In Search of the Ever-Elusive Positive Endozepine
Authors: Stephen C. Harward, James O. McNamara
Journal: Neuron (2013)

In this issue of Neuron, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar provide functional evidence for positive endozepines (positive allosteric modulators of GABAARs) within the thalamic reticular nucleus. These molecules are encoded by the Dbi gene and modulate thalamocortical oscillations. In this issue of Neuron, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar provide functional evidence for positive endozepines (positive allosteric modulators of GABAARs) within the thalamic reticular nucleus. These molecules are encoded by the Dbi gene and modulate thalamocortical oscillations. Since their initial discovery over 50 years ago, benzodiazepines have become one of the most commonly prescribed medications in the fields of Psychiatry and Neurology. Thanks to their ease of administration (orally), potency, efficacy, and low toxicity, benzodiazepines are widely used as anti-anxiety, anticonvulsant, sedative, and muscle-relaxing agents. One mechanism by which these medications mediate their effect involves increasing the duration of inhibitory postsynaptic currents (IPSCs) through GABAARs, thereby enhancing inhibitory synaptic transmission (Mody et al., 1994Mody I. De Koninck Y. Otis T.S. Soltesz I. Trends Neurosci. 1994; 17: 517-525Abstract Full Text PDF PubMed Scopus (544) Google Scholar). Biochemical studies have revealed the presence of a benzodiazepine binding site, termed the benzodiazepine receptor (BR), within GABAARs to which benzodiazepines can bind and mediate their pharmacologic effects (Braestrup and Squires, 1977Braestrup C. Squires R.F. Proc. Natl. Acad. Sci. USA. 1977; 74: 3805-3809Crossref PubMed Scopus (1179) Google Scholar, Möhler and Okada, 1977Möhler H. Okada T. Science. 1977; 198: 849-851Crossref PubMed Scopus (1392) Google Scholar). It turns out that benzodiazepines are not the only molecule able to bind to the BR within GABAARs. In fact, a diversity of small molecules can bind this site and produce a wide array of effects. Classically, these effects are divided into three categories: (1) positive allosteric modulators (PAMs) like the traditional benzodiazepines that enhance GABAR-mediated function; (2) negative allosteric modulators (NAMs), such as beta-carbolines, that reduce GABAR-mediated function; and (3) antagonists, such as flumazenil, that block the actions of both PAMs and NAMs by competing with them for access to the BR (Braestrup et al., 1980Braestrup C. Nielsen M. Olsen C.E. Proc. Natl. Acad. Sci. USA. 1980; 77: 2288-2292Crossref PubMed Scopus (525) Google Scholar, Hunkeler et al., 1981Hunkeler W. Möhler H. Pieri L. Polc P. Bonetti E.P. Cumin R. Schaffner R. Haefely W. Nature. 1981; 290: 514-516Crossref PubMed Scopus (1050) Google Scholar, Mody et al., 1994Mody I. De Koninck Y. Otis T.S. Soltesz I. Trends Neurosci. 1994; 17: 517-525Abstract Full Text PDF PubMed Scopus (544) Google Scholar). The discovery of the BR within GABAARs led to the hypothesis that the CNS produces endogenous molecules that bind to this site and serve as allosteric modulators of GABAARs—molecules that have been referred to as “endozepines” (Iversen, 1977Iversen L. Nature. 1977; 266: 678Google Scholar). This hypothesis in turn led to the discovery of a 10 kDa protein termed diazepam binding inhibitor (DBI), also known as acyl-CoA binding protein (Knudsen, 1991Knudsen J. Neuropharmacology. 1991; 30: 1405-1410Crossref PubMed Scopus (31) Google Scholar). Elimination of the gene encoding this protein has been linked to negative allosteric modulatory effects on GABAARs, one consequence of which is to promote neurogenesis postnatally in the subventricular zone (Alfonso et al., 2012Alfonso J. Le Magueresse C. Zuccotti A. Khodosevich K. Monyer H. Cell Stem Cell. 2012; 10: 76-87Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This success in identification of endogenous NAMs notwithstanding, discovery of endogenous PAMs has proven more challenging. Antagonists of the BR reduce GABA-mediated IPSCs recorded from acutely isolated hippocampal slices and cultured cortical neurons (King et al., 1985King G.L. Knox J.J. Dingledine R. Neuroscience. 1985; 15: 371-378Crossref PubMed Scopus (32) Google Scholar, Vicini et al., 1986Vicini S. Alho H. Costa E. Mienville J.M. Santi M.R. Vaccarino F.M. Proc. Natl. Acad. Sci. USA. 1986; 83: 9269-9273Crossref PubMed Scopus (55) Google Scholar). These findings are consistent with the presence of an endogenous PAM. However, these results could also be explained by negative modulatory effects of these compounds on GABAARs, thus precluding a definitive conclusion. In this issue of Neuron, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar continue the search for an endogenous PAM. Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar focus their search within a single thalamic nucleus—the reticular nucleus (nRT). The nRT plays a critical gating role in oscillatory firing between thalamic and cortical circuits (Steriade et al., 1993Steriade M. McCormick D.A. Sejnowski T.J. Science. 1993; 262: 679-685Crossref PubMed Scopus (2748) Google Scholar). Synaptic inhibition intrinsic to nRT functions to control these oscillations and a reduction of such inhibition manifests as epileptiform oscillations that promote absence seizures (Sohal and Huguenard, 2003Sohal V.S. Huguenard J.R. J. Neurosci. 2003; 23: 8978-8988PubMed Google Scholar). Interestingly, benzodiazepines can suppress these thalamocortical oscillations by enhancing inhibition within nRT (Sohal et al., 2003Sohal V.S. Keist R. Rudolph U. Huguenard J.R. J. Neurosci. 2003; 23: 3649-3657PubMed Google Scholar). Furthermore, humans with a mutation of the γ2 subunit of GABAARs that disrupts the BR commonly develop absence seizures (Wallace et al., 2001Wallace R.H. Marini C. Petrou S. Harkin L.A. Bowser D.N. Panchal R.G. Williams D.A. Sutherland G.R. Mulley J.C. Scheffer I.E. Berkovic S.F. Nat. Genet. 2001; 28: 49-52Crossref PubMed Google Scholar). Together, these observations led Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar to hypothesize that a PAM of GABAARs resides within the nRT and that it functions to enhance synaptic inhibition, thereby limiting thalamocortical oscillations. In pursuit of this hypothesis, several key findings emerged. First, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar studied mutant animals with a point mutation of the α3 subunit of GABAAR (α3(H126R)) which disrupts the BR. Whole-cell recordings from neurons within nRT revealed reduced duration of both spontaneous ISPCs (sIPSCs) and evoked IPSCs (eIPSCs) in slices from mutant animals compared to wild-type controls. Responses of outside-out patches from WT and mutant nRT cells to laser-evoked GABA uncaging were similar, arguing that differences in GABA affinity, chloride conductance, or GABAAR expression did not account for the differences observed in IPSCs. Moreover, a BR antagonist reduced duration of IPSCs in nRT cells of slices of wild-type but not mutant animals. These findings are consistent with the presence of an endogenous PAM within nRT of wild-type mice. Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar provide additional convincing evidence of a PAM residing within nRT by examining an adjacent thalamic nucleus—the ventrobasal (VB) nucleus. In contrast to neurons within nRT, a BR antagonist had no effect on the duration of IPSCs of neurons within VB. Might this be due to differences in the nature of GABAARs in the two nuclei? Or might PAM activity be present within nRT but not VB? To distinguish these possibilities, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar performed an elegant series of experiments combining “sniffer patches” with GABA uncaging. Outside-out membrane patches containing GABAARs were obtained from VB cells and then placed into either VB or nRT within thalamic slices. Moving the patches from VB to nRT resulted in an increased duration of the GABA response within nRT compared to VB. These results exclude the possibility that differences in composition of GABAARs are sufficient to account for the different responses to the BR antagonist in VB compared to nRT. Instead the results provide powerful support for the presence of a PAM within nRT. In search of the molecular identity of the PAM, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar explore DBI—a protein that is highly expressed in nRT and has previously been shown to bind the BR of GABAARs. Using a mouse lacking a 400 kb region of chromosome 1 (nm1054) containing the Dbi gene plus several others, Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar detect a reduction of sIPSC duration in the mutant animal compared to wild-type controls. These findings are similar to those observed in mice with a disrupted BR (α3(H126R)). Importantly, the reduced IPSC duration was rescued by viral expression of Dbi, demonstrating that loss of this gene in particular is sufficient to account for the reduced IPSC duration. These findings provide strong evidence that the Dbi gene encodes the endogenous PAM within nRT. The fact that inhibition within nRT plays a critical role in regulating thalamic oscillations led Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar to query whether the reduced duration of IPSCs within nRT neurons might be associated with enhanced sensitivity to absence seizures in a chemoconvulsant model. Indeed, enhanced sensitivity to chemoconvulsant-induced seizures was detected in mice lacking the Dbi gene (nm1054 mice). Similarly, mice with a disrupted BR in their GABAARs (α3(H126R)) exhibited prolonged epileptiform activity in response to the chemoconvulsant. These findings are consistent with the proposal that an endogenous PAM within nRT, specifically encoded by the Dbi gene, reduces susceptibility to absence seizures by enhancing GABAAR function. In sum, this lovely series of experiments establishes the presence of a PAM of GABAAR function that acts through the BR. Additionally, this work narrows the molecular identity of this PAM to a product of a single gene—Dbi; that said, unanswered questions persist. Christian et al., 2013Christian C.A. Herbert A.G. Holt R.L. Peng K. Sherwood K.D. Pangratz-Fuehrer S. Rudolph U. Huguenard J.R. Neuron. 2013; 78 (this issue): 1063-1074Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar’s immunohistochemical study reveals expression of the Dbi gene within both VB and nRT, yet PAM effects are detectable only within nRT. Moreover, in contrast to the PAM effects linked to the Dbi gene in the present study, NAM effects have been linked to the Dbi gene in work by Alfonso et al., 2012Alfonso J. Le Magueresse C. Zuccotti A. Khodosevich K. Monyer H. Cell Stem Cell. 2012; 10: 76-87Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar. How can products of the same gene function as both NAMs and PAMs (Figure 1)? Do distinct peptides derived from the Dbi gene mediate these opposing effects? Or might distinct posttranslational modifications of the same peptide result in opposing effects? And from what cells are these peptides released and how? Answers to these questions promise to inform how GABAARs function in the healthy nervous system. Additionally, as demonstrated in this present study, disordered function of PAMs and/or NAMs may contribute to some diseases of the nervous system. As such, these PAMs and NAMs may provide novel targets for new classes of pharmacological agents that modulate GABAAR function similar to benzodiazepines but ideally without the tolerance and dependence associated with chronic benozdiazpine use. Endogenous Positive Allosteric Modulation of GABAA Receptors by Diazepam binding inhibitorChristian et al.NeuronMay 30, 2013In BriefFunctional evidence for endogenous positive allosteric modulation mimicking benzodiazepine actions on GABAA receptors has been elusive. Christian et al. demonstrate that diazepam binding inhibitor mediates an endogenous potentiation of GABAA receptor currents in the thalamic reticular nucleus that is antiepileptic. 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