NCAM Expression Research Articles

Neural Precursors from Canine Skin: A New Direction for Testing Autologous Cell Replacement in the Brain

Recent work indicates that neural progenitors can be isolated from the skin of rodents and humans. The persistence of these cells in accessible adult tissue raises the possibility of their exploitation for research and therapeutic purposes. This study reports on the derivation, culture, and characterization of homogenous canine skin-derived neuroprecursor cells (SKiNPs) from mature animals. Canine tissue was used because naturalistic brain diseases in community-dwelling dogs are emerging as ecologically sound models for a range of neurological conditions. Adult SKiNPs were initially isolated as neurospheres and then cultured for 10-15 passages in an adherent monolayer assay. Serumfree expansion conditions contained B-27, 20 ng/mL EGF, and 40 ng/mL bFGF. Gene expressions by PCR indicated expression of nestin, CD133, NCAM, and FGF2R, but not GFAP. Highly uniform expression of nestin (76 +/- 8.3%), NCAM (84 +/- 3.3%), betaIII-tubulin (96 +/- 4.3%), and CD133 (68 +/- 13.5%) was also observed. Directed differentiation of SKiNPs in the presence of serum induced betaIIItubulin, NSE, NCAM, and MAP2 in >90% of differentiated cells by immunophenotype analysis. Our culture system rapidly induces canine skin cells into neural precursors, maintains nestin expression in more than 75% of proliferating cells, and generates an almost universal neuronal-like phenotype after 7 days of in vitro differentiation. Their biological characteristics are suggestive of transiently amplifying fate-restricted neuroprecursors rather than true neural stem cells. This system may be an effective alternative for autologous neurorestorative cell replacement in canine models for further translational research.

Repeated risperidone treatment increases the expression of NCAM and PSA-NCAM protein in the rat medial prefrontal cortex

The present study investigates whether the anti-schizophrenic drug risperidone may evoke changes in the expression of NCAM/PSA-NCAM proteins, an indispensable element in the remodeling of synaptic arrangements, in the medial prefrontal cortex (mPFC). Rats were treated with risperidone (0.2 mg/kg, i.p.) either once or repeatedly (once a day, for 21 days). The expression of NCAM and PSA-NCAM proteins was analyzed via western blot and immunohistochemistry at intervals of 3 h and 3, 6, and 9 days after the single or the last risperidone dose. Repeated (but not acute) administration of risperidone was found to increase the expression of NCAM-180, NCAM-140 and PSA-NCAM proteins at 3 or 6 days after treatment. PSA-NCAM immunoreactivity was found in cell bodies, perisomatic-like sites, and in the neuropil of the mPFC. Neither single nor repeated risperidone administration changed the number of PSA-NCAM neurons in the mPFC. In contrast, the repeated risperidone treatment increased the number of PSA-NCAM perisomatic-like sites and the length density of PSA-NCAM positive neuropil at 3 days after the last injection. The data obtained indicate that risperidone, given repeatedly, may promote the remodeling of the structure of presumably GABA-ergic interneurons and that it may evoke the rearrangement of the synaptic contact in the mPFC.

Universal expression of cell adhesion molecule NCAM in neuroblastoma in contrast to L1: implications for different roles in tumor biology of neuroblastoma?

Neuroblastoma is a biological, genetic and morphological heterogeneous tumor with a variable clinical course. NCAM is a cell adhesion molecule belonging to the immunoglobulin superfamily with structural similarities to cell adhesion molecule L1. The aim of this study was to determine the expression of NCAM in neuroblastoma and to compare the results to the findings of a previous study which examined L1 expression in the same group of patients. NCAM expression was investigated on a tissue array with 66 surgically resected neuroblastoma samples by immunohistochemistry with a monoclonal antibody clone 1B6 and peroxidase method. Strong expression of NCAM was detected in all of the 66 (100%) neuroblastoma tumors in contrast to L1 which was not expressed in all tumors. In contrast to L1, which was found to predict favorable outcome, NCAM is universally expressed in neuroblastoma. Therefore NCAM represents a marker for neuroblastomas irrespectively of their stages whereas L1 as an indicator for developing neuronal cells seems to identify more mature stages of this tumor. The high grade of NCAM expression might present a prerequisite for establishment of antibody-based therapies.

