Bioisosteres of ethyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo [1,5-a][1,4]diazepine-3-carboxylate (HZ-166) as novel alpha 2,3 selective potentiators of GABAA receptors: Improved bioavailability enhances anticonvulsant efficacy.

HZ-166 has previously been characterized as an α2,3-selective GABAA receptor modulator with anticonvulsant, anxiolytic, and anti-nociceptive properties but reduced motor effects. We discovered a series of ester bioisosteres with reduced metabolic liabilities, leading to improved efficacy as anxiolytic-like compounds in rats. In the present study, we evaluated the anticonvulsant effects KRM-II-81 across several rodent models. In some models we also evaluated key structural analogs. KRM-II-81 suppressed hyper-excitation in a network of cultured cortical neurons without affecting the basal neuronal activity. KRM-II-81 was active against electroshock-induced convulsions in mice, pentylenetetrazole (PTZ)-induced convulsions in rats, elevations in PTZ-seizure thresholds, and amygdala-kindled seizures in rats with efficacies greater than that of diazepam. KRM-II-81 was also active in the 6 Hz seizure model in mice. Structural analogs of KRM-II-81 but not the ester, HZ-166, were active in all models in which they were evaluated. We further evaluated KRM-II-81 in human cortical epileptic tissue where it was found to significantly-attenuate picrotoxin- and AP-4-induced increases in firing rate across an electrode array. These molecules generally had a wider margin of separation in potencies to produce anticonvulsant effects vs. motor impairment on an inverted screen test than did diazepam. Ester bioisosters of HZ-166 are thus presented as novel agents for the potential treatment of epilepsy acting via selective positive allosteric amplification of GABAA signaling through α2/α3-containing GABA receptors. The in vivo data from the present study can serve as a guide to dosing parameters that predict engagement of central GABAA receptors.

Genetic dissection of the olfactory bulbs of mice: QTLs on four chromosomes modulate bulb size.

Olfaction is influenced by a complex mix of environmental and genetic factors that modulate the production, migration, and maturation of cells in the olfactory bulbs. In this study we analyzed effects of sex, age, body weight, and brain weight on olfactory bulb size in sexually mature mice. We then used regression corrected values (residuals) to map quantitative trait loci (QTLs) that selectively modulate bulb weight. This biometric analysis has relied on an F2 intercross between C57BL/6J (B6) and DBA/2J (D2) inbred strains and a large sample of 35 BXD recombinant inbred (RI) strains. Bilateral bulb weight in adult mice ranges from 10 to 30 mg. Half of this remarkable variation can be predicted from differences in brain weight, sex, body weight, and age. A 100-mg difference in brain weight is associated with a 4.4-mg difference in bulb weight. Bulbs gain in weight by 0.2 mg/week--a 1% increase that continues until at least 300 days of age. Males tend to have slightly larger bulbs than females. By combining data from both related crosses (F2 and RI) we identified four QTLs with selective effects on bulb size (genomewide p < .05). Bulb4a is located on chromosome 4 (Chr 4) and Bulb6a is located on Chr 6. Alleles inherited from B6 at both of these loci increase bulb weight by 0.5-1.0 mg. Bulb11a is located on proximal Chr 11 and Bulb17a is located on the proximal part of Chr 17. In contrast to the first two QTLs, B6 alleles at these two loci decrease bulb weight by 0.5-1.0 mg. Collectively, the four loci account for 20% of the phenotypic variance in bulb weight.

Genetic control of the mouse cerebellum: identification of quantitative trait loci modulating size and architecture.

To discover genes influencing cerebellum development, we conducted a complex trait analysis of variation in the size of the adult mouse cerebellum. We analyzed two sets of recombinant inbred BXD strains and an F2 intercross of the common inbred strains, C57BL/6J and DBA/2J. We measured cerebellar size as the weight or volume of fixed or histologically processed tissue. Among BXD recombinant inbred strains, the cerebellum averages 52 mg (12.4% of the brain) and ranges 18 mg in size. In F2 mice, the cerebellum averages 62 mg (12.9% of the brain) and ranges approximately 20 mg in size. Five quantitative trait loci (QTLs) that significantly control variation in cerebellar size were mapped to chromosomes 1 (Cbs1a), 8 (Cbs8a), 14 (Cbs14a), and 19 (Cbs19a, Cbs19b). In combination, these QTLs can shift cerebellar size an appreciable 35% of the observed range. To assess regional genetic control of the cerebellum, we also measured the volume of the cell-rich, internal granule layer (IGL) in a set of BXD strains. The IGL ranges from 34 to 43% of total cerebellar volume. The QTL Cbs8a is significantly linked to variation in IGL volume and is suggestively linked to variation in the number of cerebellar folia. The QTLs we have discovered are among the first loci shown to modulate the size and architecture of the adult mouse cerebellum.

Complex trait analysis of the hippocampus: mapping and biometric analysis of two novel gene loci with specific effects on hippocampal structure in mice.

