Ernest F. Terwilliger

Enhanced partner preference in a promiscuous species by manipulating the expression of a single gene

The molecular mechanisms underlying the evolution of complex behaviour are poorly understood. The mammalian genus Microtus provides an excellent model for investigating the evolution of social behaviour. Prairie voles (Microtus ochrogaster) exhibit a monogamous social structure in nature, whereas closely related meadow voles (Microtus pennsylvanicus) are solitary and polygamous. In male prairie voles, both vasopressin and dopamine act in the ventral forebrain to regulate selective affiliation between adult mates, known as pair bond formation, as assessed by partner preference in the laboratory. The vasopressin V1a receptor (V1aR) is expressed at higher levels in the ventral forebrain of monogamous than in promiscuous vole species, whereas dopamine receptor distribution is relatively conserved between species. Here we substantially increase partner preference formation in the socially promiscuous meadow vole by using viral vector V1aR gene transfer into the ventral forebrain. We show that a change in the expression of a single gene in the larger context of pre-existing genetic and neural circuits can profoundly alter social behaviour, providing a potential molecular mechanism for the rapid evolution of complex social behaviour.

The V1a Vasopressin Receptor Is Necessary and Sufficient for Normal Social Recognition: A Gene Replacement Study

Vasopressin modulates many social and nonsocial behaviors, including emotionality. We have previously reported that male mice with a null mutation in the V1a receptor (V1aR) exhibit a profound impairment in social recognition and changes in anxiety-like behavior. Using site-specific injections of a V1aR-specific antagonist, we demonstrate that the lateral septum, but not the medial amygdala, is critical for social recognition. Reexpressing V1aR in the lateral septum of V1aR knockout mice (V1aRKO) using a viral vector resulted in a complete rescue of social recognition. Furthermore, overexpression of the V1aR in the lateral septum of wild-type (wt) mice resulted in a potentiation of social recognition behavior and a mild increase in anxiety-related behavior. These results demonstrate that the V1aR in the lateral septum plays a critical role in the neural processing of social stimuli required for complex social behavior.

Identification of a protein encoded by the vpu gene of HIV-1

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS1–3. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture4,5. The complexity of virus‐host cell interaction is reflected in the complex organization of the viral genome6–9. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3′ orf proteins, as well as a protein of unknown function from the open reading frame called R10–18. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV)19–21 that do not have the capacity to encode a similar protein.

CaMKII: a biochemical bridge linking accumbens dopamine and glutamate systems in cocaine seeking

Increases in dopamine and glutamate transmission in the nucleus accumbens independently promote the reinstatement of cocaine seeking, an animal model of relapse. Here we have tested whether cocaine reinstatement in rats depends on interactions between accumbal dopamine and glutamate systems that are mediated by Ca2+/calmodulin-mediated kinase II (CaMKII). We show that stimulation of D1-like dopamine receptors in the nucleus accumbens shell reinstates cocaine seeking by activating L-type Ca2+ channels and CaMKII. Cocaine reinstatement is associated with D1-like dopamine receptor–dependent increases in accumbens shell CaMKII phosphorylated on Thr286 and glutamate receptor 1 (GluR1) phosphorylated on Ser831 (a known CaMKII phosphorylation site), in addition to increases in cell-surface expression of GluR1-containing AMPA receptors in the shell. Consistent with these findings, cocaine reinstatement is attenuated by intra-shell administration of AAV10-GluR1-C99, a vector that impairs the transport of GluR1-containing AMPA receptors. Thus, CaMKII may be an essential link between accumbens shell dopamine and glutamate systems involved in the neuronal plasticity underlying cocaine craving and relapse.

Variation in Oxytocin Receptor Density in the Nucleus Accumbens Has Differential Effects on Affiliative Behaviors in Monogamous and Polygamous Voles

Oxytocin receptors in the nucleus accumbens have been implicated in the regulation of alloparental behavior and pair bond formation in the socially monogamous prairie vole. Oxytocin receptor density in the nucleus accumbens is positively correlated with alloparenting in juvenile and adult female prairie voles, and oxytocin receptor antagonist infused into the nucleus accumbens blocks this behavior. Furthermore, prairie voles have higher densities of oxytocin receptors in the accumbens than nonmonogamous rodent species, and blocking accumbal oxytocin receptors prevents mating-induced partner preference formation. Here we used adeno-associated viral vector gene transfer to examine the functional relationship between accumbal oxytocin receptor density and social behavior in prairie and meadow voles. Adult female prairie voles that overexpress oxytocin receptor in the nucleus accumbens displayed accelerated partner preference formation after cohabitation with a male, but did not display enhanced alloparental behavior. However, partner preference was not facilitated in nonmonogamous meadow voles by introducing oxytocin receptor into the nucleus accumbens. These data confirm a role for oxytocin receptor in the accumbens in the regulation of partner preferences in female prairie voles, and suggest that oxytocin receptor expression in the accumbens is not sufficient to promote partner preferences in nonmonogamous species. These data are the first to demonstrate a direct relationship between oxytocin receptor density in the nucleus accumbens and variation in social attachment behaviors. Thus, individual variation in oxytocin receptor expression in the striatum may contribute to natural diversity in social behaviors.

The Calcium-sensing Receptor Is Involved in Strontium Ranelate-induced Osteoclast Apoptosis NEW INSIGHTS INTO THE ASSOCIATED SIGNALING PATHWAYS

Strontium ranelate exerts both an anti-catabolic and an anabolic effect on bone cells. To further investigate the molecular mechanism whereby strontium ranelate inhibits bone resorption, we focused our attention on the effects of strontium ranelate on osteoclast apoptosis and on the underlying mechanism(s). Using primary mature rabbit osteoclasts, we demonstrated that strontium \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{Sr}_{\mathrm{o}}^{2+})\) \end{document} dose-dependently stimulates the apoptosis of mature osteoclasts. As shown previously for calcium \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{Ca}_{\mathrm{o}}^{2+})\) \end{document}, the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document}-induced effect on mature osteoclasts is mediated by the \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}-sensing receptor, CaR, which in turn stimulates a phospholipase C-dependent signaling pathway and nuclear translocation of NF-κB. Unlike \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}, however, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document}-induced osteoclast apoptosis was shown to depend on PKCβII activation and to be independent of inositol 1,4,5-trisphosphate action. As a consequence of these differences in their intracellular signaling pathways, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document} in combination were shown to exert a greater effect on mature osteoclast apoptosis than did either divalent cation by itself. Altogether, our results show that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} acts through the CaR and induces osteoclast apoptosis through a signaling pathway similar to but different in certain respects from that of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}. This difference in the respective signaling cascades enables \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} to potentiate \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document}-induced osteoclast apoptosis and vice versa. In this manner, it is conceivable that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Sr}_{\mathrm{o}}^{2+}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{o}}^{2+}\) \end{document} act together to inhibit bone resorption in strontium ranelate-treated patients.

The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis

Intracellular transduction pathways that are dependent on activation of the CaR by Cao2+ have been studied extensively in parathyroid and other cell types, and include cytosolic calcium, phospholipases C, A2, and D, protein kinase C isoforms and the cAMP/protein kinase A system. In this study, using bone marrow cells isolated from CaR−/− mice as well as DN‐CaR‐transfected RAW 264.7 cells, we provide evidence that expression of the CaR plays an important role in osteoclast differentiation. We also establish that activation of the CaR and resultant stimulation of PLC are involved in high Cao2+‐induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Cao2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF‐κB in a CaR‐ and PLC‐dependent manner. In summary, our data suggest that stimulation of the CaR may play a pivotal role in the control of both osteoclast differentiation and apoptosis in the systems studied here through a signaling pathway involving activation of the CaR, phospholipase C, and NF‐κB.— Mentaverri, R., Yano, S., Chattopadhyay, N., Petit, L., Kifor, O., Kamel, S., Terwilliger, E. F., Brazier, M., Brown, E. M. The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis. FASEB J. 20, E1945‐E1954 (2006)

Recombinant Adeno-Associated Virus Vectors Efficiently and Persistently Transduce Chondrocytes in Normal and Osteoarthritic Human Articular Cartilage

Successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. In the present study we tested the hypothesis that recombinant adeno-associated virus (rAAV) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. Using an rAAV vector carrying the Escherichia coli beta-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter/enhancer (rAAV-lacZ), transduction efficiency exceeded 70% for isolated normal human adult articular chondrocytes, and osteoarthritic human articular chondrocytes. These were comparable to the transduction efficiency obtained with neonatal bovine articular chondrocytes. Transduction of explant cultures of articular cartilage resulted in reporter gene expression within the tissue of all three cartilage types to a depth exceeding 450 microm, which remained present until 150 days. When rAAV-lacZ vectors were applied to femoral chondral defects and osteochondral defects in vivo in a rat knee model, reporter gene expression was achieved for at least 10 days after transduction. These data suggest that AAV-based vectors can efficiently transduce and stably express foreign genes in articular chondrocytes, including chondrocytes of normal and osteoarthritic human articular cartilage. The data further suggest that the same rAAV vectors are capable of transducing chondrocytes in situ within their native matrix to a depth sufficient to be of potential clinical significance. Finally, the data demonstrate that these rAAV vectors are capable of effectively delivering recombinant genes to chondral and osteochondral defects in vivo.

Cocaine-Induced Chromatin Remodeling Increases Brain-Derived Neurotrophic Factor Transcription in the Rat Medial Prefrontal Cortex, Which Alters the Reinforcing Efficacy of Cocaine

Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.

Viral vector-mediated gene transfer of the vole V1a vasopressin receptor in the rat septum: improved social discrimination and active social behaviour

This study explores the effects of enhancing vasopressin V1a receptor expression in the septum using viral vector‐mediated gene transfer on social discrimination and social interactions. Bilateral infusion of an adeno‐associated viral vector containing the prairie vole V1a receptor gene (V1aR‐AAV) regulated by a neuron‐specific enolase promoter resulted in a stable increase in V1a receptor binding density in the rat septum without affecting oxytocin receptor density. Control animals were infused with a vector expressing the lacZ gene. In a social discrimination paradigm, only V1aR‐AAV‐treated animals succeeded in discriminating a previously encountered from a novel juvenile after an interexposure interval (IEI) of more than 2 h, demonstrating the functional incorporation of the vole V1a receptor in the rat septal circuits underlying short‐term memory processes. Microdialysis administration of synthetic vasopressin during the first juvenile exposure, used to mimic intraseptal release patterns of the neuropeptide, produced similar prolongations in recognition (up to an IEI of 24 h) in both V1aR‐AAV and control animals. Septal microdialysis administration of a selective V1a, but not oxytocin, receptor antagonist in both groups prevented discrimination even after an IEI of as short as 0.5 h, confirming the specificity of the vole V1a receptor involvement in social discrimination abilities. In addition, active social interactions were found to be increased among V1aR‐AAV rats compared to controls. Viral vector‐mediated gene transfer provides a valuable tool for studies on the role of localized gene expression on behavioural parameters.