Neural differentiation arrest in embryonal carcinoma cells with forced expression of EWS-FLI1

Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET) has a characteristic chimeric oncogene EWS-FLI1, which results from chromosomal translocation t (11; 22), that is believed to initiate tumorigenesis of EWS/PNET. However, the specific details of EWS/PNET oncogenesis and exact role of EWS-FLI1 remain largely unknown. In this study we explored the role of EWS-FLI1 in tumor differentiation using an embryonal carcinoma cell line P19 as a model, with forced expression of EWS-FLI1 in these cells. EWS-FLI1 has been reported to promote neural differentiation in fibroblasts, mesenchymal stem cells and rhabdomyosarcoma cells. We show forced expression of EWS-FLI1 causes absence of retinoic acid-induced neural morphology, and decreases expression of neural-specific proteins MAPT and NCAM. Critical transcriptional factors for neural differentiation and stem cells are also altered in the presence of EWS-FLI1, including decreases in levels of Oct-3 and Pax-6, and an increase in the level of Id2, which is a target of EWS-FLI1. Increased proliferation and decreased apoptotic rates are also observed in P19 cells with forced expression of EWS-FLI1. Our results raise the possibility that arrest of neural differentiation by forced expression of EWS-FLI1 as observed in this study may result from dysregulation of the cell cycle and cell proliferation. Taken together, our results demonstrate that the modulation of neural differentiation in P19 cells which have a stem cell-like pluripotency in vitro can provide a novel model system to study the neural differentiation effects of EWS-FLI1 tumorigenesis of EWS/PNET.

Expression of the neural cell adhesion molecule (CD56) by human myeloma cells

Recent studies in multiple myeloma indicate that molecules associated with different haematopoietic lineages may be expressed aberrantly by myeloma cells. In order to investigate this phenomenon further, we studied the immunophenotype of bone marrow cells from 21 patients with multiple myeloma using a panel of monoclonal antibodies against T,B, myelomonocytic, and natural killer (NK)-cell antigens. Leu-19/NKH1 (CD56), a molecule identical to N-CAM, which is normally expressed by neuroectodermal and NK cells, was found in 13 patients (62%). Dual-parameter flow cytometry was used to correlate N-CAM positivity with DNA aneuploidy or cytoplasmic immunoglobulin expression as markers of myeloma cells. When N-CAM was found positive, other haematopoietic antigens were expressed only in three out of 13 cases (23%). In contrast, myeloma cells not expressing N-CAM frequently exhibited pre-B cell markers, myeloid antigen, and HLA-DR, respectively (seven out of eight cases, 88%). Six out of eight N-CAM-negative myelomas were of the IgG lambda isotype, otherwise no clearcut association with basic clinical and laboratory parameters was noted. We conclude that N-CAM expression is a common finding in multiple myeloma. Whether its expression and the observed antigenic heterogeneity is just a manifestation of malignancy or N-CAM may play a role in the biology of multiple myeloma regarding tumour cell spread, remains to be explained.

Astrogliosis in the hippocampus and cortex and cognitive deficits in rats with streptozotocin-induced diabetes: Effects of melatonin

We examined, using a Western blot technique, the contents and compositions of a specific neuronal protein, NCAM, and of an astrocyte marker, GFAP, in the hippocampus and cortex of rats with streptozotocin (STZ)-induced diabetes and compared these indices with those in control (intact) animals and STZ-diabetic rats treated with melatonin. Behavioral cognitive indices manifested in the passive avoidance test (PAT) and Morris water maze (MWM) learning performance were also estimated in the above groups of animals. As was found, STZ-diabetic rats demonstrated clear cognitive deficits according to the values of the retention latency in the PAT and time of reaching the escape platform in the MWM performance. In these animals, the GFAP content was elevated, and the amount of degraded products of this protein increased, as compared with the control. Simultaneously, considerable down-regulation of the NCAM expression and modifications of NCAM isoform composition were found in diabetic animals. In addition, significantly increased levels of lipid peroxidation (according to the amounts of malondialdehyde + 4-hydroxyalkenals) were measured in the cortex and hippocampus of rats with stable diabetic hyperglycemia. All the above-mentioned shifts were significantly smoothed or even nearly completely compensated in the case of treatment of STZ-diabetic rats with melatonin (10 mg/kg per day). The role of diabetes-related changes in the amount and composition of specific neural and glial proteins in the development of cognitive deficits, the involvement of oxidative stress in the mechanisms of the respective shifts, and possible mechanisms of the neuroprotective effect of melatonin with respect to diabetes-related pathological biochemical and behavioral shifts are discussed.

