Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.

Hallucinogens, including mescaline, psilocybin, and lysergic acid diethylamide (LSD), profoundly affect perception, cognition, and mood. All known drugs of this class are 5-HT(2A) receptor (2AR) agonists, yet closely related 2AR agonists such as lisuride lack comparable psychoactive properties. Why only certain 2AR agonists are hallucinogens and which neural circuits mediate their effects are poorly understood. By genetically expressing 2AR only in cortex, we show that 2AR-regulated pathways on cortical neurons are sufficient to mediate the signaling pattern and behavioral response to hallucinogens. Hallucinogenic and nonhallucinogenic 2AR agonists both regulate signaling in the same 2AR-expressing cortical neurons. However, the signaling and behavioral responses to the hallucinogens are distinct. While lisuride and LSD both act at 2AR expressed by cortex neurons to regulate phospholipase C, LSD responses also involve pertussis toxin-sensitive heterotrimeric G(i/o) proteins and Src. These studies identify the long-elusive neural and signaling mechanisms responsible for the unique effects of hallucinogens.

Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex.

The majority of neurons in the adult neocortex are produced embryonically during a brief but intense period of neuronal proliferation. The radial glial cell, a transient embryonic cell type known for its crucial role in neuronal migration, has recently been shown to function as a neuronal progenitor cell and appears to produce most cortical pyramidal neurons. Radial glial cell modulation could thus affect neuron production, neuronal migration, and overall cortical architecture; however, signaling mechanisms among radial glia have not been studied directly. We demonstrate here that calcium waves propagate through radial glial cells in the proliferative cortical ventricular zone (VZ). Radial glial calcium waves occur spontaneously and require connexin hemichannels, P2Y1 ATP receptors, and intracellular IP3-mediated calcium release. Furthermore, we show that wave disruption decreases VZ proliferation during the peak of embryonic neurogenesis. Taken together, these results demonstrate a radial glial signaling mechanism that may regulate cortical neuronal production.

Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.

Precise patterns of cell division and migration are crucial to transform the neuroepithelium of the embryonic forebrain into the adult cerebral cortex. Using time-lapse imaging of clonal cells in rat cortex over several generations, we show here that neurons are generated in two proliferative zones by distinct patterns of division. Neurons arise directly from radial glial cells in the ventricular zone (VZ) and indirectly from intermediate progenitor cells in the subventricular zone (SVZ). Furthermore, newborn neurons do not migrate directly to the cortex; instead, most exhibit four distinct phases of migration, including a phase of retrograde movement toward the ventricle before migration to the cortical plate. These findings provide a comprehensive and new view of the dynamics of cortical neurogenesis and migration.

Terpene trilactones from Ginkgo biloba are antagonists of cortical glycine and GABA(A) receptors.

Glycine and gamma-aminobutyric acid, type A (GABA(A)) receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory synaptic transmission in the adult central nervous system. During development, the activation of these receptors leads to membrane depolarization. Ligands for the two receptors have important implications both in disease therapy and as pharmacological tools. Terpene trilactones (ginkgolides and bilobalide) are unique constituents of Ginkgo biloba extracts that have various effects on the central nervous system. We have investigated the relative potency of these compounds on glycine and GABA(A) receptors. We find that most of the ginkgolides are selective and potent antagonists of the glycine receptor. Bilobalide, the single major component in G. biloba extracts, also reduces glycine-induced currents, although to a lesser extent. Both ginkgolides and bilobalide inhibit GABA(A) receptors, with bilobalide demonstrating a more potent effect. Additionally, we provide evidence that open channels are required for glycine receptor inhibition by ginkgolides. Finally, we employ molecular modeling to elucidate the similarities and differences in the structure of the terpene trilactones to account for the pharmacological properties of these compounds and demonstrate a striking similarity between ginkgolides and picrotoxinin, a GABA(A) and recombinant glycine alpha-homomeric receptor antagonist.

Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells.