Healthy and diseased corticospinal motor neurons are selectively transduced upon direct AAV2-2 injection into the motor cortex.

Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1(G93A) transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken ß-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1(G93A) transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases.

Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization.

PURPOSE: Breast cancer is thought to develop from noninvasive precursor lesions, although the earliest steps of neoplastic transformation are still undefined. Usual ductal hyperplasia (UDH) is considered to represent a benign proliferation of ductal epithelial cells, whereas atypical ductal hyperplasia (ADH) may represent the first clonal neoplastic expansion of these cells. The aim of this study was to examine genetic alterations in UDH and ADH and to determine the relationship between these lesions in the same breast biopsy. EXPERIMENTAL DESIGN: Comparative genomic hybridization analysis was used to define copy number alterations in DNA extracted from archival sections of 18 patients. Nine patients showed ADH with adjacent UDH, and nine showed pure UDH. None showed evidence of invasive cancer or ductal carcinoma in situ. RESULTS: Five of the nine ADH lesions showed chromosome copy number alterations. 16q loss (five cases) and 17p loss (two cases) were the most frequent changes. The associated UDH lesions in these five patients also showed copy number alterations, always a subset of the changes present in the paired ADH. In one other patient, the UDH showed eight chromosomal alterations, whereas the paired ADH showed no changes. Only one of nine cases with pure UDH showed comparative genomic hybridization abnormalities. CONCLUSIONS: These data support the likelihood that UDH is a precursor of ADH, at least in some cases representing neoplastic growth. The frequencies of 16q and 17p losses suggest that alterations of candidate genes located in these chromosomal regions may play a role early in breast carcinogenesis.

Genomic alterations in tubular breast carcinomas.

Tubular carcinoma of the breast is a well-differentiated variant of invasive ductal carcinoma and has been shown to have an exceptionally favorable prognosis, as manifested by a low incidence of lymph node metastases and an excellent overall survival. It is unknown whether this subtype represents an early step along the continuum of development to a more aggressive, poorly differentiated ductal cancer, or whether these cancers are destined to remain well differentiated with limited metastatic potential. We undertook an analysis of 18 pure tubular carcinomas of the breast using comparative genomic hybridization to evaluate the chromosomal changes in these tumors. An average of 3.6 chromosomal alterations of the genome were identified per case. The most frequent change involved loss of 16q (in 78% of tumors) and gain of 1q (in 50% of tumors). All but one case with 1q gain also exhibited a concomitant 16q loss. Other frequent changes involved 16p gain in 7 of 18 cases (39%) and distal 8p loss in 5 of 18 cases (28%). Comparison with known genomic alterations in a mixed group of invasive cancers shows tubular cancer to have fewer overall chromosomal changes per tumor (P <.01), higher frequency of 16q loss (P <.001), and lower frequency of 17p loss (P =.007). These results strongly suggest that tubular carcinomas are a genetically distinct group of breast cancers.

Loss of chromosome 16q in lobular carcinoma in situ.

Lobular carcinoma in situ (LCIS) and infiltrating lobular carcinoma may represent different forms of the same disease based on their frequent clinical association and similar histologic features. Patients with LCIS are at increased risk of multicentric and bilateral disease. Thus, LCIS may represent both a precursor to infiltrating lobular carcinoma and a marker of risk for breast cancer. To identify genomic alterations in LCIS, comparative genomic hybridization was performed on 17 cases without concurrent invasive carcinoma. Loss involving chromosome 16q was present in 88% of cases and was the sole detected alteration in 29%. Gain involving 1q was second in frequency, occurring in 41% of tumors, and in all cases was associated with loss of 16q. Other recurrent changes were loss involving 17p (18%), 8p (12%), and 12q24 (12%). E-cadherin immunohistochemistry was performed on all LCIS cases to evaluate the correlation of loss involving 16q22, the site of the E-cadherin gene, and altered protein expression. Most cases with 16q22 loss showed altered E-cadherin expression (12 of 13). These results in LCIS are similar to changes reported in infiltrating lobular cancer, confirming a genetic relationship between them. HUM PATHOL 32:292-296.

Comparative genomic hybridization.

Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome-wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.

Chromosomal alterations in ductal carcinomas in situ and their in situ recurrences.

