Light and electron microscopic examination of endothelial cells from bovine retinal vessels in long-term cultures

The aim of the present work was to examine and compare the ultrastructure of bovine retinal endothelial cells (BRECs) in vitro during several passages in a medium selective for endothelial cells. The identity of the endothelial cells was confirmed immunohistochemically, up to the tenth passage. Changes in their ultrastructure in comparison to endothelial cells in vivo occurred at the onset of culturing and not progressively with repeated passages. The cultured BRECs show high metabolic activity in all passages. While retaining their identity as endothelial cells, they modify their lipid metabolism, so that lipids are stored. This change in lipid metabolism was induced by the medium. Received: 19 May 1999 / Accepted: 4 August 1999     

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated,several publications have addressed this important issue to improve cultureconditions for cattle reproductive biotechnologies, with the ultimate goal ofproducing in vitro embryos similar in quality to those developingin vivo. Here, we review general aspects of bovine embryometabolism in vitro and in vivo, and discuss theuse of metabolic analysis of embryos produced in vitro to assessviability and predict a viable pregnancy after transference to the female tract.     

Expression of PTHrP and PTHR (PTH/PTHrP-r) mRNAs and polypeptides in bovine ovary and stimulation of bovine blastocyst development in vitro following PTHrP treatment during oocyte maturation

Parathyroid hormone related protein (PTHrP) and its receptor have well-established roles in the development and regulation of many tissues, including bone and mammary gland. The objectives of this study were: (1) to characterize the distribution of mRNAs encoding parathyroid hormone (PTH)-related protein (PTHrP) and receptor (PTHR) in bovine ovary; (2) to characterize the distribution of PTHrP and PTHR polypeptides in bovine ovary; (3) to examine the influences of PTHrP (1–141) treatment during bovine oocyte maturation in vitro on blastocyst development. mRNAs encoding PTHrP and PTHR were detected by in situ hybridization methods in oocytes, and granulosa cells in all follicles from primordial to large antral. PTHrP and PTHR polypeptides displayed distinct distribution patterns with PTHrP polypeptides primarily confined to oocytes from primordial to large antral follicles. PTHrP polypeptides were detectable but at a reduced level in ovarian stroma and in granulosa and thecal layers. PTHR polypeptides were detected in oocytes of all follicular stages but were predominantly found in ovarian stroma, granulosa and theca follicular layers. Supplementation of serum-free cSOFMaa oocyte maturation medium with PTHrP (1–141) resulted in a concentration-dependent increase in development to the blastocyst stage in vitro. The results suggest that granulosa cells may be a primary site of PTHrP production and release. Oocytes from all follicular stages stained strongly for PTHrP polypeptides and PTHrP enhanced development to the blastocyst stage in vitro. Accepted: 12 October 2000     