Stress downregulates hippocampal expression of the adhesion molecules NCAM and CHL1 in mice by mechanisms independent of DNA methylation of their promoters

Stress is an important physiological regulator of brain function in young and adult mammals. The mechanisms underlying regulation of the consequences of stress, and in particular severe chronic stress, are thus important to investigate. These consequences most likely involve changes in synaptic function of brain areas being part of neural networks that regulate responses to stress. Cell adhesion molecules have been shown to regulate synaptic function in the adult and we were thus interested to investigate a regulatory mechanism that could influence expression of three adhesion molecules of the immunoglobulin superfamily (NCAM, L1 and CHL1) after exposure of early postnatal and adult mice to repeated stress. We hypothesized that reduction of adhesion molecule expression after chronic stress, as observed previously in vivo, could be due to gene silencing of the three molecules by DNA methylation. Although adhesion molecule expression was reduced after exposure of C57BL/6 mice to stress, thus validating our stress paradigm as imposing changes in adhesion molecule expression, we did not observe differences in methylation of CpG islands in the promoter regions of NCAM, L1 and CHL1, nor in the promoter region of the glucocorticoid receptor in the hippocampus, the expression of which at the protein level was also reduced after stress. We must therefore infer that severe stress in mice of the C57BL/6 strain downregulates adhesion molecule levels by mechanisms that do not relate to DNA methylation.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development

BackgroundCongenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice.Methods and resultsWe analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos.ConclusionIt is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

Targeting the neural cell adhesion molecule in cancer

NCAM is a tumour associated antigen expressed on small cell lung cancer, neuroblastoma, rhabdomyosarkoma, brain tumours, multiple myelomas and acute myeloid leukaemia. Constant and strong expression of NCAM is a prerequisite for the development of antibody-based immunotherapies. From the spectrum of existing anti-NCAM compounds, radioimmunoconjugates and immunotoxins represent the clinically most advanced and successful strategies. Here we provide an overview of the evolving field of anti-NCAM immunotherapy for cancer and discuss its indications and limitations.

Are renal proximal tubular epithelial cells constantly prepared for an emergency? Focus on “The proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells”

a human kidney contains approximately one million functional units (the nephrons) that consist of a filter (the glomerulus) and a processing portion (the proximal, intermediate, and distal tubule). The glomeruli produce ∼180 liters of primary filtrate every day of which only 1 to 2 liters are

Papillary carcinoma of the thyroid: low expression of NCAM (CD56) is associated with downregulation of VEGF‐D production by tumour cells

The expression of NCAM was investigated in tissue sections of 61 cases of papillary carcinoma and in 14 lymph node metastases using immunohistochemistry. Tumour cells of 18 primary tumours were not stained, whereas in the remaining 43 cases, NCAM was expressed in less than 5% tumour cells. Similar results were obtained when NCAM expression was evaluated at the RNA level. Reduced expression of NCAM is an early event since 6/15 cases (40%) of micro-carcinoma were NCAM-negative. NCAM-positive tumour cells were more often located at the invasion front of the tumour. It has been reported that NCAM expression may affect lymphangiogenesis. In tissue sections immunostained for podoplanin, it was found that lymphatic vessels were extremely rare inside the body of the tumour, and were mostly associated with foci of chronic inflammation and/or of reparative fibrosis. Lymphangiogenesis is sustained by VEGF-C, VEGF-D, and FGF2. Analysis of micro-dissected samples of the tumour and of the paired normal thyroid tissue revealed that RNA transcripts for VEGF-D were significantly less numerous in the tumour tissue (p = 0.001). The potential role of NCAM in tumour cell biology was investigated by silencing the NCAM gene in the TPC1 thyroid papillary carcinoma cell line. It was found that NCAM down-regulation caused a significant reduction (p < 0.05) in the expression of both VEGF-C and VEGF-D mRNAs. In addition, NCAM-silenced TPC-1 cells were more adhesive to different extracellular matrix components, and were less efficient in cell migration (59% reduction; p < 0.05) and invasiveness (68% reduction). These latter results confirm that modifications of NCAM expression cause profound alterations in the adhesive and migratory properties of tumour cells, but are in apparent discrepancy with the observation that loss of NCAM is usually associated with increased tumour invasiveness in vivo.

