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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.

Mortality in infants with hypoplastic left heart syndrome (HLHS) is strongly correlated with right ventricle (RV) dysfunction. Cell therapy has demonstrated potential improvements of RV dysfunction in animal models related to HLHS, and neonatal human derived c-kit

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 (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.

The spinal locomotor central pattern generator (CPG) generates rhythmic activity with alternating flexion and extension phases. This rhythmic pattern is likely to result from inhibitory interactions between neural populations representing flexor and extensor half-centers. However, it is unclear whether the flexor-extensor CPG has a quasi-symmetric organization with both half-centers critically involved in rhythm generation, features an asymmetric organization with flexor-driven rhythmogenesis, or comprises a pair of intrinsically rhythmic half-centers. There are experimental data that support each of the above concepts but appear to be inconsistent with the others. In this theoretical/modeling study, we present and analyze a CPG model architecture that can operate in different regimes consistent with the above three concepts depending on conditions, which are defined by external excitatory drives to CPG half-centers. We show that control of frequency and phase durations within each regime depends on network dynamics, defined by the regime-dependent expression of the half-centers' intrinsic rhythmic capabilities and the operating phase transition mechanisms (escape vs. release). Our study suggests state dependency in locomotor CPG operation and proposes explanations for seemingly contradictory experimental data. NEW & NOTEWORTHY Our theoretical/modeling study focuses on the analysis of locomotor central pattern generators (CPGs) composed of conditionally bursting half-centers coupled with reciprocal inhibition and receiving independent external drives. We show that this CPG framework can operate in several regimes consistent with seemingly contradictory experimental data. In each regime, we study how intrinsic dynamics and phase-switching mechanisms control oscillation frequency and phase durations. Our results provide insights into the organization of spinal circuits controlling locomotion.

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 adeno-associated viral vectors (AAV2/1) to deliver intrabody genes to 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 over a sustained period of time in this HD model, even when delivery is initiated after disease onset.

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 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.

Cocaine abuse is a significant problem not only in the general population but also among pregnant women. Since cocaine readily crosses the placenta and is metabolized slowly in fetuses, they can be exposed to significant levels of cocaine for long periods. In humans the most common consequences of cocaine abuse during pregnancy include premature birth, lower birth weight, respiratory distress, bowel infarctions, cerebral infarctions, reduced head circumference, and increased risk of seizures. Behaviorally these newborns show an increased degree of "tremulousness," crying and irritability, and are over-reactive to environmental stimuli. Within a month these behaviors have recovered dramatically, but not to normal levels. Thus while there are a number of abnormalities associated with cocaine-exposed neonates, they are not imminently debilitating or life-threatening. However, the long-term consequences of this prenatal cocaine exposure remain to be elucidated. We have examined a rat model for neurochemical, neuroanatomical and behavioral changes resulting from prenatal cocaine exposure. Since cocaine is known to act by blocking the inactivation of the neurotransmitters dopamine, serotonin and norepinephrine, our studies have focused on brain dopamine (DA) and serotonin (5-HT) pathways. In this model system we have found neurochemical changes that are present at birth and that return to normal as the rat ages--similar to the recovery observed in infants. However, there are other neurochemical, anatomical and behavioral changes that persist after birth which may provide insights into the long-term consequences. It is hoped that by understanding the changes occurring in this rat model we will be better prepared to devise pharmacological interventions to circumvent the secondary consequences of prenatal cocaine exposure. These consequences might include increased susceptibility to drug addiction, seizures, depression, schizophrenia, Parkinson's disease, etc.

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.

To investigate the role of neurotrophins in the initial formation of striatal patch versus matrix, the spatial and temporal expression of trkB receptors was examined using immunohistochemistry. Polyclonal antibodies, against the C-terminus or the tyrosine kinase domain, revealed trkB-immunoreactive cells and fibers localized to patches beginning on embryonic day 19 in the rat, which co-localized with patchy dopamine fibers, substance P-immunoreactive neurons and glutamate receptors. Patchy striatal trkB expression was maintained after lesioning the nigrostriatal dopamine system. The patchy trkB distribution persisted through postnatal day 14, then became more homogeneous at the same time that nigrostriatal afferents become homogeneous. Later in development, trkB immunoreactivity was most intense in a subpopulation of large striatal cells that were similar in size and frequency to those immunoreactive for choline acetyltransferase. The spatiotemporal expression of trkB receptor in phenotypically distinct striatal patches, as well as evidence that neurotrophins regulate expression of neuronal phenotypic markers during development, may indicate a convergence of neurotrophins and afferent innervation on to future patch cells that may regulate the establishment of striatal compartmentalization.

Neurons in the visual cortex exhibit heterogeneity in feature selectivity and the tendency to generate action potentials synchronously with other nearby neurons. By examining visual responses from cat area 17 we found that, during gamma oscillations, there was a positive correlation between each unit's sharpness of orientation tuning, strength of oscillations, and propensity towards synchronisation with other units. Using a computational model, we demonstrated that heterogeneity in the strength of rhythmic inhibitory inputs can account for the correlations between these three properties. Neurons subject to strong inhibition tend to oscillate strongly in response to both optimal and suboptimal stimuli and synchronise promiscuously with other neurons, even if they have different orientation preferences. Moreover, these strongly inhibited neurons can exhibit sharp orientation selectivity provided that the inhibition they receive is broadly tuned relative to their excitatory inputs. These results predict that the strength and orientation tuning of synaptic inhibition are heterogeneous across area 17 neurons, which could have important implications for these neurons' sensory processing capabilities. Furthermore, although our experimental recordings were conducted in the visual cortex, our model and simulation results can apply more generally to any brain region with analogous neuron types in which heterogeneity in the strength of rhythmic inhibition can arise during gamma oscillations.

Rats that had been prenatally exposed to cocaine were tested later in life for their sensitivity to cocaine-kindled seizures and acute cocaine-induced seizures. When treated daily with cocaine, beginning at one month of age, males prenatally exposed to 40 mg/kg cocaine developed seizures in a fewer number of days than those prenatally exposed to saline. Prenatally cocaine-treated females did not seize more rapidly than controls in the cocaine kindling paradigm; however, they were more susceptible to seizures in response to an acute high dose of cocaine. These results suggest that rats prenatally cocaine-treated are more sensitive to the seizure-producing effects of cocaine later in life, and this enhanced sensitivity is differentially expressed in males and females.

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.

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.

To assess the importance of brain cytochrome P450 (P450) activity in 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. © 2010 Nature America, Inc. All rights reserved.

Nigrostriatal dopamine (DA) projections terminate in distinct patches during the late prenatal and early postnatal period in the rat. During the first postnatal week, patches of DA fibers overlap with clusters of striatal neurons that share several identified characteristics. The early segregation of striatal cell types into either these patches or the surrounding matrix becomes a permanent organizational feature of the striatum. In order to determine whether the heterogeneous distribution of DA influences the formation of cellular patches, the developmental organization of chemically identifiable cell types was examined in normal rats and in rats DA depleted as infants (0 or 3 d) or in utero (embryonic days 17–18). During the first postnatal week, corresponding patches of DA afferents and substance P (SP)- immunoreactive neurons existed in the striatum of normal animals, and AChE-positive zones overlapped these patches in the lateral striatum. Injection of 6-hydroxydopamine into the lateral ventricles of fetal or infant rats produced a dramatic loss of striatal DA terminals. Neither the patchy distribution of SP-immunoreactive neurons nor the distinctive pattern of AChE staining present during the first 2 postnatal weeks was disrupted. During the third postnatal week, cells immunoreactive for leu-enkephalin or calbindin-D28k were confined to the matrix compartment, and this compartmentalization was also not noticeably changed by pre- or postnatal DA depletion. In adult animals, overlapping patches of leu-enkephalin- and SP-immunoreactive fibers were observed, regardless of whether any DA terminals remained. Thus, the basic organization of the striatal patch and matrix compartments develops normally in the absence of DA innervation through much of the formative period. Although these observations do not completely dismiss the possibility that the first DA afferents to appear in the striatal primordia influence contracted striatal cells to develop the patch phenotype, they suggest that the patchy distribution of DA afferents may be secondary to the early clustering of striatal neurons forming the patch compartment.

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.

Infant rats given bilateral dopamine-depleting brain lesions and unilateral transplants of embryonic nigral tissue develop turning in response to both amphetamine and stress. However, stress-induced turning did not develop unless animals were previously exposed to amphetamine, and was greatest in animals exposed early to the drug. These findings suggest that amphetamine alters certain properties of the transplanted cells so as to enhance their functional capacity.

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.

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.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.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.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.

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.

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.

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. Immunocyto-chemical 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.

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. [16] 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.

We previously reported that prenatal cocaine exposure (40 mg/kg s.c., E10-E20) increased susceptibility to convulsant-induced seizures later in life, with female rats becoming more sensitive to seizures induced by cocaine and pentylenetetrazol (PTZ), and males more sensitive to PTZ-induced seizures (Snyder-Keller and Keller, 1995, 2000). In order to determine the locus of enhanced seizure susceptibility in the brains of prenatally cocaine-treated rats, we examined the distribution and density of Fos-immunoreactive cells after cocaine- and PTZ-induced seizures in mature rats. Subconvulsive cocaine doses induced c-fos in cortical areas as well as densely dopamine-innervated regions such as striatum and nucleus accumbens. Following cocaine-induced seizures, intense c-fos induction was observed in piriform cortex, amygdala, and hippocampus. Quantification of the number of Fos-immunoreactive cells in the brains of prenatally cocaine-treated versus prenatally saline-treated rats revealed differences in piriform cortex and amygdala that were indicative of a lower threshold in prenatally cocaine-treated female rats. Following PTZ-induced seizures, the same pattern of limbic structures were recruited with increasing seizure severity. Only females exhibited changes in the number of Fos-immunoreactive cells as a result of prenatal cocaine treatment. Pretreatment with the noncompetitive NMDA antagonist MK-801 blocked both cocaine- and PTZ-induced seizures, and Fos expression in limbic areas was also blocked. The dopamine D1 antagonist SCH 23390 blocked cocaine-induced seizures and associated c-fos induction, but not PTZ-induced seizures or Fos. Examination of the pattern of Fos expression at 15-20 min postseizure revealed that the initial site of c-fos induction associated with PTZ-induced seizures appeared to be the piriform cortex, whereas cocaine-induced seizures induced early expression in both piriform cortex and lateral amygdala. These findings suggest that neural alterations residing in the piriform cortex and amygdala are likely to account for the increased seizure susceptibility of prenatally cocaine-treated rats.

We previously reported that prenatal cocaine exposure increased susceptibility to cocaine-induced seizures later in life. Here we examine whether this enhanced susceptibility to seizures generalizes to other chemoconvulsants, and whether postnatal cocaine treatment similarly increases susceptibility. Following prenatal cocaine treatment (40 mg/kg; E10-20), both male and female rats were more likely to seize to a dose of 30 mg/kg pentylenetetrazol (PTZ) at 2 months of age, although the severity of the seizures observed was increased only in females. Daily cocaine injections (10-20 mg/kg SC) during the first 10 days after birth also produced effects that were dependent on the sex of the animal. Postnatally cocaine-treated female rats showed no greater incidence of seizures in response to an acute high dose of cocaine, but did exhibit an increased susceptibility to cocaine-kindled seizures. Male, but not female, postnatally cocaine-treated rats were more susceptible to PTZ-induced seizures. The increased susceptibility to seizures induced by two different chemoconvulsants after prenatal cocaine treatment suggests that developmental cocaine exposure, particularly during the second trimester equivalent, alters the balance between excitation and inhibition in the brain.

Induction of the immediate-early gene c-fos by the stimulants cocaine and amphetamine (AMPH) was analyzed by Fos immunocytochemistry at different ages in the brains of prenatally cocaine-treated and control rats. Cocaine and AMPH induced c-fos in patches of striatal neurons during the first postnatal week, and thereafter produced a progressively more homogeneous pattern that was more dense medially. Quantification of Fos-immunoreactive cells in older rats revealed differences related to sex and prenatal cocaine treatment. Both cocaine and AMPH produced dose-dependent increases in the number of Fos-immunoreactive cells in striatum. Prenatal cocaine exposure resulted in increased Fos in males in response to AMPH (2 mg/kg) at P18 and cocaine (10 mg/kg) at 1-2 months. In females, prenatal cocaine treatment resulted in a reduced response to cocaine at 1-2 months. Increased c-fos induction was observed in control females compared to control males in response to low doses of stimulants; no such sex difference was observed in prenatally cocaine-treated rats. The dopamine D1 antagonist SCH23390 blocked cocaine-mediated c-fos induction in all groups. The NMDA antagonist MK-801 blocked cocaine-mediated c-fos induction in the medial striatum. In females only, MK-801 pretreatment resulted in a dramatic increase in the number of Fos-immunoreactive cells in lateral striatum. These findings indicate differences in the neural basis of c-fos induction in males and females, and changes in stimulant-mediated c-fos induction resulting from prenatal cocaine exposure.