BACKGROUND: Ductal carcinoma in situ (DCIS) recurs in the same breast following breast-conserving surgery in 5%-25% of patients, with the rate influenced by the presence or absence of involved surgical margins, tumor size and nuclear grade, and whether or not radiation therapy was performed. A recurrent lesion arising soon after excision of an initial DCIS may reflect residual disease, whereas in situ tumors arising after longer periods are sometimes considered to be second independent events. The purpose of this study was to determine the clonal relationship between initial DCIS lesions and their recurrences. METHODS: Comparative genomic hybridization (CGH) was used to compare chromosomal alterations in 18 initial DCIS lesions (presenting in the absence of invasive disease) and in their subsequent ipsilateral DCIS recurrences (detected from 16 months to 9.3 years later). RESULTS: Of the 18 tumor pairs, 17 showed a high concordance in their chromosomal alterations (median = 81%; range = 65%-100%), while one case showed no agreement between the paired samples (having two and 20 alterations, respectively). Morphologic characterization of the DCIS pairs showed clear similarities. The mean number of CGH changes was greater in the recurrent tumors than in the initial lesions (10.7 versus 8.8; P =.019). The most common changes in both the initial and the recurrent in situ lesions were gains involving chromosome 17q and losses involving chromosomes 8p and 17p. The degree of concordance was independent of the time interval before recurrence and of the presence of positive surgical margins. CONCLUSIONS: In this study, DCIS recurrences were clonally related to their primary lesions in most cases. This finding is consistent with treatment paradigms requiring wide surgical margins and/or postoperative radiation therapy.

Genetic alterations in ERBB2-amplified breast carcinomas.

Amplification of the ERBB2 oncogene has recently received attention as a target for antibody-based therapies and as a predictor of response to adjuvant chemotherapy. Modification of treatment strategies based on ERBB2 status has led to further interest in the genetic alterations that accompany ERBB2 gene amplification or overexpression. In this study, chromosome alterations that are associated with ERBB2 amplification were defined by comparative genomic hybridization (CGH). Additionally, fluorescence in situ hybridization (FISH) was used to validate gene amplification, and protein expression was detected immunohistochemically. ERBB2-amplified tumors as detected by FISH, immunohistochemistry (IHC), or CGH had twice as many CGH-defined chromosomal alterations (means of 11.8, 11.0, and 12.7, respectively) as the nonamplified tumors (means of 6.8, 7.0, and 5.6, respectively). ERBB2 positivity correlated with the total number of genetic events. A wide spectrum of copy number gains and losses was seen by CGH in all of the tumors. An increased number of losses of 18q and gains of 20q was found in ERBB2-positive tumors. Other common aberrations for all of the tumors were copy number gains of 1q (58%), 8q (52%), 20q (30%), and losses of 18q (39%), 13q (39%), and 3p (33%). A high degree of concordance was observed among the three methods in 33 primary breast cancers. The concurrence for ERBB2 detection between FISH and IHC was 90%, between FISH and CGH was 82%, and between IHC and CGH was 84%. This study shows that breast tumors showing erbB2 overexpression or gene amplification are genetically distinct from erbB2-negative tumors. These differences may relate to the mechanisms underlying altered response to adjuvant therapies and may define the responsiveness to erbB2-directed immunotherapy.

Genetic alterations in primary bladder cancers and their metastases.

Bladder cancer progression is thought to be associated with sequential genetic events. To search for the specific genetic changes associated with the metastatic process, comparative genomic hybridization was performed on 22 primary tumors and 24 metastases (10 distant and 14 nodal metastases) from 17 patients with stage pT2-4 bladder cancer. There was a striking similarity between the genetic alterations present in the primary and metastatic tumor samples from the same patient. The mean number of genetic changes/tumor was 12.2 for primary tumors and 11.7 for metastases. There was a strong concordance in the specific aberrations present in each patient's primary and metastatic lesions (mean, 75%). Concordance was also high among multiple sites from an individual primary tumor (mean, 96%) and multiple metastases from the same patient (mean, 75%). There were no specific genetic changes overrepresented in the metastases compared with their primary tumors. Genetic alterations present in more than 40% of tumors included gains on 6p, 8q, 10q, and 17q and losses involving 8p, 10q, and Y. Two regions of high-level amplification were common: (a) 10q22.1-q23.1 (32.6%); and (b) 17q11-21.3 (23.9%; the locus of erbB-2). A summary statistic was developed to quantitate the degree of clonal relationships between biopsies from the same patient. These data support a model in which minimal clonal evolution occurs in the metastatic tumor cell population after the metastatic event. When comparing primary cancers from patients with and without metastases, however, several unique genetic changes were identified in those cancers with metastases, suggesting that these loci may harbor genes important to the metastatic process.

Overcoming cellular senescence in human cancer pathogenesis.

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and -3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P = 0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11-q12 (0.99) and +8p22-pter (0.94) in the immortal muscle invasive TCCs, and of -9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13-p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P = 0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P = 0.04) than was p16 or pRb alteration (P = 0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11-q12, -3p13-p14, or -8p21-pter.

Genetic alterations in primary breast cancers and their metastases: direct comparison using modified comparative genomic hybridization.

Breast tumor development and progression are thought to be driven by an accumulation of genetic alterations, but little is known about the specific changes that occur during the metastatic process. We analyzed pairs of primary breast cancers and their matched lymph node metastases from 11 patients, pairs of primaries and distant metastases from three patients, and pairs of primaries, and local recurrences from two patients by using comparative genomic hybridization (CGH). Simultaneous hybridization analysis of primary versus matched lesion DNAs from 11 patients was also performed (modified CGH). This modified approach was useful not only for confirming CGH results but also for demonstrating quantitative differences between aberrations present at both sites. Frequent chromosomal changes present at both sites (> 35% of 16 cases) were 1q, 8q, and 17q gains and 6q, 8p, 9q, 13q, 16q, 17p, and Xp losses. The total number of aberrations detected exclusively in the lymph nodes or distant metastases was higher than that in the primary tumors (2.5 vs. 0.7, P < 0.05). We found high-level amplifications in four metastases (two lymph nodes and two distant metastases), but none in any primary tumor. These findings suggest that progression from primary breast cancer to metastasis may be associated with the acquisition of further genetic changes. Although further investigations are required, it was of interest that 3 of 11 patients (27%) showed 18q loss solely in their lymph node metastases.

20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells.

Breast, bladder, colon, and ovarian carcinomas show frequent low level 20q gain and less frequently high level 20q13.2 amplification, but the significance of these 20q amplifications in transformation has not been defined. Using karyotypic and comparative genomic hybridization (CGH) analyses, chromosome losses and gains were analysed in six newly immortalized human uroepithelial cell (HUC) lines transformed by Human Papillomavirus 16 (HPV16) E7. Results showed clonal chromosomes with 20q11->qter gain in all six lines. CGH revealed a peak of 20q13.2 amplification in two cell lines. FISH with whole chromosome 20 paint showed expanded chromosome regions (ECRs) and double minute chromosomes (DMs) that contained chromosome 20 material in cell lines with 20q13.2 amplification. FISH with probes from the center of the 20q13.2 human breast cancer amplicon showed as many as 24 signals in cells with 20q13.2 amplification. The acquisition of genome instability in these E7-HUCs did not correlate with TP53 mutation, as all E7-HUCs contained only wildtype TP53. These results suggest that low level 20q gain is associated with overcoming cellular senescence in E7 transformed cells (P-value=2 x 10(-7)), but does not confer genome instability, while high level 20q13.2 amplification is associated with chromosome instability. Loss of 10p (P-value = 3 x 10(-5)) was also important in immortalization of E7-transformed HUCs. Thus, these results have profound implications for interpreting the significance of high versus low level 20q gains in human cancers.

Identification of gains and losses of DNA sequences in primary bladder cancer by comparative genomic hybridization.

Comparative genomic hybridization (CGH) makes it possible to detect losses and gains of DNA sequences along all chromosomes in a tumor specimen based on the hybridization of differentially labeled tumor and normal DNA to normal human metaphase chromosomes. In this study, CGH analysis was applied to the identification of genomic imbalances in 26 bladder cancers in order to gain information on the genetic events underlying the development and progression of this malignancy. Losses affecting 11p, 11q, 8p, 9, 17p, 3p, and 12q were all seen in more than 20% of the tumors. The minimal common region of loss in each chromosome was identified based on the analysis of overlapping deletions in different tumors. Gains of DNA sequences were most often found at chromosomal regions distinct from the locations of currently known oncogenes. The bands involved in more than 10% of the tumors were 8q21, 13q21-q34, 1q31, 3q24-q26, and 1p22. In conclusion, these CGH data highlight several previously unreported genetic alterations in bladder cancer. Further detailed studies of these regions with specific molecular genetic techniques may lead to the identification of tumor suppressor genes and oncogenes that play an important role in bladder tumorigenesis.

Influence of age and gender on the presence of coronary calcium detected by ultrafast computed tomography.