Expression of various cellular markers in genitourinary rhabdomyosarcoma: immunohistochemical study using tma methodology

Purpose Immunohistochemical analysis of the expression of various cellular markers in rhabdomyosarcoma (RMS) may improve the design of therapeutic schedules according to individualized risk-based data and may even define specific therapeutic targets in RMS. The aim of this study was to evaluate the extent of the expression of various cellular markers in RMS, using tissue microarray (TMA) technique. Material and methods TMA paraffin embedded block was constructed of 8 samples of RMS from different patients. Each tumour sample was presented in the block by several cores, 2 mm in diameter, containing tumour tissue with adjacent transitional epithelium and normal detrusor which served as positive and negative controls. After serial slicing of 4 μm thickness, the histological slides were stained with hematoxylin & eosin (H&E) and immunostained with antibodies against Erb-B2, p53, c-kit, Ki 67, Caspase 3, EGFR, N-CAM, TOPO-II and BCL-10. The immunostaining was graded semi quantitatively by the percent of positively staining cells and the intensity of stain. Results Four cellular markers were expressed in RMS including Erb-B2, Ki 67, EGFR and N-CAM. Significant positive staining (grades 2-3), for at least one of these markers, was noted in 5 of the cases. Conclusions The expression of Erb-B2, Ki 67, EGFR and N-CAM in genitourinary RMS may have potential therapeutic implications. Our preliminary results may promote further studies to assess the potential role of these markers to serve as therapeutic targets for specific therapeutic agents and thus decrease the overall treatment burden in children suffering from genitourinary RMS.

Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos

The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, “tet-on” and “tet-off”, works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes.

Evaluation of NCAM and DKK1 Expression in Multiple Myeloma and MGUS by Gene and Tissue Microarrays: Expression Accompanies Progression.

