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.

GABAergic cell transplants in the anterior cingulate cortex reduce neuropathic pain aversiveness.

Dysfunction of inhibitory circuits in the rostral anterior cingulate cortex underlies the affective (aversive), but not the sensory-discriminative features (hypersensitivity) of the pain experience. To restore inhibitory controls, we transplanted inhibitory interneuron progenitor cells into the rostral anterior cingulate cortex in a chemotherapy-induced neuropathic pain model. The transplants integrated, exerted a GABA-A mediated inhibition of host pyramidal cells and blocked gabapentin preference (i.e. relieved ongoing pain) in a conditioned place preference paradigm. Surprisingly, pain aversiveness persisted when the transplants populated both the rostral and posterior anterior cingulate cortex. We conclude that selective and long lasting inhibition of the rostral anterior cingulate cortex, in the mouse, has a profound pain relieving effect against nerve injury-induced neuropathic pain. However, the interplay between the rostral and posterior anterior cingulate cortices must be considered when examining circuits that influence ongoing pain and pain aversiveness.

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.

Synaptic activity unmasks dopamine D2 receptor modulation of a specific class of layer V pyramidal neurons in prefrontal cortex.

Dopamine D2 receptors (D2Rs) play a major role in the function of the prefrontal cortex (PFC), and may contribute to prefrontal dysfunction in conditions such as schizophrenia. Here we report that in mouse PFC, D2Rs are selectively expressed by a subtype of layer V pyramidal neurons that have thick apical tufts, prominent h-current, and subcortical projections. Within this subpopulation, the D2R agonist quinpirole elicits a novel afterdepolarization that generates voltage fluctuations and spiking for hundreds of milliseconds. Surprisingly, this afterdepolarization is masked in quiescent brain slices, but is readily unmasked by physiologic levels of synaptic input which activate NMDA receptors, possibly explaining why this phenomenon has not been reported previously. Notably, we could still elicit this afterdepolarization for some time after the cessation of synaptic stimulation. In addition to NMDA receptors, the quinpirole-induced afterdepolarization also depended on L-type Ca(2+) channels and was blocked by the selective L-type antagonist nimodipine. To confirm that D2Rs can elicit this afterdepolarization by enhancing Ca(2+) (and Ca(2+)-dependent) currents, we measured whole-cell Ca(2+) potentials that occur after blocking Na(+) and K(+) channels, and found quinpirole enhanced these potentials, while the selective D2R antagonist sulpiride had the opposite effect. Thus, D2Rs can elicit a Ca(2+)-channel-dependent afterdepolarization that powerfully modulates activity in specific prefrontal neurons. Through this mechanism, D2Rs might enhance outputs to subcortical structures, contribute to reward-related persistent firing, or increase the level of noise in prefrontal circuits.

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.

Investigation of food and environmental exposures relating to the epidemiology of Campylobacter coli in humans in Northwest England.

This study uses multilocus sequence typing (MLST) to investigate the epidemiology of Campylobacter coli in a continuous study of a population in Northwest England. All cases of Campylobacter identified in four Local Authorities (government administrative boundaries) between 2003 and 2006 were identified to species level and then typed, using MLST. Epidemiological information was collected for each of these cases, including food and recreational exposure variables, and the epidemiologies of C. jejuni and C. coli were compared using case-case methodology. Samples of surface water thought to represent possible points of exposure to the populations under study were also sampled, and campylobacters were typed with multilocus sequence typing. Patients with C. coli were more likely to be older and female than patients with C. jejuni. In logistic regression, C. coli infection was positively associated with patients eating undercooked eggs, eating out, and reporting problems with their water supply prior to illness. C. coli was less associated with consuming pork products. Most of the cases of C. coli yielded sequence types described elsewhere in both livestock and poultry, but several new sequence types were also identified in human cases and water samples. There was no overlap between types identified in humans and surface waters, and genetic analysis suggested three distinct clades but with several "intermediate" types from water that were convergent with the human clade. There is little evidence to suggest that epidemiological differences between human cases of C. coli and C. jejuni are a result of different food or behavioral exposures alone.