Fine structure of the choroidal coat of the avian eye

 To clarify further the functional anatomy of the avian choroid, including its innervation, 12 adult White-Leghorn chickens were studied by standard electron microscopy and immunoelectron microscopy with somatostatin antibody. The endothelial cells of the blood vessels in the choriocapillaris have fenestrations only facing the retina, while the nuclei are situated toward the sclera. In addition to tight junctions and zonulae adherentes, adjoining endothelial cells form gap junctions and dense plaques with attached filaments resembling those of smooth muscle cells. The fine structure of arteries and veins is similar to that of the vasculature described in other organs. The supporting tissue is organized in trabeculae, i.e., bridges of cellular and fibrous elements that surround and sustain blood and lymphatic vessels. This tissue consists primarily of a system of fusiform or star-shaped smooth muscle cells, connected to each other and to those in the vessels’ walls through macular junctions of the adherent type, less prominent than desmosomes, and perhaps also punctiform gap junctions. Occasionally, trabecular smooth muscle cells approach the lymphatic vessels, which lack a muscular tunica, and abut their endothelium with spinous appendages. This stromal muscle tissue may act as a pump for moving the lymph. The suprachoroidea consists of large lymphatic lacunae and the multilayered membrana fusca. The elongated fuscal cells form adherent junctions, tight junctions, and perhaps also gap junctions, suggesting that the membrana fusca exerts complex functions. Nerves containing myelinated axons reach the choroid and divide into smaller branches, a few of which innervate the membrana fusca. Numerous, thin nerve branches reach both the walls of arteries and veins and the trabeculae, and synaptic terminals abut the outer muscular layers of the vessel’s wall and the smooth muscle cells of the supporting tissue. Immunocytochemistry reveals the presence of numerous somatostatin-positive and somatostatin-negative axons and synaptic terminals within both trabeculae and vascular tunica media. The somatostatin-positive axons are presumed to be cholinergic axons of the choroid neurons residing in the ciliary ganglion. Taken together, these observations indicate that the avian choroid is a highly vascularized muscular sheath that may be endowed with degrees of motility and elasticity higher than those of the mammalian choroid and may therefore play an important role in compensation for experimental defocus. Accepted: 21 January 1997     

Fine structure of the semicompact formation of the nucleus ambiguus of the rat

 We investigated the fine structure of the semicompact formation of the nucleus ambiguus (AmS), which was identified by retrogradely labeled pharyngeal (PH) motoneurons. When cholera toxin subunit B-conjugated horseradish peroxidase was injected into the lower pharyngeal muscle, many retrogradely labeled PH neurons were found throughout the AmS. Besides the PH neurons, two types of neurons were recognized in the AmS: unlabeled medium-sized neurons and unlabeled small neurons. The PH neuron was large (27.6 × 44.1 μm) and polygonal, and contained many Nissl bodies and well-developed cell organelles with a prominent spherical nucleus. The medium-sized neuron was dark and oval (19.3 × 33.2 μm), and contained many free ribosomes and much swollen rough endoplasmic reticulum with a distorted oval nucleus. The small neuron was spindle-shaped (12.3 × 20.2 μm), and had poorly developed cell organelles with an irregularly shaped nucleus. The average number of axosomatic terminals in a sectional plane was largest in the PH neurons (32.8), smaller in the medium-sized neurons (23.1), and smallest in the small neurons (6.3). The number of axo-somatic terminals containing round vesicles (Gray’s type I) was almost equal to that of terminals containing pleomorphic vesicles (Gray’s type II) in the PH neuron, and slightly smaller in the small and the medium-sized neurons. About 60% of the axodendritic terminals were Gray’s type I, and 40% were type II. These results indicate that there are two different types of interneurons besides the PH motoneurons in the AmS. Accepted: 24 June 1997     

Epidermal growth factor, vascular endothelial growth factor and progesterone promote placental development in rat whole-embryo culture

 The development and survival of rat embryos in whole-embryo culture is limited by the lack of any maternal blood circulation in a purely fetal placenta. If the resulting placental insufficiency could be overcome for some time by an increase of the placental exchange area, a prolonged culture period would result and facilitate the development of embryos. In the present study, several attempts to stimulate proliferation and growth of the fetal placenta were made by the addition of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and progesterone to the culture medium. Rat embryos were routinely explanted with their embryonic membranes at 10.5 days of gestation. Decidua, parietal yolk sac, Reichert’s membrane and the layer of superficial trophoblastic giant cells were removed. The explants were cultured and gassed continously for 24 h in rotating plastic tubes containing rat serum, diluted to 50% with modified COON’s F12 medium. Either of the two growth factors or progesterone were added to each culture tube and a control group was cultured without any factor. After the addition of each of these factors the stimulatory effect on placental growth was assessed by morphometric evaluation of several placental parameters from semithin cross-sections: On adding each of the factors the whole cross-sectional area of the placenta significantly increased, as did the area of the fetal placental mesenchyme. VEGF also increased the area of the trophoblast, and the area of the blood vessels enclosed within the trophoblast, by an average of 9.4% and 23.6%, respectively. Thus, VEGF treatment resulted in a measurable extension of the exchange area of the fetal placenta. Accepted: 4 February 1998     

Growth hormone (GH) action in early embryogenesis: expression of a GH-response gene in sites of GH production and action

Growth hormone (GH) may act as a local growth factor in early embryonic development, since GH- and GH-receptor (GHR) immunoreactivity is present in all tissues and most cells of embryonic chicks during organogenesis. However, as GHR-immunoreactivity could, alternatively, reflect the presence of GH-binding proteins (GHBPs) rather than authentic receptors linked to signal transduction mechanisms, GHR immunoreactivity may not be indicative of GH target sites. The possibility that GH may act as an autocrine or paracrine factor during embryogenesis was therefore assessed in the present study by determining the presence and cellular localization of mRNA for a GH-responsive gene. The mechanism of GH action involves the induction of a number of specific GH-response genes. In chickens a novel GH-responsive gene (GHRG-1) has been identified as a marker of GH action. In situ hybridization, using a 860 bp probe for GHRG-1 mRNA, demonstrated widespread expression of the GHRG-1 gene in embryonic tissues known to contain GH- and GHR-immunoreactivity (e.g. in the spinal cord, skin, heart, liver, muscle, bone and lung). GHRG-1 mRNA was not, however, present in all cells of each tissue. It was, furthermore, not present in subepithelial cells of the esophagus and bronchus and was lacking in many spinal cord ependyma, which are also known to lack GH immunoreactivity. These results therefore support the possibility that GH acts as an autocrine/paracrine factor during early chick embryogenesis, which was hitherto thought to be a ”growth-without-GH” syndrome. Accepted: 26 September 2001     

In vitro and in vivo approaches to study angiogenesis in the pathophysiology and therapy of endometriosis

Endometriosis represents one of the most common gynaecological disorders. According to the implantation theory, angiogenesis is a major prerequisite for the initiation and progression of the disease. Thus, during the last decade, many studies have focused on the mechanisms regulating angiogenesis in endometriotic lesions. For this purpose, sophisticated in vitro and in vivo approaches have been established, which are highlighted in this review. Enzyme-linked immunosorbent assays demonstrate the imbalance of pro- and anti-angiogenic growth factors in isolated peritoneal fluid from endometriosis patients. Histological, immunohistochemical and gene expression analyses of endometriotic tissue provide detailed information on the angio-architecture of endometriotic lesions and the different growth factor expression by various cell populations. Moreover, cell culture systems are useful tools for the identification of hormonal and immunological factors involved in the angiogenic process. Finally, sophisticated in vivo models, such as rodent models of peritoneal endometriosis as well as the chorioallantoic membrane assay and the dorsal skinfold chamber, allow for the detailed analysis of blood vessel development in ectopic endometrium and the efficacy of angiogenesis inhibitors. The findings resulting from all these approaches will help to provide better insights into the pathophysiology of endometriosis and to establish new anti-angiogenic treatment strategies for the future.     

Ablation of axial structures activates apoptotic pathways in somite cells of the chick embryo