OBJECTIVES: This study sought to determine the relation between coronary calcification detected with ultrafast computed tomography and lumen narrowing defined with angiography and evaluated whether this relation is influenced by age and gender. BACKGROUND: Ultrafast computed tomography has been shown to be a sensitive method for detection of coronary calcification associated with atherosclerotic disease, but the relation between the extent of coronary calcification and degree of lumen narrowing and the possible influence of gender or age, or both, on this relation have not been clarified. METHODS: Seventy men and 70 women were studied with ultrafast computed tomography for analysis of coronary calcification and coronary angiography. Coronary atherosclerosis was considered present if any lumen irregularity was noted on angiography, and obstructive coronary artery disease was defined as a lumen diameter narrowing > or = 70%. RESULTS: Coronary calcification had a sensitivity of 88% for identification of patients with atherosclerotic disease and 97% for those with obstructive disease, with corresponding specificities of 55% and 41%, respectively. The sensitivity of coronary calcium for detection of atherosclerotic disease in women < 60 years old was 50%, significantly less than the 97% sensitivity in women > 60 years old and the 87% sensitivity in men < 60 years old (p < 0.05 for each comparison). Logistic regression analysis revealed a 1.81-fold increase in the likelihood of detecting coronary calcification in the atherosclerotic lesions of men compared with those in women (95% confidence interval 1.12 to 2.93, p = 0.016) when controlled for age and severity of coronary disease by angiography. CONCLUSIONS: Atherosclerotic lesions in women are less likely to have coronary calcium than lesions with a similar degree of lumen narrowing in men. Differences in the pattern of coronary calcification between men and women may provide insight into the gender differences observed in the clinical development of symptomatic coronary artery disease.

Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.

Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples.

Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells.

Parameters of genome instability and morphological alterations associated with cell transformation were studied in an isogeneic set of clonal human uroepithelial cell (HUC) lines immortalized by the human papilloma virus 16 (HPV16) E6 and/or E7 gene(s). HPV16 E6 binds p53, leading to rapid degradation of p53, whereas E7 binds and alters pRb and other proteins. We report that two independent E7-immortalized HUC lines showed minimal phenotypic or genotypic alterations, except that both lines contained amplification of 20q DNA sequences and a greater polyploidization at an early passage. The E7-immortalized HUC line resembled normal HUC lines, except that they failed to senesce. In contrast, the E6-immortalized HUC lines were morphologically altered, contained numerous random chromosome aberrations, and showed unstable evolving karyotypes with passage in culture. No amplified DNA sequences were detected in E6-immortalized HUC lines. Instead, clonal losses of chromosome regions (i.e., -3p, -6q, -9p), putatively containing tumor suppressor or senescence genes, accompanied the E6-HUC immortalization event. E6-immortalized HUC lines showed transformed phenotypes similar to E6/E7-HUC lines. The difference in genome stability between E6- and E7-immortalized HUC was highly significant statistically (p-value < 10(-6). Thus, the HPV16 E7 gene led to HUC immortalization by a pathway that blocked cellular senescence, but did not disrupt genome stability. These results implicate p53 loss, but not pRb alteration, in genome destabilization.

Carcinogen-induced amplification of SV40 DNA inserted at 9q12-21.1 associated with chromosome breakage, deletions, and translocations in human uroepithelial cell transformation in vitro.

The fate of integrated SV40 viral genome in SV40-immortalized human uroepithelial cells (SV-HUC) during multistep chemical transformation in vitro was studied. We previously reported that exposure of SV-HUC at passage (P) 15 to the chemical carcinogens 3-methylcholanthrene (MCA), 4-aminobiphenyl (ABP), or the N-hydroxy metabolites of ABP causes tumorigenic transformation and/or neoplastic progression. We report now that these same chemical carcinogens induce amplification of SV40 DNA in SV-HUC. We used fluorescence in situ hybridization (FISH) to show that this amplification occurs at the SV40 integration site, which was mapped near a common fragile site at 9q12-21.1 on the der(9)t(8;9) chromosome that is present in all SV-HUC at the earliest passage studied. Karyotypic analysis, along with FISH, also revealed that all carcinogen-induced tumors (T-SV-HUCs) had breaks at 9q12-21.1, deletions of 9q12-21.1-->pter, and new derivative chromosomes containing SV40 in the segment 9q12-21.1-->9q34::8q22-->8qter. Southern blot analysis, along with FISH, confirmed SV40 genome rearrangements in T-SV-HUCs. In contrast, no 9q12-21.1 breaks were observed in control SV-HUC. Thus, these results associate 9q12-21.1-->pter alterations with HUC tumorigenic transformation. In addition, these results indicate for the first time that (carcinogen-induced) amplification of chromosome-integrated viral genes may create sites that are prone to breakage, deletions, and translocations. These results suggest a new mechanism by which chemical carcinogens in synergy with a DNA tumor virus could initiate a cascade of events that contribute to the genomic instability associated with tumorigenesis.

