Distinct properties of layer 3 pyramidal neurons from prefrontal and parietal areas of the monkey neocortex

In primates, working memory function depends on activity in a distributed network of cortical areas that display different patterns of delay task-related activity. These differences are correlated with, and might depend on, distinctive properties of the neurons located in each area. For example, layer 3 pyramidal neurons (L3PNs) differ significantly between primary visual and dorsolateral prefrontal (DLPFC) cortices. However, to what extent L3PNs differ between DLPFC and other association cortical areas is less clear. Hence, we compared the properties of L3PNs in monkey DLPFC versus posterior parietal cortex (PPC), a key node in the cortical working memory network. Using patch-clamp recordings and biocytin cell filling in acute brain slices, we assessed the physiology and morphology of L3PNs from monkey DLPFC and PPC. The L3PN transcriptome was studied using laser microdissection combined with DNA microarray or quantitative PCR. We found that in both DLPFC and PPC, L3PNs were divided into regular spiking (RS-L3PNs) and bursting (B-L3PNs) physiological subtypes. Whereas regional differences in single-cell excitability were modest, B-L3PNs were rare in PPC (RS-L3PN:BL3PN, 94:6), but were abundant in DLPFC (50:50), showing greater physiological diversity. Moreover, DLPFC L3PNs display larger and more complex basal dendrites with higher dendritic spine density. Additionally, we found differential expression of hundreds of genes, suggesting a transcriptional basis for the differences in L3PN phenotype between DLPFC and PPC. These data show that the previously observed differences between DLPFC and PPC neuron activity during working memory tasks are associated with diversity in the cellular/ molecular properties of L3PNs.Fil: Gonzalez Burgos, Guillermo. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Miyamae, Takeaki. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Krimer, Yosef. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Gulchina, Yelena. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Pafundo, Diego Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Krimer, Olga. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Bazmi, Holly. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Arion, Dominique. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Enwright, John F.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Fish, Kenneth N.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Lewis, David A.. Univeristy of Pittsburgh. School of Medicine; Estados Unido

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Phosphonoformate (foscarnet) is a pyrophosphate (PP(i)) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP(i) binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP(i) or a nucleotide such as ATP) which also interacts with the PP(i) binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3′-azido-3′-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PP(i), and ATP. Alternatively, the binding position or orientation of PP(i), ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not

Multiple, Linked Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Treatment-Experienced Patients Are Missed by Standard Genotype Analysis

To investigate the extent to which drug resistance mutations are missed by standard genotyping methods, we analyzed the same plasma samples from 26 patients with suspected multidrug-resistant human immunodeficiency virus type 1 by using a newly developed single-genome sequencing technique and compared it to standard genotype analysis. Plasma samples were obtained from patients with prior exposure to at least two antiretroviral drug classes and who were on a failing antiretroviral regimen. Standard genotypes were obtained by reverse transcriptase (RT)-PCR and sequencing of the bulk PCR product. For single-genome sequencing, cDNA derived from plasma RNA was serially diluted to 1 copy per reaction, and a region encompassing p6, protease, and a portion of RT was amplified and sequenced. Sequences from 15 to 46 single viral genomes were obtained from each plasma sample. Drug resistance mutations identified by single-genome sequencing were not detected by standard genotype analysis in 24 of the 26 patients studied. Mutations present in less than 10% of single genomes were almost never detected in standard genotypes (1 of 86). Similarly, mutations present in 10 to 35% of single genomes were detected only 25% of the time in standard genotypes. For example, in one patient, 10 mutations identified by single-genome sequencing and conferring resistance to protease inhibitors (PIs), nucleoside analog reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors (NNRTIs) were not detected by standard genotyping methods. Each of these mutations was present in 5 to 20% of the 20 genomes analyzed; 15% of the genomes in this sample contained linked PI mutations, none of which were present in the standard genotype. In another patient sample, 33% of genomes contained five linked NNRTI resistance mutations, none of which were detected by standard genotype analysis. These findings illustrate the inadequacy of the standard genotype for detecting low-frequency drug resistance mutations. In addition to having greater sensitivity, single-genome sequencing identifies linked mutations that confer high-level drug resistance. Such linkage cannot be detected by standard genotype analysis

DPC 817: a Cytidine Nucleoside Analog with Activity against Zidovudine- and Lamivudine-Resistant Viral Variants

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents