the neonatal rat: influence of hypothermia.

Ischemia is a potent modulator of gene expression. Differential expression of transcription factors after focal ischemia may reflect the potential for neuronal recovery in peri-ischemic regions. Previously, we demonstrated that hypothermia reduces the volume of damage in a model of neonatal focal ischemia. In the present study, immunocytochemistry was used to assess the temporal and spatial profiles of the transcription factors Fos and pCREB under normal and hypothermic conditions in this neonatal model of focal ischemia. At 7 days of age, rat pups underwent a permanent middle cerebral artery occlusion (MCAo) coupled with a temporary 1-h occlusion of the common carotid artery (CCAo). They were maintained at 37 degrees C throughout ischemia and reperfusion (Normothermic), or given 1 h of hypothermic conditions (28 degrees C) either during the occlusion (Intraischemic Hypothermia) or during the second hour of reperfusion (postischemic hypothermia). In normothermic pups, Fos immunoreactivity peaked at early time points (4-8 h post-ischemia) in a narrow band in peri-ischemic regions. By later stages of reperfusion (12-24 h), there was a more widespread induction in peri-ischemic regions including the ipsilateral cortex. In contrast with Fos, the constitutive transcription factor pCREB was reduced in core regions at all time points examined. Both the c-fos induction in peri-ischemic regions and the reduction of pCREB in the core were attenuated by intraischemic hypothermia. Postischemic hypothermia altered the distribution of Fos immunoreactivity without significantly changing the number of Fos- and pCREB-immunoreactive cells compared to normothermic rats. Both intra- and postischemic hypothermia reduced the number of caspase-immunoreactive cells. Thus, focal ischemia in the P7 rat produces different distributions of Fos and pCREB than what has been observed in adult rats subjected to focal ischemia, and expression of these transcription factors can be altered by hypothermia.

Pattern of corticostriatal innervation in organotypic cocultures is dependent on the age of the cortical tissue.

The patch-matrix organization of the striatum is defined by the selective expression of neuronal markers and a semisegregated pattern of afferents and efferents that develops before birth in all mammals. Differential projections from 'limbic' and 'somatomotor' cortices contribute to the selective circuitry of patch ("striosome") and matrix compartments. Organotypic cultures were used to determine the pattern of early corticostriatal innervation as a first step toward understanding the role of cortical innervation in the development of striatal patch-matrix organization. Perinatal striatum (E19-P4) was cocultured with the cortex obtained from same-age or different-age rats in the presence or absence of substantia nigra obtained from E14-15 fetuses. After 4-21 days in vitro, crystals of biocytin were placed directly onto the cortical piece to trace cortical projections into the striatal piece. Cortex obtained from fetuses (E19-22) or neonatal (P0-1) rats gave rise to a dense innervation of both prenatal and postnatal striatal slices; however, the pattern of biocytin-labeled fibers was found to be highly dependent on the age of the cortical tissue used. Cortex derived from rats between E20 and P1 gave rise to a heterogeneous distribution of fibers indicative of striatal patches when combined with striatal slices from same-age or younger (E18-19) fetuses. Cortex from E18-19 fetuses produced a homogeneous innervation even when cocultured with older striatal tissue in which the striatal patches were already present. The postnatal cortex (P2-P5) gave rise to little to no innervation of striatum of all ages. Similar findings were obtained with the use of either prelimbic or somatosensory cortex. In double- and triple-labeled cultures, the distribution of corticostriatal fibers overlapped substantially with patches of developing striatal neurons, as revealed by DARPP-32 immunocytochemistry. Dopaminergic innervation present when the substantia nigra was included in the cocultures also distributed preferentially to the developing patch compartment, but it did not substantially alter the pattern of corticostriatal innervation. These findings suggest that the cortex provides directive signals to the developing striatum rather than simply responding to the presence of patches that have already formed.

Blockade of D1 dopaminergic transmission alleviates c-fos induction and cleaved caspase-3 expression in the brains of rat pups exposed to prenatal cocaine or perinatal asphyxia.

Hypoxia due to uterine vasoconstriction may be an important cause of the teratogenic consequences of prenatal cocaine exposure. We used immediate-early gene and cleaved caspase-3 expression patterns to monitor fetal brain regions affected by intrauterine hypoxia and prenatal cocaine and pretreatment with the D1 dopamine receptor antagonist SCH 23390 to determine how much of the induction observed was due to dopamine. Both cocaine binge (3 x 15 mg/kg) and perinatal asphyxia on embryonic day 22 (E22) induced c-fos in the striatum as well as in several other brain regions within 3 h after treatment. Maternal administration of a D1 dopamine antagonist, SCH 23390, before either cocaine or asphyxia exposure dramatically reduced the numbers of Fos-immunoreactive cells in the striatum as well as in many other brain regions. Cells immunoreactive for cleaved caspase-3 expression were more numerous after perinatal asphyxia than after prenatal cocaine exposure in most brain regions 24 h after C-section. SCH 23390 decreased caspase-3 expression after both birth insults, indicating that the increased incidence of apoptosis is related to overactivation of dopaminergic pathways.

c-fos and cleaved caspase-3 expression after perinatal exposure to ethanol, cocaine, or the combination of both drugs.

