A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell-type-, and subcellular compartment-specific manner. We therefore developed a novel approach combining cell-specific physiological and anatomical characterization with superresolution imaging, and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically-projecting GABAergic interneurons possess increased CB1 receptor number, active-zone complexity, and receptor/effector ratio compared to dendritically-projecting interneurons, in agreement with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked dramatic CB1-downregulation in a dose-dependent manner. Full receptor recovery required several weeks after cessation of Δ9-tetrahydrocannabinol treatment. These findings demonstrate that cell-type-specific nanoscale analysis of endogenous protein distribution is possible in brain circuits, and identify novel molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.

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The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, is active in phagocytosis and delivers antigen for presentation. We have previously shown that transgenic (tg) mice that have been created to overexpress bovine FcRn (bFcRn) demonstrate increased half-life of mouse IgG, significantly increased antigen-specific IgG in serum and augmented expansion of antigen-specific B cells and plasma cells after immunization. One of the interesting questions surrounding this enhanced immune response is whether these tg mice could effectively induce immune response to weakly immunogenic antigens. To address this question, we immunized these bFcRn tg mice with a conserved hemagglutinin subunit 2 (HA2)-based synthetic peptide that was recently found to be effectively targeted by neutralizing antibodies. Using an ELISA system, we found that, whereas wild-type mice showed a weak immune response and developed only a de minimis amount of antibody against the epitope, FcRn over-expressing animals mounted a robust reaction expressed in specific antibody titers on day 28 that continued to rise through day 50. Consistent with our previous data, the enhanced immune response resulting from the FcRn overexpression was also associated with a substantial increase in the number of spleen derived B cells, dendritic cells, granulocytes and plasma cells. Based on this evidence, we propose that tg mice that overexpress bFcRn offer major advantages in monoclonal antibody production because the tg mice would allow the generation of antibodies (hybridomas) to weakly immunogenic antigens that otherwise would be difficult or even impossible to make.

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.

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The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress the bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In the current study, we analyzed the localization of the bFcRn in secondary lymphoid organs, and focused to demonstrate the in vivo impact of its overexpression in the spleen on the course of antibody production. bFcRn was highly expressed by red pulp macrophages and marginal zone macrophages in the spleen and by subcapsular sinus macrophages and macrophage-like cells in the interfollicular areas in the lymph node cortex. We also demonstrated that splenic dendritic cells of Tg mice express bFcRn and intraperitoneal immunization of these mice with T-dependent antigens led to more than threefold increase in the number of antigen-specific activated T helper cells with increased size and numbers of germinal centers compared to wild-type controls. bFcRn expression in splenic B cells was also detected and that may also contribute to the enhanced B cell activation. Finally, we demonstrated that these Tg mice developed efficient immune response against very low dose of antigen, reflecting another important practical benefit of these Tg mice.

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.