Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.     

Brain single-photon emission computed tomography and magnetic resonance imaging in Machado-Joseph disease

BACKGROUND: Machado-Joseph disease (MJD) is one of the most frequently encountered spinocerebellar ataxias. However, few reports on brain single-photon emission computed tomographic (SPECT) imaging (BSI) with hexylmethylpropylene amineoxine labled with technetium Tc 99m and magnetic resonance imaging (MRI) have been performed for the evaluation of patients with MJD. OBJECTIVES: To investigate possible abnormalities with BSI and MRI in patients with MJD and to correlate these findings with the duration of symptoms; cerebellar, extrapyramidal, and pyramidal syndromes; and the molecular characteristics of the MJD mutation. PATIENTS AND METHODS: Twelve patients (8 males and 4 females [mean age, 39 years]) with genetically proven MJD were studied. The patients underwent BSI and MRI on the same day. Brain SPECT imaging was performed after an intravenous injection of 99mTc-hexylmethylpropylene amineoxine. The transaxial, coronal, and sagittal BSIs obtained were submitted to visual and semiquantitative analyses. Magnetic resonance imaging was obtained in a 2-T system with coronal, sagittal, transaxial, and 3-dimensional (volumetric) acquisitions. The volumes of the cerebellar hemispheres and vermis were calculated. Control groups for BSI (22 female and 20 male subjects [mean age, 33 years]) and MRI (13 female and 4 male subjects [mean age, 32.2 years]) were included for comparison. RESULTS: Correlation was observed between the perfusion abnormalities identified by visual analysis in the BSI with the structural abnormalities observed on MRI in the parietal lobes and vermis. Brain SPECT imaging identified (by visual analysis) more perfusion abnormalities in the inferior portion of the frontal lobes, mesial and lateral portions of the temporal lobes, basal ganglia, and cerebellar hemispheres. Magnetic resonance imaging identified more abnormalities in the pons and superior portions of the frontal lobes. Olivary atrophy was identified by MRI. Semiquantitative analysis showed a statistically significant difference of perfusion in the inferior and superior portions of the frontal lobes, lateral portion of the temporal lobes, parietal lobes, left basal ganglia, cerebellar hemispheres, and vermis when compared with the control group. A significant difference was noted between the vermis and cerebellar volumes on MRI when compared with the control group. A significant relationship was observed between the perfusion of the left parietal lobe (P =.05) and extrapyramidal syndrome. There was a tendency toward an inverse relationship between the duration of symptoms and the perfusion of the cerebellar hemispheres (rho = -0.37; P =.24) and volume of the vermis (rho = -0.30; P =.34); between the length of the expanded (CAG)n repeat and the perfusion of the left parietal lobe (rho = -0.32; P =.36), vermis (rho = -0.28; P =.43), and pons (rho = -0.28; P =.42). A direct association was observed between the length of the expanded (CAG)n repeat and the perfusion of the lateral portion of the right temporal lobe (rho = 0.67; P =.03). CONCLUSIONS: Brain SPECT imaging and MRI were capable of identifying subclinical abnormalities in individuals with MJD. These findings may be helpful for a better understanding of the pathophysiology of this disease.     

CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1.

Human CD4 (HuCD4) is the principal receptor for human immunodeficiency virus type 1 (HIV-1) in human cell infection. Susceptibility of rabbit cell lines to infection with HIV-1 raised questions concerning whether a CD4 homolog serves as HIV-1 receptor on rabbit cells. Sequence comparisons of rabbit CD4 (RbCD4) cloned from a rabbit thymus cDNA library showed that 6 of the 18 residues implicated in HIV-1 binding by CD4 differ between the human and rabbit proteins. No correlation between RbCD4 expression by rabbit cell lines and their ability to support HIV-1 infection was seen. Transfection of RbCD4-negative, HTLV-I-transformed cell lines with HuCD4 significantly enhanced HIV-1 infectivity, suggesting that these lines lack a receptor present on other RbCD4-negative lines that produce high levels of p24 in their native state. Inhibition of HIV-1 infection with soluble HuCD4 was demonstrated for all rabbit lines tested, but complete inhibition was obtained only with a rabbit T-cell line expressing RbCD4 and with HuCD4 transfectants. The results suggest that HIV-1 infection of the RbCD4-positive line proceeds through a receptor similar to HuCD4 but that an additional receptor or receptors may serve this purpose in RbCD4-negative lines.     

SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling

Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins. Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role. SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait. N-terminal Cbl interacts with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent manner. We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells. In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain. Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity. We have evidence that SLAP forms homodimers through its C-terminal region. Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity. The in vivo association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.     

Correlation between lower urinary tract symptoms and erectile dysfunction in men presenting for prostate cancer screening

Lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) are age-related conditions that may have a profound impact on the quality of life. The relationship between LUTS and ED is not completely understood. In this study, we assessed this relationship in men over 45 years of age during a prostate cancer screening program. LUTS and ED were evaluated in 1267 men aged 45-75 years (mean 58.2+/-8.2 years). Patients completed the International Prostate Symptom Score (IPSS) and the International Index of Erectile Function-5 (IIEF-5). The association between LUTS and ED was analyzed and the influence of age in the results was tested. We also evaluated the influence of the intensity of LUTS in the ED severity. A total of 514 (40.6%) patients were considered symptomatic of LUTS (24.8% with mild, 11.8% with moderate and 4% with severe LUTS). ED was present in 758 (59.9%) men and was considered mild in 25.0%, moderate in 18.3% and severe in 16.7%. The IIEF-5 score had a negative correlation with both the IPSS score (r=-0.33, P<0.001) and age (r=-0.31 and P<0.001). Age was positively associated with the IPSS score (r=0.14 and P<0.001). A significant correlation was observed between LUTS and ED, with 57.6% of the men with LUTS presenting ED as opposed to 29.7% of the asymptomatic population (odds ratio=3.32; 95% CI =2.57-4.29, P<0.001). Age-adjusted univariate analysis revealed a significant and independent influence of LUTS on the incidence of ED (odds ratio=2.72; 95% CI=2.08-3.57, P<0.001). IIEF scores varied significantly according to the severity of the urinary symptoms. Our findings in a prostate cancer screening population confirm that LUTS is an age-independent predictor of ED. Furthermore, they demonstrate that not only the presence of LUTS increases the likelihood of developing ED, but the severity of LUTS is associated with the intensity of ED.