It has been known for some time that ablation of the neural tube and/or the notochord in the chick embryo leads to a massive wave of cell death in the adjacent somites. It is postulated that in the normal embryo, survival signals emanate from the neural tube and/or notochord that suppress apoptosis in the cells of the somites, except for a small population of sclerotome cells that are programmed to die naturally. In this study we show that axial ablation results in the death of sclerotome and not somitic neural crest cells, and we have examined the apoptotic response of these cells to the ablation. We show that several elements of the apoptotic cascade become detectable in somite cells in response to the withdrawal of survival signals. We demonstrate the down-regulation of bcl-2 protein in the somites adjacent to, and caudal to, the site of ablation, corresponding to the region that displays an elevated level of cell death. Although caspase-9 appeared to be activated in somites at all levels of the trunk, caspase-2 showed a clear response to the ablation of the axial structures. Removal of the neural tube and notochord produced an up-regulation of caspase-2 activity in somites in the region of the operation. Cleavage of two down-stream substrates of these caspases was examined. The cleavage of poly (ADP-ribose) polymerase (PARP) was apparent in somites at all levels of the trunk, and showed only a modest up-regulation after ablation. By contrast, the cleavage of DNA fragmentation factor (DFF45) showed a marked up-regulation in response to ablation, suggesting that this is a primary substrate for a caspase-dependent apoptotic mechanism. Evidence was also found for a caspase-independent mechanism, since the expression of apoptosis-inducing factor (AIF) was found to be very sensitive to, and up-regulated in somites by, axial ablation. Because the wave of apoptosis that is precipitated in somites by removal of the axial structures may be mediated by BMP-4, we examined the levels of BMP-4 in somites in response to axial ablation. BMP-4 expression was clearly up-regulated in somites adjacent to, or close to, the site of operation. Accepted: 9 July 2001     

Cocaine administration in pregnant rabbits alters cortical structure and function in their progeny in the absence of maternal seizures

 Previous studies have reported that cocaine exposure in utero results in structural and functional alterations in the development of the anterior cingulate cortex (ACC). In the present study, the effects of maternal cocaine dosage and of cocaine-elicited maternal seizures on the progeny were studied. The incidence of maternal generalized tonic clonic seizures (GTCSs) elicited by cocaine was recorded. No GTCSs were elicited in pregnant rabbits by doses of 2 or 3 mg/kg of cocaine, but GTCSs were sometimes elicited by the highest dose (4 mg/kg per injection). We analyzed the offspring of cocaine-exposed and control animals using three assays of ACC development: (i) the structure of apical dendrites of pyramidal neurons, (ii) the distribution of a calcium binding protein (parvalbumin) in the dendrites of GABAergic neurons, and (iii) coupling of D₁-like receptors and their G proteins. In all progeny of rabbits exposed to 3 or 4 mg/kg of cocaine during pregnancy, there was a significant change in the structure of apical dendrites, a significant increase in the number of dendrites of GABAergic neurons which were parvalbumin immunoreactive, and a significant reduction in D₁/G protein coupling. In assays of apical dendrites, the effects on offspring of rabbits given 2 mg/kg cocaine were as pronounced as in offspring of rabbits given 3 or 4 mg/kg, but the effects on parvalbumin immunoreactivity and D₁/G protein coupling were reduced at this low dose. Thus, previous findings of ACC developmental abnormalities in offspring of rabbits given a dose of 4 mg/kg were replicated, the effects were shown to be dose-related and to be independent of maternal seizures. A mechanism by which dysfunction of the D₁receptor system could mediate cocaine-associated changes in all three parameters of ACC structure and function is discussed. Received: 16 August 1996 / Accepted: 8 November 1996     

Patterns of cell death during gastrulation in chick and mouse embryos

 We have examined the distribution of cells at an early stage of the cell death process in gastrulating chick and mouse embryos, using a DNA nick end-labelling technique to label nuclei that are undergoing DNA fragmentation in situ. In the chick embryo, the incidence of nuclei showing DNA fragmentation was mapped by digitizing the occurrence of these nuclei from sections, and reconstructing the three separate layers of the entire embryo at several stages of gastrulation. In the chick, DNA fragmentation was found in nuclei throughout the embryo, in cells of all three germ layers, but most especially in the epiblast in the rostral germinal crescent and in the lateral marginal zones. This region of greatest cell death formed an arc rostrally and laterally in the epiblast, and was consistent through gastrulation and into the early neurulation stage. While the extensive cell death in the chick embryo may be due to cell redundancy, it is also possible that the pattern of death observed could be related to the compression of the embryo against the barrier of yolk at the periphery of the area pellucida during expansion. In a number of cases in the chick, local regions of elevated cell death were also observed in the primitive streak. This may be associated with the changing cell-cell and cell-matrix interactions experienced by cells traversing the primitive streak. In the gastrulating mouse embryo, by contrast, nuclei undergoing DNA fragmentation showed no consistent regions of elevated incidence, in any of the embryonic layers. DNA fragmentation in these embryos was, however, observed in nuclei of cells in the visceral endoderm and in the epiblast. The lack of any clear pattern of DNA fragmentation in the mouse embryo at this stage of development leaves the roles of the dying cells enigmatic. The death may, however, be lineage-related or be a reflection of a cellular redundancy necessary in a developing system that is undergoing extensive cell rearrangement and cellular adhesive change. Accepted: 29 July 1996     

Developmental change in the voltage-dependence of the pacemaker current, if, in rat ventricle cells

 Myocytes were isolated from newborn and adult rat ventricle. Using the whole-cell patch clamp, the two cell populations were compared for the presence of the hyperpolarization-activated pacemaker current i_f. As in other mammalian species, the threshold voltage in acutely dissociated adult rat myocytes was extremely negative (–113 ± 5 mV; n=12). In contrast, threshold in newborn cells was relatively positive, regardless of whether measured in acutely dissociated (–72 ± 2 mV; n=6) or cultured cells (–70 ± 2 mV; n=9). Current density was not reduced in the adult. These results suggest that with development the ventricle assumes its non-pacemaker function, at least in part, by a shift of the voltage dependence of i_f outside the physiological range. Received: 8 August 1996 / Received after revision and accepted: 29 October 1996     

Oocyte ultrastructure in bovine primordial to early tertiary follicles

 The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 μm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 μm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules. Accepted: 8 October 1996     

Expression of a 45K subunit of platelet-activating factor acetylhydrolase in the developing mouse cerebellum

 The 45K subunit of platelet-activating factor acetylhydrolase (PAFAH-45K) is the product of a candidate gene for Miller-Dieker lissencephaly. We studied the expression of this protein in the developing mouse cerebellar cortex by immunochemical and immunohistochemical methods. Western blotting studies indicated that PAFAH-45K is more abundant in the fetal than the postnatal period. Immunohistochemical studies revealed developmental changes in the localization of PAFAH-45K-immunoreactivity, which shifted from the somata of Purkinje cells to the neuropil of the molecular layer. Our findings indicate that PAFAH expression is developmentally regulated and suggest its role in histogenetic processes in the cerebellar cortex other than neuronal migration. Accepted: 25 November 1997     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Reversal of the relative expression of cardiac and skeletal alpha1 subunit isoforms of L-type calcium channel during in vitro myogenesis

 Cardiac and skeletal type of excitation-contraction coupling (ECC) are quite different. Those differences could be explained by structural ones in the molecular entities involved in ECC, ie dihydropyridines (DHP) receptors (α1 subunit of L-type calcium channels) of the sarcolemma or ryanodine receptors of the sarcoplasmic reticulum membrane. As previously demonstrated by means of electrophysiology, the two types of ECC coexist during the first stages of in vitro development of skeletal muscle, whereas the skeletal type predominates at the later ones. In order to see whether evolution of ECC could be correlated with the one of α1 subunit expression, we determined by Northern Blotting which isoforms of α1 subunit are expressed during the in vitro myogenesis. mRNA corresponding to the cardiac isoform are present in myoblasts (before fusion), but patch-clamp experiments showed that they are not functional. After fusion, skeletal and cardiac mRNA are coexpressed in myotubes, with different intensities: whereas expression of skeletal mRNA (which are the more intensive) stabilized at the later stages tested, cardiac mRNA decreased. We conclude that evolution in mRNA α1 subunit isoforms expression could partly explained evolution of ECC features during in vitro myogenesis. Received: 24 July 1996 / Received after revision and accepted: 30 September 1996     