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.

Rapid evolution and the importance of recombination to the gastroenteric pathogen Campylobacter jejuni.

Responsible for the majority of bacterial gastroenteritis in the developed world, Campylobacter jejuni is a pervasive pathogen of humans and animals, but its evolution is obscure. In this paper, we exploit contemporary genetic diversity and empirical evidence to piece together the evolutionary history of C. jejuni and quantify its evolutionary potential. Our combined population genetics-phylogenetics approach reveals a surprising picture. Campylobacter jejuni is a rapidly evolving species, subject to intense purifying selection that purges 60% of novel variation, but possessing a massive evolutionary potential. The low mutation rate is offset by a large effective population size so that a mutation at any site can occur somewhere in the population within the space of a week. Recombination has a fundamental role, generating diversity at twice the rate of de novo mutation, and facilitating gene flow between C. jejuni and its sister species Campylobacter coli. We attempt to calibrate the rate of molecular evolution in C. jejuni based solely on within-species variation. The rates we obtain are up to 1,000 times faster than conventional estimates, placing the C. jejuni-C. coli split at the time of the Neolithic revolution. We weigh the plausibility of such recent bacterial evolution against alternative explanations and discuss the evidence required to settle the issue.

Identification of potential environmentally adapted Campylobacter jejuni strain, United Kingdom.

In a study of Campylobacter infection in northwestern England, 2003-2006, C. jejuni multilocus sequence type (ST)-45 was associated with early summer onset and was the most prevalent C. jejuni type in surface waters. ST-45 is likely more adapted to survival outside a host, making it a key driver of transmission between livestock, environmental, and human settings.

Tracing the source of campylobacteriosis.

Campylobacter jejuni is the leading cause of bacterial gastro-enteritis in the developed world. It is thought to infect 2-3 million people a year in the US alone, at a cost to the economy in excess of US $4 billion. C. jejuni is a widespread zoonotic pathogen that is carried by animals farmed for meat and poultry. A connection with contaminated food is recognized, but C. jejuni is also commonly found in wild animals and water sources. Phylogenetic studies have suggested that genotypes pathogenic to humans bear greatest resemblance to non-livestock isolates. Moreover, seasonal variation in campylobacteriosis bears the hallmarks of water-borne disease, and certain outbreaks have been attributed to contamination of drinking water. As a result, the relative importance of these reservoirs to human disease is controversial. We use multilocus sequence typing to genotype 1,231 cases of C. jejuni isolated from patients in Lancashire, England. By modeling the DNA sequence evolution and zoonotic transmission of C. jejuni between host species and the environment, we assign human cases probabilistically to source populations. Our novel population genetics approach reveals that the vast majority (97%) of sporadic disease can be attributed to animals farmed for meat and poultry. Chicken and cattle are the principal sources of C. jejuni pathogenic to humans, whereas wild animal and environmental sources are responsible for just 3% of disease. Our results imply that the primary transmission route is through the food chain, and suggest that incidence could be dramatically reduced by enhanced on-farm biosecurity or preventing food-borne transmission.

Campylobacter jejuni multilocus sequence types in humans, northwest England, 2003-2004.

Detailed understanding of the epidemiology of Campylobacter is increasingly facilitated through use of universal and reproducible techniques for accurate strain differentiation and subtyping. Multilocus sequence typing (MLST) enables discriminatory subtyping and grouping of isolate types into genetically related clonal complexes; it also has the advantage of ease of application and repeatability. Recent studies suggest that a measure of host association may be distinguishable with this system. We describe the first continuous population-based survey to investigate the potential of MLST to resolve questions of campylobacteriosis epidemiology. We demonstrate the ability of MLST to identify variations in the epidemiology of campylobacteriosis between distinct populations and describe the distribution of key subtypes of interest.