Methods to increase the development and sustained function of embryonic mesencephalic dopamine cells after transplantation into dopamine (DA)-depleted striatum are currently under investigation. Elements that are crucial for the maturation and connectivity of neurons during normal development of the brain may also play a role in the development and integration of grafted embryonic tissue. Based on in vitro and in vivo observations of the enhancing effects of striatal tissue on nigral dopaminergic cell development and survival, we demonstrate that inclusion of embryonic striatal cells, specifically from the lateral ganglionic eminence (LGE), produces dopaminergic transplants with augmented functional effects. Rats neonatally DA-depleted and co-transplanted with embryonic nigral and LGE cells developed improved functional outcome when compared with animals receiving only nigral cells, and they required the transplantation of fewer nigral cells to produce a strong behavioral effect. Anatomically, the inclusion of LGE cells produced increased DA cell survival, a higher density of reinnervation into the DA-depleted host striatum, and patches of DA fibers within the co-transplants. There were also an increased number of host striatal cells which induced the immediate-early gene c-fos in co-transplanted animals compared to animals receiving nigral cells alone, indicating a higher degree of host-cell activation. The ability to enhance function, cell survival, reinnervation, and host activation with nigral-striatal co-transplants in the presence of fewer nigral cells supports the hypothesis of a trophic influence of striatal cells on nigral DA cells.

Microdialysis studies were conducted on prenatally saline-treated and prenatally cocaine-treated rats, either as pups (10-30 days old) or young adults (40-190 days old), to study the effects of prenatal cocaine exposure on the mesolimbic dopamine (DA) system. In the n. accumbens of saline-treated rats, basal dialysate concentrations of DA were similar in pups and adults; however, the levels of DA metabolites, DOPAC, HVA, and the serotonin metabolite, 5-HIAA, were markedly lower in pups. In pups, prenatal cocaine exposure led to basal dialysate levels of DA in the n. accumbens that were twice control levels; however, there was no difference in response to a period of intermittent tail pinch or an acute injection of cocaine (20 mg/kg). In the adult, basal levels of DA, DOPAC, HVA and 5-HIAA in n. accumbens did not differ across prenatal treatments. However, in prenatally cocaine-treated adults a cocaine injection led to an enhanced rise in extracellular DA compared to controls. In frontal cortex of adult rats, basal levels of DA, DOPAC and HVA did not differ across prenatal treatments; however, basal levels of 5-HIAA in this region were significantly elevated in prenatal-cocaine rats. No group differences were observed in the frontal cortex in response to either tail pinch or cocaine. Thus prenatal cocaine exposure produces an increase in basal extracellular DA in the n. accumbens of pups which returns to normal with aging. While this initial difference normalizes, prenatal cocaine exposure induces other persistent changes in adulthood.

The antimitotic drug methylazoxymethanol was used to destroy striatal patch neurons during their three-day-period of neurogenesis in the rat. Single or multiple injections of methylazoxymethanol were given during embryonic days 13-15, the period when patch neurons are known to undergo their final cell division. Methylazoxymethanol treatments produced a dramatic reduction in striatal volume. Immunocytochemical analysis revealed the continued presence of patches of neurons that were substance P-immunoreactive and devoid of calbindin and enkephalin immunoreactivity. Both the number of patches and relative volume occupied by patches was reduced in methylazoxymethanol-treated striata. Patch neurons could also be labelled by an intrastriatal injection of FluoroGold during the first postnatal week. The early ingrowth of nigrostriatal dopamine afferents was less noticeably patchy in the methylazoxymethanol-treated animals, in part owing to an overall increase in density. Large reductions in the number of neurons immunoreactive for choline acetyltransferase were observed, whereas NADPH diaphorase-stained neurons were not reduced unless methylazoxymethanol was given on embryonic day 15. Injections of bromo-deoxy-uridine, either during or after the 24 h that each methylazoxymethanol injection was considered to be effective, revealed that (i) some patch neurons continued to be generated in the 24-h period following methylazoxymethanol administration, and (ii) many patch neurons were generated after the effects of methylazoxymethanol had worn off. These findings demonstrate that it was impossible to completely eliminate the patches using methylazoxymethanol injections during the period of patch neurogenesis. However, methylazoxymethanol treatment during this time did produce a dramatic loss of cells and a relatively greater reduction in patch volume. Despite this disruption, the appropriate compartmentalization of neuroactive substances appeared to be maintained.

