Does chronic systemic injection of the DREADD agonists clozapine-N-oxide or Compound 21 change behavior relevant to locomotion, exploration, anxiety, and depression in male non-DREADD-expressing mice?

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are chemogenetic tools commonly-used to manipulate brain activity. The most widely-used synthetic DREADD ligand, clozapine-N-oxide (CNO), is back-metabolized to clozapine which can itself activate endogenous receptors. Studies in non-DREADD-expressing rodents suggest CNO or a DREADD agonist that lacks active metabolites, such as Compound 21 (C21), change rodent behavior (e.g. decrease locomotion), but chronic injection of CNO does not change locomotion. However, it is unknown if chronic CNO changes behaviors relevant to locomotion, exploration, anxiety, and depression, or if chronic C21 changes any aspect of mouse behavior. Here non-DREADD-expressing mice received i.p. Vehicle (Veh), CNO, or C21 (1 mg/kg) 5 days/week for 16 weeks and behaviors were assessed over time. Veh, CNO, and C21 mice had similar weight gain over the 16-week-experiment. During the 3rd injection week, CNO and C21 mice explored more than Veh mice in a novel context and had more open field center entries; however, groups were similar in other measures of locomotion and anxiety. During the 14th-16th injection weeks, Veh, CNO, and C21 mice had similar locomotion and anxiety-like behaviors. We interpret these data as showing chronic Veh, CNO, and C21 injections given to male non-DREADD-expressing mice largely lack behavioral effects. These data may be helpful for behavioral neuroscientists when study design requires repeated injection of these DREADD agonists.

Analyses with double knockouts of the Bmpr1a and Bmpr1b genes demonstrate that BMP signaling is involved in the formation of precerebellar mossy fiber nuclei derived from the rhombic lip.

Bone morphogenetic proteins (BMPs) have been hypothesized to specify distinct dorsal neural fates. During neural development, BMPs are expressed in the roof plate and adjacent neuroepithelium. Because several hindbrain nuclei that form the proprioceptive/vestibular/auditory sensory network originate from the rhombic lip, near the roof plate, BMP signaling may regulate the development of these nuclei. To test this hypothesis genetically, we have examined the development of the hindbrain in BMP type I receptor knockout mice. Our results demonstrate that BMP signaling is involved in the formation of precerebellar mossy fiber nuclei, which give rise to cerebellar mossy fibers, but is not required for the development of the inferior olivary nucleus, which gives rise to cerebellar climbing fibers.

Otic mesenchyme expression of Cre recombinase directed by the inner ear enhancer of the Brn4/Pou3f4 gene.

Brn4/Pou3f4 is a POU-domain transcription factor expressed in the otic mesenchyme that is required for the normal development of the inner ear. In this report, we describe the isolation of an otic mesenchyme enhancer in the Brn4 gene. Subsequently, this enhancer was used to drive the expression of Cre recombinase in the otic mesenchyme of transgenic mice. When intercrossed with the ROSA reporter strain, R26R, ss-galactosidase expression is detected in several inner ear structures derived from otic mesenchyme, including the temporal bone, spiral ligament, spiral limbus, and mesenchyme underlying sensory epithelium of the utricle, saccule and semicircular canals. Thus, this Cre pedigree can induce conditional rearrangement of genes in the otic mesenchyme, and will serve as a powerful genetic tool to characterize the function of genes in the mesenchymal tissues of the inner ear.

Genetic analyses demonstrate that bone morphogenetic protein signaling is required for embryonic cerebellar development.

The cerebellum has been a useful model for studying many aspects of neural development because of its relatively simple cytoarchitecture and developmental program. Yet, the genetic mechanisms underlying early differentiation and patterning of the cerebellum are still poorly characterized. Cell expression studies and culture experiments have suggested the importance of bone morphogenetic proteins (BMPs) in development of specific populations of cerebellar neurons. Here, we examined mice with targeted mutations in the BMP type I receptor genes Bmpr1a and Bmpr1b, to genetically test the hypothesis that BMPs play an inductive role in the embryogenesis of cerebellar granule cells. In Bmpr1a;Bmpr1b double knock-out mice, severe cerebellar patterning defects are observed resulting in smaller cerebella that are devoid of foliation. In mutants containing either single BMP receptor gene mutation alone, cerebellar histogenesis appears normal, thereby demonstrating functional redundancy of type I BMP receptors during cerebellar development. Loss of BMP signaling in double mutant animals leads to a dramatic reduction in the number of cerebellar granule cells and ectopic location of many of those that remain. Molecular markers of granule cell specification, including Math1 and Zic1, are drastically downregulated. In addition, Purkinje cells are disorganized and ectopically located, but they appear to be correctly specified. Consistent with the interpretation that granule cells alone are affected, phosphorylated Smad1/5/8 is immunolocalized predominantly to granule cell precursors and not appreciably detected in Purkinje cell precursors. This study demonstrates that BMP signaling plays a crucial role in the specification of granule cells during cerebellar development.

Signaling through BMP type 1 receptors is required for development of interneuron cell types in the dorsal spinal cord.

During spinal cord development, distinct classes of interneurons arise at stereotypical locations along the dorsoventral axis. In this paper, we demonstrate that signaling through bone morphogenetic protein (BMP) type 1 receptors is required for the formation of two populations of commissural neurons, DI1 and DI2, that arise within the dorsal neural tube. We have generated a double knockout of both BMP type 1 receptors, Bmpr1a and Bmpr1b, in the neural tube. These double knockout mice demonstrate a complete loss of D1 progenitor cells, as evidenced by loss of Math1 expression, and the subsequent failure to form differentiated DI1 interneurons. Furthermore, the DI2 interneuron population is profoundly reduced. The loss of these populations of cells results in a dorsal shift of the dorsal cell populations, DI3 and DI4. Other dorsal interneuron populations, DI5 and DI6, and ventral neurons appear unaffected by the loss of BMP signaling. The Bmpr double knockout animals demonstrate a reduction in the expression of Wnt and Id family members, suggesting that BMP signaling regulates expression of these factors in spinal cord development. These results provide genetic evidence that BMP signaling is crucial for the development of dorsal neuronal cell types.