Notable differences in hippocampal structure are associated with intriguing differences in development and behavioral capabilities. We explored genetic and environmental factors that modulate hippocampal size, structure, and cell number using sets of C57BL/6J (B6) and DBA/2J (D2) mice; their F1 and F2 intercrosses (n = 180); and 35 lines of BXD recombinant inbred (RI) strains. Hippocampal weights of the parental strains differ by 20%. Estimates of granule cell number also differ by approximately 20%. Hippocampal weights of RI strains range from 21 to 31 mg, and those of individual F2 mice range from 23 to 36 mg (bilateral weights). Volume and granule cell number are well correlated (r = 0.7-0.8). Significant variation is associated with differences in age and sex. The hippocampus increases in weight by 0.24 mg per month, and those of males are 0.55 mg heavier (bilateral) than those of females. Heritability of variation is approximately 50%, and half of this genetic variation is generated by two quantitative trait loci that map to chromosome 1 (Hipp1a: genome-wide p < 0.005, between 65 and 100 cM) and to chromosome 5 (Hipp5a, p < 0.05, between 15 and 40 cM). These are among the first gene loci known to produce normal variation in forebrain structure. Hipp1a and Hipp5a individually modulate hippocampal weight by 1.0-2.0 mg, an effect size greater than that generated by age or sex. The Hipp gene loci modulate neuron number in the dentate gyrus, collectively shifting the population up or down by as much as 200,000 cells. Candidate genes for the Hipp loci include Rxrg and Fgfr3.

Greater song complexity is associated with augmented song system anatomy in zebra finches.

We revisited the relationship between brain anatomy and song behavior in zebra finches. Consistent with previous studies in other songbirds, we find that differences in volume of the telencephalic song control nucleus HVc is predictive of differences in repertoire size and phrase duration in zebra finches. We extend the study of brain and behavior correlations in song birds by showing that repertoire size in zebra finches can be predicted by variance in several brain nuclei, providing the first demonstration that volumetric differences across multiple components of a neural network are predictive of song behavior.

Song, sexual selection, and a song control nucleus (HVc) in the brains of European sedge warblers.

Female sedge warblers select males that have more complex songs as mates. This study tests two predictions concerning HVc, a telencephalic nucleus that is essential for song learning and production: first, that males with more complex songs will have a larger HVc, and second that males who pair successfully will have a larger HVc than unpaired males. Data on song composition and pairing status were collected from wild sedge warblers breeding in Hungary. We found significant positive correlations between three song attributes (repertoire size, song complexity, and song length) and the size of HVc. Males that paired successfully also had more complex songs (repertoire size and song complexity, though not song length) than males that did not. However, we find no direct evidence that males who paired successfully had a larger HVc than unpaired males. These findings are discussed in relation to the possible functions of HVc and also to current views on sexual selection and the evolution of the song control pathway.

Greater song complexity is associated with augmented song system anatomy in zebra finches.

We revisited the relationship between brain anatomy and song behavior in zebra finches. Consistent with previous studies in other songbirds, we find that differences in volume of the telencephalic song control nucleus HVc is predictive of differences in repertoire size and phrase duration in zebra finches. We extend the study of brain and behavior correlations in song birds by showing that repertoire size in zebra finches can be predicted by variance in several brain nuclei, providing the first demonstration that volumetric differences across multiple components of a neural network are predictive of song behavior.

Differences in the complexity of song tutoring cause differences in the amount learned and in dendritic spine density in a songbird telencephalic song control nucleus.

In our search for relations between vocal learning and neuron structure in the song control nuclei of songbird forebrains, we tested whether differential experience that leads to differences in adult song repertoire would affect dendritic spine density in HVc (also called high vocal center) and RA (robustus archistriatalis). We tape-tutored juvenile Eastern marsh wrens (Cistothorus palustris) with either 5 or 45 song types. As adults, the small repertoire group had learned mostly 5 or 6 song types, and the large repertoire group had learned 36 to 47. Wrens that learned the large song repertoires had a greater dendritic spine density for the most spiny neurons present in HVc (mean difference, 36%), but not in RA. Recent physiological evidence describes HVc as a premotor area coding syllables, motifs, and higher-order song patterns, and our data now clearly reveal that differences in the size of the song repertoire that is experienced lead to differences both in song learning and in the density of dendritic spines in HVc. In the forebrain song nuclei of these songbirds, as in some other vertebrate systems, differences in learning and performance are associated with differences in synaptic anatomy specifically in the region that organizes the learned pattern.

Variation in the volume of zebra finch song control nuclei is heritable: developmental and evolutionary implications.

In many songbird species, females prefer males that sing a larger repertoire of syllables. Males with more elaborate songs have a larger high vocal centre (HVC) nucleus, the highest structure in the song production pathway. HVC size is thus a potential target of sexual selection. Here we provide evidence that the size of the HVC and other song production nuclei are heritable across individual males within a species. In contrast, we find that heritabilities of other nuclei in a song-learning pathway are lower, suggesting that variation in the sizes of these structures is more closely tied to developmental and environmental differences between individuals. We find that evolvability, a statistical measure that predicts response to selection, is higher for the HVC and its target for song production, the robustus archistriatalis (RA), than for all other brain volumes measured. This suggests that selection based on the functions of these two structures would result in rapid major shifts in their anatomy. We also show that the size of each song control nucleus is significantly correlated with the song related nuclei to which it is monosynaptically connected. Finally, we find that the volume of the telencephalon is larger in males than in females. These findings begin to join theoretical analyses of the role of female choice in the evolution of bird song to neurobiological mechanisms by which the evolutionary changes in behaviour are expressed.