When embryonic ventral mesencephalic tissue containing nigral dopamine (DA) neurons is transplanted into adult DA-depleted striatum, synaptic connections form and behavioral effects are observed. This study investigated the cotransplantation of embryonic striatal tissue as a means of enhancing the innervation, survival, and functional effects of nigral transplants. Rats neonatally DA-depleted, via bilateral intraventricular injections of 6-hydroxydopamine, developed turning in response to amphetamine and stress following unilateral transplantation of either nigral or combination nigral-striatal cell suspensions. Animals with cotransplants developed higher levels of turning to both stimuli and maintained these responses for a longer period of time post-transplantation, when compared with animals receiving transplants of nigral cells alone. In addition, these combination transplants required fewer dopaminergic cells to produce a strong behavioral effect on the host. Dense patches of tyrosine hydroxylase (TH)-immunoreactive fibers were observed within the cotransplants, yet no greater outgrowth of DA fibers into host striatum was detected. Amphetamine produced widespread induction of the immediate-early gene c-fos in cells of host striatum that extended beyond the transplant-derived DA innervation. After both amphetamine and stress, Fos protein was found within both types of transplants, but these F os-immunoreactive cells did not colocalize with TH-immunoreactive cells nor dense TH-immunoreactive patches within the grafts. Thus, cotransplanted embryonic striatal tissue augments the effects of ventral mesencephalic transplants, possibly by providing a trophic influence that enhances the function of the DA cells without increasing cell survival.

Topographic patterns of pure-tone responses in inferior colliculus (IC) of Wistar rats were mapped using immunohistochemical staining for the nuclear protein Fos, the translation product of the c-fos proto-oncogene. Patterns were compared in ICs of immature and mature rats and in mature rats which experienced auditory deprivation beginning on day 14, an age near the developmental onset of hearing. Neonatal hearing losses, caused here by exposure to potentially deafening noise, are known to result in audiogenic seizure susceptibility in neonatal rats. These seizures can be triggered only by high-frequency stimuli and are believed to be initiated in IC. Thus, it seemed possible that susceptibility might depend on derangements of topographic frequency representation due to neonatal auditory deprivation. The band-like frequency-response domains, characteristic of adult IC, were found to be poorly differentiated in ICs of immature rats. On day 12, only lower-frequency stimuli induced discrete bands of Fos immunoreactivity while responses to higher frequencies remained exceptionally diffuse within ventral portions of IC. Only after day 24 did responses to the highest frequencies also appear mature. Furthermore, most significantly, adult rats which were transiently deafened on day 14, retained the more voluminous response patterns which were characteristic of immature IC. Because frequency selectivity in cochlea also develops by a low-to-high frequency sequence, results are consistent with a hypothesis that topographic organization arises in IC by an activity-dependent process. Whereas neonatal noise exposure also conferred audiogenic seizure susceptibility, it appears the arrest of tonotopic organization of IC is the probable basis of this reflex epilepsy.

The effect of prenatal cocaine on the anatomical development of the striatum was examined. The distribution and density of dopaminergic innervation of the striatum of animals exposed to cocaine during the second and third week of gestation was not noticeably different from prenatally saline-injected or untreated controls at any age. The patch/matrix organization of the striatum also appeared unaltered: neurons exhibiting dense substance P staining were localized to patches that overlapped dopamine terminal patches early in development, and enkephalin- and calbindin-immunoreactive neurons were found segregated to the matrix. Histochemical staining for acetylcholinesterase and NADPH diaphorase also revealed no differences between prenatally cocaine-treated and control brains. Whereas prenatal cocaine treatment failed to modify the basic compartmental organization of the striatum, it did lead to a hyperinnervation of serotonin-immunoreactive fibers which developed slowly after birth. Thus prenatal exposure to cocaine is capable of altering the ingrowth of serotonergic projections to the striatum while producing no change in the organization of neurons intrinsic to the striatum.