Introduction: Expression NCAM1, a cell adhesion molecule involved in neuron-neuron adhesion, is also expressed by multiple myeloma (MM PC). Osteolytic bone lesions are a hallmark of MM and elevated expression of NCAM in MM PC has been correlated with this process (Ely and Knowles, 2003). We have previously reported that MM PC express DKK1 and MM blocks osteoblast differentiation in a DKK1-specific manner, suggesting that secretion of the Wnt signaling inhibitor plays a role in MM bone disease. Herein we used gene expression microarrays and tissue microarrays (TMAs) to investigate the simultaneous expression of DKK1 and NCAM in MM and MGUS.Methods: The study population consisted of 198 newly diagnosed MM and 44 MGUS. RNA from CD138-selected plasma cells was hybridized to Affymetrix U133Plus microarrays and data processed with Affymetrix Microarray Suite GCOS1.1 software. TMAs were constructed from formalin-fixed, paraffin embedded bone marrow biopsies. Serial sections of TMA were immunostained for CD138, NCAM and DKK1. TMAs were scanned using ScanScope using 20x lens, assessed using TMA lab software (Aperio Technologies) and scored as an average number of cells in the context of CD138 staining.Results: When put in context of a recently described molecular classification (Zhan et al., 2006), NCAM and DKK1 were found significantly co-over-expressed (DKK1+/NCAM+) in HYPERDIPLOID MM (P < 0.01); NCAM+/DKK1− was typical for MMSET-spike MM (P<0.001); NCAM−/DKK1+ DKK1 was characteristic of MM in MAF-spike disease (P < 0.001), CCND1 spikes with co-expression of CD20 (P<0.01), and the so-called MYELOID subgroup (P<0.001). In contrast, virtually all cases of MGUS were DKK1−/NCAM-. On TMAs, DKK1 expression varied in CD138-positive cells in MM, and in osteocytes and megakaryocytes in MM and MGUS, but was clearly negative in osteoblasts/lining cells. NCAM expression, was also variably expressed in PC of MM cases and in virtually 100% of osteoblasts/lining cells in both MM and MGUS. Osteocytes were distinctly negative for NCAM in both diseases. Of 195 analyzable MM biopsies, PC were DKK1+ in 1% to > 90% of PC in 191 (98%); of these, 94 were also NCAM+ in 5% to > 90% of PC; 1 case was positive for NCAM+/DKK− and 3 were NCAM−/DKK−. There was no significant difference in clinical parameters and survival in a comparison of NCAM+/DKK+ and NCAM−/DKK+ groups. DKK1 gene expression was higher in DKK1+/NCAM+ than in DKK1+/NCAM− MM (P=0.006); NCAM gene expression was higher in DKK1+/NCAM+ than in DKK+/NCAM− MM (P<0.0001) and NCAM was the number one SAM-defined gene over-expressed in DKK1+/NCAM+ relative to DKK1+/NCAM− disease. Consistent with GEP data, DKK1+/NCAM+ MM was over-represented in HYPERDIPLOID MM (32% v. 10%; P<0.001), while DKK1+/NCAM− disease was overrepresented in MAF-spike (15% v. 2%; P=0.003) and MYELOID subgroups (32% v. 18%; P=0.03). DKK1+/NCAM+ MM was completely absent in CCND1-spike /CD20-negative disease, with only 4% of DKK1+/NCAM− cases in this subgroup. No difference was observed for the other subtypes.Conclusion: DKK1 is expressed in all cases of MM with half also expressing NCAM. Neither gene is expressed in MGUS PC. These data suggests that expression of these genes in plasma cells accompanies disease progression.

BMP and LIF signaling coordinately regulate lineage restriction of radial glia in the developing forebrain

The earliest radial glia are neural stem cells that guide neural cell migration away from ventricular zones. Subsequently, radial glia become lineage restricted during development before they differentiate into more mature cell types in the CNS. We have previously shown that subpopulations of radial glial cells express markers for glial and neuronal restricted precursors (GRPs and NRPs) in expression patterns that are temporally and spatially regulated during CNS development. To characterize further the mechanism of this regulation in rat forebrain, we tested whether secreted factors that are present during development effect lineage restriction of radial glia. We show here that in radial glial cultures LIF/CNTF up-regulates, whereas BMP2 down-regulates GRP antigens recognized by monoclonal antibodies A2B5/4D4. These activities combined with secretion of BMPs dorsally and LIF/CNTF from the choroid plexus provide an explanation for the graded distribution pattern of A2B5/4D4 in dorso-lateral ventricular regions in vivo. The regulation by LIF/CNTF of A2B5/4D4 is mediated through the JAK-STAT pathway. BMP2 promotes expression on radial glial cells of the NRP marker polysialic acid most likely by regulating N-CAM expression itself, as well as at least one polysialyl transferase responsible for synthesis of polysialic acid on N-CAM. Taken together, these results suggest that generation of lineage-restricted precursors is coordinately regulated by gradients of the secreted factors BMPs and LIF/CNTF during development of dorsal forebrain.

NCAM expression by immature and tumor-derived endothelial cells and in vivo targeting

NCAM expression by immature and tumor-derived endothelial cells and in vivo targeting

No effect of prolonged corticosterone over-exposure on NCAM, SGK1, and RGS4 mRNA expression in rat hippocampus

Prolonged over-exposure of rats to corticosterone attenuates 5-HT 1A-receptor-mediated responses in hippocampal CA1 cells through an unknown mechanism, not involving downregulation of 5-HT 1A receptor expression. We here tested if corticosterone changes 5-HT 1A receptor function indirectly, by altering hippocampal mRNA expression of NCAM, SGK1, or RGS4, which all modulate 5-HT 1A receptor function. We found that the expression of none of these candidates was affected by corticosterone treatment.

Effects of letrozole on catecholaminergic neurotransmitter levels, NCAM expression and cognitive functions in intact female rats

Effects of letrozole on catecholaminergic neurotransmitter levels, NCAM expression and cognitive functions in intact female rats

Effects of ethanol and transforming growth factor beta (TGF beta) on neuronal proliferation and nCAM expression.

BACKGROUND Developmental events targeted by ethanol are cell proliferation, neuronal migration, and neurite outgrowth; the latter processes being mediated by neural cell adhesion molecule (nCAM). TGFbeta1 affects all three of these events. Therefore, the effects of ethanol on transforming growth factor (TGF) beta1 mediated activities in neocortical neurons in vitro were examined. METHODS Primary cultures of cortical neurons were obtained from 16-day-old fetuses and were treated with TGFbeta1 (0 or 10 ng/ml) and ethanol (0 or 400 mg/dl) for 48 hr. The effects of these substances on cell numbers, [ H]thymidine incorporation, and the expression of nCAM were determined. RESULTS Both cell growth (the change in cell numbers over time) and cell proliferation were inhibited by TGFbeta1 and ethanol. The action of these two anti-mitogenic factors was additive. In contrast, TGFbeta1 also promoted the expression of three isoforms of nCAM. Likewise, ethanol also up-regulated nCAM expression. On the other hand, ethanol blocked TGFbeta1-mediated nCAM expression, particularly of the 120 and 180 kDa isoforms. CONCLUSIONS TGFbeta ligands inhibit neuronal proliferation and stimulate the expression of cell adhesion proteins that promote the movement of postmitotic neurons and process outgrowth. Ethanol alters these phenomena as well. Thus, in neurons, as in astrocytes, TGFbeta1 and ethanol may interact.

Dopamine D2 receptor binding, Drd2 expression and the number of dopamine neurons in the BXD recombinant inbred series: genetic relationships to alcohol and other drug associated phenotypes.

It has not been established to what extent the natural variation in dopamine systems contribute to the variation in ethanol response. The current study addresses this issue by measuring D2 dopamine (DA) receptor binding, the expression of Drd2, the number of midbrain DA neurons in the BXD recombinant inbred (RI) series and then compares these strain means with those previously reported for a variety of ethanol and other drug-related phenotypes. Data were collected for 21 to 23 of the BXD RI strains and the parental strains. D2 DA receptor autoradiography was performed using 125I-epidepride as the ligand [ Kanes S, Dains K, Cipp L, Gatley J, Hitzemann B, Rasmussen E, Sanderson S, Silverman S, Hitzemann R (1996) Mapping the genes for haloperidol-induced catalepsy. J Pharmacol Exp Ther 277:1016-1025]. Drd2 expression was measured using the Affymetrix oligoarray system. Immunocytochemical techniques were used to determine the number of midbrain DA neurons [Hitzemann B, Dains K, Hitzemann R (1994) Further studies on the relationship between dopamine cell density and haloperidol response. J Pharmacol Exp Ther 271:969-976]. The range of difference in receptor binding for the RI strains was approximately 2-fold in all regions examined, the core, the shell of the nucleus accumbens (NAc) and the dorsomedial caudate-putamen (CPu); heritability in all regions was moderate--(h2 approximately 0.35). Drd2 expression in forebrain samples from the RI and parental strains ranged 1.5- to h2-fold and was moderate-0.47. Variation in the number of tyrosine hydroxylase (TH) positive neurons was moderate, 41% and 26% and h2 was low--0.19 and 0.15 for the ventral tegmental area (VTA) and substantia nigra compacta (SNc), respectively. Significant correlations were found between D2 DA receptor binding and the low dose (1.33 g/kg) ethanol stimulant response. (p < 0.002) and between expression and conditioned place preference (CPP) (p < 0.0005). No significant correlations were detected between ethanol preference and either receptor binding or Drd2 expression; however, a significant correlation was found between preference and Ncam expression. Ncam is approximately 0.2 Mb from Drd2. Overall, the data suggest ethanol preference and CPP are associated with the expression of Drd2 or closely linked genetic loci.