Ontogeny of NADPH-diaphorase in rat forebrain and midbrain

 This study characterizes the developmental expression of NADPH-diaphorase from embryo to adulthood in the forebrain, midbrain and cerebellum of rat brain via histochemical staining. On embryonic day 12 no neurons stained. Labeling was observed in certain nuclei from E15 through the postnatal period to adulthood. Labeling in neurons increased or maintained a constant level with increased age. The embryo demonstrated substantial labeling in neurons of the caudate putamen, bed nucleus of the stria terminalis, preoptic area, lateral hypothalamic area, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, magnocellular nucleus posterior commissure, and periaqueductal central gray. Additional neuronal labeling was observed postnatally in the olfactory bulb, cerebral cortex, amygdala, various nuclei of the thalamus, interpeduncular nucleus, linear nucleus of the raphe, pretectal area and superior colliculus. In the cerebellum, labeling appeared only after P14 in cells of the molecular cell layer and granular cell layer. The sizes of labeled neurons developed significantly from P4 to P14 in several nuclei. The distinctive temporal and spatial expression pattern of NADPH-diaphorase implies that the NO/cGMP system may play an important role in physiological and developmental functions. Accepted: 8 September 1997     

In vitro evidence that the reduction in mesencepalic dopaminergic neurons in the weaver heterozygote is not due to a failure in target cell interaction

 The murine weaver (wv) mutation is characterized by a genetically determined loss of several neuronal populations, which include the nigrostriatal dopaminergic neurons. Animals homozygous for the wv gene exhibit marked deficits in dopaminergic morphological and neurochemical parameters. The wv gene shows incomplete dominance in that heterozygous (wv/+) mice exhibit moderate reductions in midbrain dopaminergic neuron number. It is unclear whether the dopaminergic neuronal loss in homozygous and heterozygous animals results from an effect of the wv gene solely on the dopaminergic neurons or is due to a failure of interaction of dopaminergic neurons with target cells of the striatum. This issue has been addressed utilizing three-dimensional reaggregate tissue cultures to determine whether the wv gene acts directly on the mesencephalic dopaminergic neurons. Embryonic mesencephalon and striatum from wv/+ and wild-type (+/+) brains were dissociated and the cells recombined into four mesencephalic-striatal aggregate combinations: (1) mesencephalic_(+/+)-striatal_(+/+)aggregates; (2) mesencephalic _(wv/+) -striatal _(wv/+) aggregates; (3) mesencephalic _(wv/+) -striatal_(+/+)aggregates; and (4) mesencephalic_(+/+)-striatal _(wv/+) aggregates. At 29 days and 57 days of culture, the number of dopaminergic neurons and dopamine content from mesencephalic-striatal aggregates consisting of mixed genotype or from only wv/+ tissue were quantitated and compared with that from mesencephalic-striatal cultures prepared from +/+ tissue alone. At both culture time points, aggregates containing wv/+ mesencephalon coaggregated with either wv/+ or +/+ striatum contained fewer dopaminergic neurons than mesencephalic-striatal cultures composed of only +/+ cells. Coaggregation of +/+ mesencephalon with wv/+ striatum did not have a detrimental effect on dopaminergic cell number. The findings demonstrate that the difference in the number of mesencephalic dopaminergic neurons between wv/+ and +/+ animals seen in vivo can be reproduced in three-dimensional reaggregate culture. Since the coculture of +/+ striatum with wv/+ mesencephalon did not appear to rescue wv/+ dopaminergic neurons in the aggregates as compared to wv/+ striatum and, wv/+ striatum proved to be a perfectly adequate target for +/+ mesencephalic dopaminergic neurons, it appears that the effect of the wv gene is on the dopaminergic neurons themselves. Received: 7 August 1996 / Accepted: 20 December 1996