In rats made susceptible to audiogenic seizures by exposing them to an intense noise at a critical time during development, subsequent noise exposure elicited seizures and induced the proto-oncogene c-fos in auditory regions of the brain. Cells showing Fos-like immunoreactivity were especially dense in dorsal and external cortices of the inferior colliculus, and were nearly absent after pretreatment with the N- methyl- d -aspartate (NMDA) antagonist MK-801. Noise exposure alone (i.e. no seizure) produced a localized zone of c-fos induction within the inferior colliculus, but only when presented during the time period when susceptibility to audiogenic seizures can be most effectively induced.

Induction of the proto-oncogene c-fos occurred in cells of the striatum in response to stimuli that are known to release dopamine (DA). As revealed by fos immunocytochemistry, amphetamine (AMPH) produced c-fos induction in many cells of the medial two-thirds of the striatum of normal rats, with patchy labeling in the lateral third. The lateral patches were found to be coincident with patches of striatal neurons lacking calbindin immunoreactivity. In animals DA-depleted at birth, few fos-immunoreactive neurons were present in response to AMPH. In those with unilateral transplants of DA-rich mesencephalic tissue, c-fos induction was greater on the transplanted side. The DA D1 antagonist SCH 23390 completely blocked c-fos induction in all animals. The N-methyl-D-aspartate antagonist MK-801 also blocked c-fos induction by AMPH within the medial striatum, but intensified c-fos induction laterally in those animals with DA innervation. A second set of experiments examined the functional importance of c-fos induction in the AMPH sensitization of turning behavior that occurs in these animals. Both AMPH and stress produced turning, but only AMPH produced widespread c-fos induction, and stress-induced turning only occurred after exposure to AMPH. Treatment with MK-801 prior to AMPH administration blocked the subsequent development of stress-induced turning. Whereas a high dose of MK-801 (1.0 mg/kg) completely blocked c-fos induction, a lower dose (0.1 mg/kg) blocked c-fos induction in controls, but left patches of fos-immunoreactive neurons in lesioned animals given transplants. Thus the sensitization of transplant-related behaviors is NMDA dependent and associated with c-fos induction in host striatal neurons.

Cell suspensions derived from the ventral mesencephalon of CD-1 mice were unilaterally transplanted into the striatum of neonatal Sprague-Dawley rats that had been bilaterally dopamine depleted. Thirty-eight percent of the grafts survived. Tyrosine-hydroxylase-immunoreactive neurons within the transplant innervated the host striatum with a dense fiber plexus. The grafts appeared to exert some degree of functional control over motor behavior in that these animals made contralateral rotations in response to amphetamine and tail pinch. In order to provide additional evidence that the motor behavior is associated with the transplant itself, the graft was removed. This was achieved by using a mouse skin graft to provoke an immunological response against the transplanted neural tissue. The immunological response resulted in the specific loss of the transplant with little or no damage to the surrounding neural tissue. The amount of rotation observed after tail pinch and amphetamine injection was severely affected by neural graft rejection. The loss of turning was associated most directly with the loss of tyrosine hydroxylase immunoreactivity within the transplant rather than with the massive reduction of tyrosine-hydroxylase-positive fibers in the ipsilateral host striatum. These data suggest that dopamine cells in mouse nigral grafts play an essential role in eliciting rotational behavior in neonatally dopamine depleted rats. They also show the value of skin grafting as a technique for specifically removing neural xenografts.

Patches of densely-labelled striatal neurons were observed following intrastriatal injection of the fluorescent dye Fluoro-Gold into rats younger than 10 days of age. These cell clusters showed precise overlap with islands of dense dopamine innervation seen only during the first week of life. The patches of labelled cells were still visible in adulthood, and were not abolished by 6-hydroxydopamine treatment before or after the Fluoro-Gold injection. Neonatal Fluoro-Gold injection may be a unique way of marking the patch compartment of the striatum that remains visible in adulthood.

Neonatally dopamine-depleted rats fail to show the deficits seen in similarly lesioned adults, and no longer appear to require dopamine. Nevertheless, unilateral nigral transplants provided extensive reinnervation and were capable of modifying motor patterns. Both amphetamine and external stressors elicited contraversive turning. This raises the question of whether extrinsic dopamine innervation supplements or overrides alternative compensatory mechanisms in the neonate.