Induction of dendritic spines by ß2-containing nicotinic receptors.

Glutamatergic synapses are located mostly on dendritic spines in the adult nervous system. The spines serve as postsynaptic compartments, containing components that mediate and control the synaptic signal. Early in development, when glutamatergic synapses are initially forming, waves of excitatory activity pass through many parts of the nervous system and are driven in part by a class of heteropentameric ß2-containing nicotinic acetylcholine receptors (ß2*-nAChRs). These ß2*-nAChRs are widely distributed and, when activated, can depolarize the membrane and elevate intracellular calcium levels in neurons. We show here that ß2*-nAChRs are essential for acquisition of normal numbers of dendritic spines during development. Mice constitutively lacking the ß2-nAChR gene have fewer dendritic spines than do age-matched wild-type mice at all times examined. Activation of ß2*-nAChRs by nicotine either in vivo or in organotypic slice culture quickly elevates the number of spines. RNA interference studies both in vivo and in organotypic culture demonstrate that the ß2*-nAChRs act in a cell-autonomous manner to increase the number of spines. The increase depends on intracellular calcium and activation of calcium, calmodulin-dependent protein kinase II. Absence of ß2*-nAChRs in vivo causes a disproportionate number of glutamatergic synapses to be localized on dendritic shafts, rather than on spines as occurs in wild type. This shift in synapse location is found both in the hippocampus and cortex, indicating the breadth of the effect. Because spine synapses differ from shaft synapses in their signaling capabilities, the shift observed is likely to have significant consequences for network function.

Glutamatergic synapse formation is promoted by α7-containing nicotinic acetylcholine receptors.

Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type (WT) mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in WT organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover, the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction.

Overcoming in vivo barriers to targeted nanodelivery.

Nanoparticles have been investigated as promising nanocarriers for delivery of imaging and therapeutic agents for several decades, but have met with limited success. Although enormous progress in the fields of nanotechnology and nanoscience has been achieved, basic discoveries have not yet translated into effective targeted therapies. Nanoparticles can potentially improve the pharmacokinetics and pharmacodynamics of drugs; however, the complexity of in vivo systems imposes multiple barriers that severely inhibit efficiency and have to be overcome to fully exploit the theoretical potential of nanoparticles. Here, we address two major challenges to effective systemic nanodelivery. Both limited penetration across the vascular endothelium and uptake by the reticuloendothelial system (RES) substantially impede effectiveness of nanoparticle delivery into tissues. Although the design of nanoparticles with extended circulation half-life is essential, it is not sufficient for effective penetration of nanoparticles across the formidable barrier formed by the vascular endothelium. Current nanodelivery systems rely on passive transvascular exchange and tissue accumulation. They require high dosages to create large concentration gradients that drive nanoparticles passively across the blood-tissue interface. However, passive accumulation has resulted in only a fractional dosage of nanoparticles penetrating into target tissue. This inevitably diminishes therapeutic efficacy and aggravates potential side effects. Although there are multiple ways to augment passive delivery, active delivery of targeted nanoparticles across the vascular endothelium could significantly increase the therapeutic index and decrease side effects of nanoparticle-based drug delivery systems. Use of active transendothelial transport pathways, such as caveolae, may provide an effective solution to both target and deliver nanoparticles.

Caveolae and cancer.

All blood vessels are lined by a layer of endothelial cells that help to control vascular permeability. The luminal surface of vascular endothelial cells is studded with transport vesicles called caveolae that are directly in contact with the blood and can transport molecules into and across the endothelium. The vasculature within distinct tissue types expresses a unique array of proteins that can be used to target intravenously injected antibodies directly to that tissue. When the tissue-specific proteins are concentrated in caveolae, the antibodies can be rapidly pumped out of the blood and into the tissue. Tumors appear to be a distinct tissue type with their own unique marker proteins. Targeting accessible proteins at the surface of tumor vasculature with radiolabeled antibodies destroys tumors and drastically increases animal survival. One day, it may be possible to specifically pump targeted molecules into tumors. This could increase therapeutic efficacy and decrease side effects because most of the drug would accumulate specifically in the tumor. Thus, targeting caveolae may provide a universal portal to pump drugs, imaging agents, and gene vectors out of the blood and into underlying tissue.

Targeting and imaging signature caveolar molecules in lungs.

A major goal of molecular medicine is to target imaging agents or therapeutic compounds to a single organ. Targeting imaging agents to a single organ could facilitate the high-resolution, in vivo imaging of molecular events. In addition, genetic and acquired diseases primary to a single organ, such as cystic fibrosis, tuberculosis, lung cancer, pulmonary fibrosis, pulmonary hypertension, and acute respiratory distress syndrome, could be specifically targeted in the lung. By targeting and concentrating imaging agents or therapeutics to the lungs, deleterious side effects can be avoided with greater efficacy at much lower dosages. Pathologic changes can be identified earlier and followed over time. In addition, therapeutics that have been abandoned due to toxicities may find renewed utility when coupled with specific targeting agents such as antibodies. To achieve these goals, distinct molecular signatures must be found for each organ or disease-state.

BDNF up-regulates alpha7 nicotinic acetylcholine receptor levels on subpopulations of hippocampal interneurons.

In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing alpha7 subunits (alpha7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of alpha7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABA(A) receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased alpha7-nAChR clusters were most prominent on interneuron subtypes known to directly innervate excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling alpha7-nAChR levels.

Nicotinic activity stabilizes convergence of nicotinic and GABAergic synapses on filopodia of hippocampal interneurons.

Nicotinic acetylcholine receptors containing alpha7 subunits occupy pre- and postsynaptic sites in the adult hippocampus. We find that embryonic hippocampal slices in culture display the receptors most prominently on interneurons where they form clusters localized in part on filopodia. The receptors often co-distribute specifically with GABAA receptors. In septal-hippocampal co-cultures, the filopodia become co-innervated by cholinergic and GABAergic terminals abutting the receptor clusters. Nicotinic transmission appears to stabilize the cholinergic contacts: pharmacological blockade of the alpha7-containing nicotinic receptors increases the rate of filopodia movement and decreases the incidence of the clusters being adjacent to cholinergic terminals. Immunostaining fresh hippocampal slices from neonatal rat pups confirms that cholinergic and GABAergic terminals contact alpha7-containing nicotinic receptor clusters in vivo, and the clusters appear to include filopodial sites. The results indicate a convergence of nicotinic and GABAergic input at specific sites on developing hippocampal interneurons and suggest that synaptic activity helps stabilize the nicotinic contribution.