Poly-drug abuse during pregnancy is a major public health concern. The combined effects of cocaine and ethanol may be more injurious to the fetal nervous system than either drug alone. In order to identify areas of the brain vulnerable to concurrent exposure, we examined the expression of the immediate-early gene (IEG), c-fos, and cleaved caspase-3, the 'executioner' caspase in apoptosis. Pregnant rats were treated with either ethanol diet, cocaine binge, or both. At birth, the brains of fetuses exposed to cocaine exhibited an increase in Fos immunoreactivity in many brain regions. Prenatal exposure to ethanol did not increase Fos expression above that observed in control rats at early points after birth. However, Fos expression at 24 h after birth was higher after ethanol diet treatment in several brain regions, such as the amygdala, ventromedial hypothalamus, and medial thalamus. Only in the striatum did the combination of ethanol and cocaine cause greater Fos expression than either prenatal cocaine or ethanol alone. Increased cleaved caspase-3 expression was observed at the 24-h time point for both ethanol- and cocaine-exposed brains, most notably in the septum, retrosplenial cortex, and the hippocampus. Concurrent ethanol and cocaine exposure did not elevate cleaved caspase-3 expression beyond that of either drug alone. Analysis of the extent of c-fos and caspase-3 induction did not indicate a consistent relationship of expression in any of the drug treatment groups nor in any brain region. These results indicate that both prenatal cocaine and prenatal ethanol exposure increase Fos and cleaved caspase-3 expression in the developing brain in a time- and region-dependent manner, but that the combination of low-dose, chronic ethanol, and binge cocaine does not cause greater apoptosis.

Basal EGR-1 (zif268, NGFI-A, Krox-24) expression in developing striatal patches: role of dopamine and glutamate.

Egr-1 (also known as zif268, NGFI-A, or Krox 24) is an immediate-early gene of the zinc finger family that exhibits relatively high constitutive expression in the brain, as well as inducibility by seizure activity, stimulants, and salient physiological stimuli. Immunocytochemical detection of the Egr-1 protein in the developing striatum revealed that in the late prenatal and early postnatal period, Egr-1 protein was expressed selectively in patches of striatal neurons under basal conditions. Egr-1 immunoreactivity was co-expressed with known markers of striatal patch neurons, indicating that expression was greatest in the striatal patch compartment. This patchy expression of Egr-1 transitioned to a nearly homogeneous pattern of Egr-1-immunoreactive cells by postnatal day 10, at which time most striatal neurons appeared to be Egr-1-immunoreactive. The dopamine D1 antagonist SCH23390 (0.5-1.0 mg/kg) reduced Egr-1 expression during the first week postnatal, but it was no longer effective at postnatal day 10. On the other hand, the noncompetitive NMDA antagonist MK-801 (0.5-1.0 mg/kg) became more effective at reducing Egr-1 expression with age. Neonatal destruction of nigrostriatal dopamine afferents reduced the basal pattern of Egr-1 expression for 2-3 days after the lesion, but then Egr-1 expression returned. Thus, Egr-1 expression in the developing striatum appears to be driven first by dopaminergic afferents, and then later in development by excitatory glutamatergic afferents.

Immediate-early gene expression in concurrent prenatal ethanol- and/or cocaine-exposed rat pups: intrauterine differences in cocaine levels and Fos expression.

Concurrent use of cocaine and ethanol is a common mode of abuse. Cocaine and ethanol have distinctive pharmacologies but both have been shown to cause uterine vasoconstriction and fetal hypoxia. We developed a paradigm of chronic ethanol exposure via liquid diet coupled with binge cocaine exposure on the last day of gestation. Lipton et al. demonstrated unequal segregation of cocaine in rat fetuses as a function of proximal-distal location in the uterus, indicating a differential vasoconstriction of the two main arteries supplying the uterus in rats receiving cocaine. By performing C-sections after exposure to cocaine, we were able to measure the cocaine content and immediate-early gene (IEG) induction in the brains of fetuses according to their intrauterine position and assess the potentially vasoconstrictive effect of ethanol. HPLC analysis of fetal brains exposed to cocaine supported the study of Lipton et al.: fetuses from the proximal (lower) end of the uterus had more cocaine than fetuses from the distal (upper) end. Concurrent ethanol decreased the amount of cocaine reaching the fetuses and diminished the proximal-distal gradient. There were increased numbers of Fos-immunoreactive cells in fetuses exposed to both ethanol and cocaine compared to cocaine binge only. Additionally, the gradient of c-fos induction observed as a function of intrauterine position in cocaine-treated rats was in the opposite direction: most distal fetuses generally had the most Fos-immunoreactive cells. These results indicate that IEG induction in fetal brains exposed to cocaine and ethanol may be more related to hypoxic consequences of prenatal drug exposure.