Primary parathyroid gland hyperplasia in dogs: six cases (1982-1991).

Persistent hypercalcemia attributable to parathyroid gland hyperplasia was identified in 6 dogs with primary hyperparathyroidism. Clinical signs included polydipsia (n = 4), polyuria (n = 4), and signs caused by cystic calculi (n = 3). Abnormal clinical pathologic findings included hypercalcemia (mean, 13.6 mg/dl; range, 12.6 to 14.7 mg/dl; n = 6), hypophosphatemia (mean, 2.2 mg/dl; range, 1.4 to 2.9 mg/dl; n = 6), high serum alkaline phosphatase activity (mean, 222 IU/L; range, 161 to 286 IU/L; n = 3), and isosthenuria (mean, 1.012; range, 1.006 to 1.017; n = 6). Serum parathyroid hormone concentration was within the reference range or high (mean, 23 pmol/L; range, 7 to 119 pmol/L; reference range, 1.5 to 13 pmol/L) in all dogs. At surgery, the number of large parathyroid glands was variable, being limited to 1 gland in 3 dogs, 2 glands in 2 dogs, and 4 glands in 1 dog. All visibly large parathyroid glands were surgically removed from each dog. Serum calcium concentration decreased into or below the reference range within 72 hours of surgery in all dogs, confirming the diagnosis of primary parathyroid disease. Multiple nodules of adenomatous hyperplasia were identified in each dog. All 6 dogs were treated with vitamin D and calcium carbonate following surgery. The dog from which all 4 parathyroid glands were removed has remained eucalcemic for more than 1 year with vitamin D supplementation. Vitamin D and calcium administration was discontinued within 4 to 12 weeks of surgery in the remaining 5 dogs. These dogs remained eucalcemic without vitamin D supplementation.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of an electrical synapse between solitary pairs of catfish horizontal cells by dopamine and second messengers.

1. Retinas from channel catfish were dissociated and the cells maintained in culture. Horizontal cells that normally receive input from cone photoreceptors were identified. The conductance of the electrical junction formed between a pair of 'cone' horizontal cells was measured by controlling the membrane voltage of each cell with a voltage clamp maintained through either a micropipette or a patch pipette. The two techniques yielded similar results. 2. Transjunctional current was measured while transjunctional voltage was stepped to values between +/- 60 mV. The current (measured 5 ms after a step) was proportional to voltage over the range tested. For steps to voltages greater than +/- 45 mV, the current exhibited a slight time-dependent decline. 3. Dopamine decreased junctional conductance in a dose-dependent fashion. A 50% reduction was obtained with 10 nM-dopamine. The D1 agonist fenoldopam (100 nM) also decreased junctional conductance. The uncoupling produced by either agent was rapid and reversible. 4. The introduction of 100 microM-cyclic AMP into one cell of a pair decreased junctional conductance by, on average, 40%. Forskolin (1-10 microM), an activator of adenylate cyclase, decreased junctional conductance 50-90%. 5. The introduction of 80 microM-cyclic GMP into one cell of a pair decreased junctional conductance by, on average, 40%. Nitroprusside (1-10 microM), an activator of guanylate cyclase, reduced junctional conductance 40-65%. 6. The introduction of a peptide inhibitor specific for the cyclic AMP-dependent protein kinase reversed a decrease in junctional conductance produced by superfusion with either dopamine (1 microM), fenoldopam (100 nM) or forskolin (5-10 microM). 7. Intracellular Ca2+ concentration was measured with the fluorescent indicator Fura-2. The intracellular Ca2+ concentration was increased by activation of a Ca2+ current. Junctional conductance remained constant as the internal Ca2+ concentration changed from 100 to 700 nM. 8. Intracellular pH was measured with the fluorescent indicator bis-carboxyethylcarboxyfluorescein. The application of acetate (2.5 mM) reduced intracellular pH by 0.2-0.3 units and decreased junctional conductance by approximately 50%. A subsequent application of fenoldopam did not alter intracellular pH, but decreased junctional conductance by more than 50%. 9. The sensitivity of the junctional conductance between isolated horizontal cells to dopamine is consistent with dopamine having a direct effect on coupling in intact retina. Dopamine regulates the activity of a cyclic AMP-dependent protein kinase which in turn modulates junctional conductance. Changes in intracellular pH and Ca2+ concentration are not involved in mediating the effect of dopamine on coupling. Cyclic GMP and intracellular pH may participate in regulatory pathways independent of that used by cyclic AMP.