Summary Forebrain circuits rely upon a relatively small but remarkably diverse population of GABAergic interneurons to bind and entrain large principal cell assemblies for network synchronization and rhythmogenesis. Despite the high degree of heterogeneity across cortical interneurons, members of a given subtype typically exhibit homogeneous developmental origins, neuromodulatory response profiles, morphological characteristics, neurochemical signatures, and electrical features. Here we report a surprising divergence amongst hippocampal oriens-lacunosum moleculare (O-LM) projecting interneurons that have hitherto been considered a homogeneous cell population. Combined immunocytochemical, anatomical, and electrophysiological interrogation of Htr3a-GFP and Nkx2-1-cre:RCE mice revealed that O-LM cells parse into caudal ganglionic eminence-derived 5-HT3AR-expressing, and medial ganglionic eminence- derived 5-HT3AR-lacking subpopulations. These two cohorts differentially participate in network oscillations with 5-HT3AR-containing O-LM cell recruitment dictated by serotonergic tone. Thus, members of a seemingly uniform interneuron population can exhibit unique circuit functions and neuromodulatory properties dictated by disparate developmental origins.

Nicotinic acetylcholine receptors are implicated in several neuropsychiatric disorders, including nicotine addiction, Alzheimer's, schizophrenia, and depression. Therefore, they represent a critical molecular target for drug development and targeted therapeutic intervention. Understanding the molecular mechanisms by which allosteric modulators enhance activation of these receptors is crucial to the development of new drugs. We used the substituted cysteine accessibility method to study conformational changes induced by the positive allosteric modulator N- (5-chloro-2,4-dimethoxyphenyl)-NЈ-(5-methyl-3-isoxazolyl)-urea (PNU-120596) in the extracellular ligand binding domain of ␣7 nicotinic receptors carrying the L247T mutation. PNU-120596 caused changes in cysteine accessibility at the inner beta sheet, transition zone, and agonist binding site. These changes in accessibility are similar to but not identical to those caused by ACh alone. In particular, PNU-120596 induced changes in MTSEA accessibility at N170C (in the transition zone) that were substantially different from those evoked by acetylcholine (ACh). We found that PNU-120596 induced changes at position E172C in the absence of allosteric modulation. We identified a cysteine mutation of the agonist binding site (W148C) that exhibited an unexpected phenotype in which PNU-120596 acts as a full agonist. In this mutant, ACh-evoked currents were more sensitive to thiol modification than PNUevoked currents, suggesting that PNU-120596 does not bind at unoccupied agonist-binding sites. Our results provide evidence that binding sites for PNU-120596 are not in the agonist-binding sites and demonstrate that positive allosteric modulators such as PNU-120596 enhance agonist-evoked gating of nicotinic receptors by eliciting conformational effects that are similar but nonidentical to the gating conformations promoted by ACh.Nicotinic acetylcholine receptors (nAChRs) are the prototypical member of the Cys-loop family of ligand-gated ion channels that also includes GABA A , serotonin type 3 (5-HT 3 ), and glycine receptors. This family of receptors assembles as heteromeric or homomeric pentamers around a central pore (Karlin, 2002). Each subunit contains an extracellular ligand-binding domain (LBD), an ␣-helical transmembrane domain (TMD), a transition zone that couples the LBD to the TMD, and an intracellular domain (Gay and Yakel, 2007).Neuronal nAChRs are expressed diffusely throughout most of the central nervous system; ␣7-containing receptors show the highest levels of expression (Orr-Urtreger et al., 1997). Of the neuronal nicotinic receptors, the homomeric ␣7 receptor is implicated in neurological diseases such as schizophrenia, Alzheimer's Disease, and anxiety disorders (Gotti and Clementi, 2004). Therefore, the ␣7 nicotinic receptor represents an important therapeutic target.

Allosteric modulation of membrane receptors is a widespread mechanism by which endogenous and exogenous agents regulate receptor function. For example, several members of the nicotinic receptor family are modulated by physiological concentrations of extracellular calcium ions. In this paper, we examined conformational changes underlying this modulation and compare these with changes evoked by ACh. Two sets of residues in the 7 acetylcholine receptor extracellular domain were mutated to cysteine and analyzed by measuring the rates of modification by the thiolspecific reagent 2-aminoethylmethane thiosulfonate. Using Ba 2+ as a surrogate for Ca 2+ , we found a divalent-dependent decrease the modification rates of cysteine substitutions at M 37 and M 40 , residues at which rates were also slowed by ACh. In contrast, Ba 2+ had no significant effect at N 52 C, a residue where ACh increased the rate of modification. Thus divalent modulators cause some but not all of the conformational effects elicited by agonist. Cysteine substitution of either of two glutamates (E 44 or E 172 ), thought to participate in the divalent cation binding site, caused a loss of allosteric modulation, yet Ba 2+ still had a significant effect on modification rates of these residues. In addition, the effect of Ba 2+ at these residues did not appear to be due to direct occlusion. Our data demonstrate that modulation by divalent cations involves substantial conformational changes in the receptor extracellular domain. Our evidence also suggests the modulation occurs via a binding site distinct from one which includes either (or both) of the conserved glutamates at E 44 or E 172 .

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