Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1ß2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram ß-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

D3 Receptors Regulate Excitability in a Unique Class of Prefrontal Pyramidal Cells.

The D3 dopamine receptor, a member of the Gi-coupled D2 family of dopamine receptors, is expressed throughout limbic circuits affected in neuropsychiatric disorders, including prefrontal cortex (PFC). These receptors are important for prefrontal executive function because pharmacological and genetic manipulations that affect prefrontal D3 receptors alter anxiety, social interaction, and reversal learning. However, the mechanisms by which D3 receptors regulate prefrontal circuits and whether D3 receptors regulate specific prefrontal subnetworks remains unknown. Here, we combine dopamine receptor reporter lines, anatomical tracing techniques, and electrophysiology to show that D3 receptor expression defines a novel subclass of layer 5 glutamatergic pyramidal cell in mouse PFC (either sex). D3-receptor-expressing pyramidal neurons are electrophysiologically and anatomically separable from neighboring neurons expressing D1 or D2 receptors based on their dendritic morphology and subthreshold and suprathreshold intrinsic excitability. D3-receptor-expressing neurons send axonal projections to intratelencephalic (IT) targets, including contralateral cortex, nucleus accumbens, and basolateral amygdala. Within these neurons, D3 receptor activation was found to regulate low-voltage-activated CaV3.2 calcium channels localized to the axon initial segment, which suppressed action potential (AP) excitability, particularly when APs occurred at high frequency. Therefore, these data indicate that D3 receptors regulate the excitability of a unique, IT prefrontal cell population, thereby defining novel circuitry and cellular actions for D3 receptors in PFC.SIGNIFICANCE STATEMENT The D3 dopamine receptor, a member of the Gi-coupled D2 family of dopamine receptors, are expressed throughout limbic circuits, including prefrontal cortex (PFC). They are of broad interest as a site for therapeutic intervention in serious mental illness, yet we know very little about their distribution or function within PFC. Here, we show that D3 receptors define a unique population of glutamatergic principal cells in mouse PFC that largely lack expression of D1 or D2 receptors. Within these cells, we find that D3 receptors regulate the ability to generate high-frequency action potential bursts through mechanisms not supported by other dopamine receptors. These results define unique circuitry and cellular actions for D3 receptors in regulating PFC networks.

Distinct cortical and striatal actions of a ß-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed ß-arrestin2 (ßarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific ßarr2-KO mice, we show that the antipsychotic-like effects of a ßarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-ßarr2 signaling. Furthermore, ßarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of ßarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that ßarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

Pyramidal neurons in prefrontal cortex receive subtype-specific forms of excitation and inhibition.

Layer 5 pyramidal neurons comprise at least two subtypes: thick-tufted, subcortically projecting type A neurons, with prominent h-current, and thin-tufted, callosally projecting type B neurons, which lack prominent h-current. Using optogenetic stimulation, we find that these subtypes receive distinct forms of input that could subserve divergent functions. Repeatedly stimulating callosal inputs evokes progressively smaller excitatory responses in type B but not type A neurons. Callosal inputs also elicit more spikes in type A neurons. Surprisingly, these effects arise via distinct mechanisms. Differences in the dynamics of excitatory responses seem to reflect differences in presynaptic input, whereas differences in spiking depend on postsynaptic mechanisms. We also find that fast-spiking parvalbumin interneurons, but not somatostatin interneurons, preferentially inhibit type A neurons, leading to greater feedforward inhibition in this subtype. These differences may enable type A neurons to detect salient inputs that are focused in space and time, while type B neurons integrate across these dimensions.

Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.

VIDEO ABSTRACT: The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry.

Alterations in cortical excitation and inhibition in genetic mouse models of Huntington's disease.

Previously, we identified progressive alterations in spontaneous EPSCs and IPSCs in the striatum of the R6/2 mouse model of Huntington's disease (HD). Medium-sized spiny neurons from these mice displayed a lower frequency of EPSCs, and a population of cells exhibited an increased frequency of IPSCs beginning at approximately 40 d, a time point when the overt behavioral phenotype begins. The cortex provides the major excitatory drive to the striatum and is affected during disease progression. We examined spontaneous EPSCs and IPSCs of somatosensory cortical pyramidal neurons in layers II/III in slices from three different mouse models of HD: the R6/2, the YAC128, and the CAG140 knock-in. Results revealed that spontaneous EPSCs occurred at a higher frequency, and evoked EPSCs were larger in behaviorally phenotypic mice whereas spontaneous IPSCs were initially increased in frequency in all models and subsequently decreased in R6/2 mice after they displayed the typical R6/2 overt behavioral phenotype. Changes in miniature IPSCs and evoked IPSC paired-pulse ratios suggested altered probability of GABA release. Also, in R6/2 mice, blockade of GABA(A) receptors induced complex discharges in slices and seizures in vivo at all ages. In conclusion, altered excitatory and inhibitory inputs to pyramidal neurons in the cortex in HD appear to be a prevailing deficit throughout the development of the disease. Furthermore, the differences between synaptic phenotypes in cortex and striatum are important for the development of future therapeutic approaches, which may need to be targeted early in the development of the phenotype.