Components of Endocannabinoid Signaling System Are Expressed in the Perinatal Mouse Cerebellum and Required for Its Normal Development.

Endocannabinoid (eCB) signaling system (ECS), encompassing cannabinoid receptors and enzymes involved in the synthesis and degradation of the endogenous cannabinoid signaling lipids, is highly expressed in the cerebellar cortex of adult humans and rodents. In addition to their well-established role in neuromodulation, eCBs have been shown to play key roles in aspects of neurodevelopment in the fore- and mid-brain, including neurogenesis, cell migration, and synapse specification. However, little is known about the role of ECS in cerebellar development. In this study, we conducted immunohistochemical characterization of ECS components through key stages of cerebellar development in mice using antibodies for 2-arachidonoylglycerol (2-AG) synthetizing and degrading enzymes and the major brain cannabinoid receptor, cannabinoid receptor 1 (CB1), in combination with cerebellar cell markers. Our results reveal a temporally, spatially, and cytologically dynamic pattern of expression. Production, receptor binding, and degradation of eCBs are tightly controlled, thus localization of eCB receptors and the complementary cannabinoid signaling machinery determines the direction, duration, and ultimately the outcome of eCB signaling. To gain insights into the role of eCB signaling in cerebellar development, we characterized gross anatomy of cerebellar midvermis in CB1 knock-out (CB1 KO) mice, as well as their performance in cerebellar-influenced motor tasks. Our results show persistent and selective anatomic and behavioral alterations in CB1 KOs. Consequently, the insights gained from this study lay down the foundation for investigating specific cellular and molecular mechanisms regulated by eCB signaling during cerebellar development.

Loss of GABAergic neurons in the hippocampus and cerebral cortex of Engrailed-2 null mutant mice: implications for autism spectrum disorders.

The homeobox-containing transcription factor Engrailed-2 (En2) is involved in patterning and neuronal differentiation of the midbrain/hindbrain region, where it is prominently expressed. En2 mRNA is also expressed in the adult mouse hippocampus and cerebral cortex, indicating that it might also function in these brain areas. Genome-wide association studies revealed that En2 is a candidate gene for autism spectrum disorders (ASD), and mice devoid of its expression (En2(-/-) mice) display anatomical, behavioral and clinical "autistic-like" features. Since reduced GABAergic inhibition has been proposed as a possible pathogenic mechanism of ASD, we hypothesized that the phenotype of En2(-/-) mice might include defective GABAergic innervation in the forebrain. Here we show that the Engrailed proteins are present in postnatal GABAergic neurons of the mouse hippocampus and cerebral cortex, and adult En2(-/-) mice show reduced expression of GABAergic marker mRNAs in these areas. In addition, reduction in parvalbumin (PV), somatostatin (SOM) and neuropeptide Y (NPY) expressing interneurons is detected in the hippocampus and cerebral cortex of adult En2(-/-) mice. Our results raise the possibility of a link between altered function of En2, anatomical deficits of GABAergic forebrain neurons and the pathogenesis of ASD.

Spatially restricted and developmentally dynamic expression of engrailed genes in multiple cerebellar cell types.

The cerebellum is a highly organized structure partitioned into lobules along the anterior-posterior (A-P) axis and into striped molecular domains along the medial-lateral (M-L) axis. The Engrailed (En) homeobox genes are required for patterning the morphological and molecular domains along both axes, as well as for the establishment of the normal afferent topography required to generate a fully functional cerebellum. As a means to understand how the En genes regulate multiple levels of cerebellum construction, we characterized En1 and En2 expression around birth and at postnatal day (P) 21 during the period when the cerebellum undergoes a remarkable transformation from a smooth ovoid structure to a highly foliated structure. We show that both En1 and En2 are expressed in many neuronal cell types in the cerebellum, and expression persists until at least P21. En1 and En2 expression, however, undergoes profound changes in their cellular and spatial distributions between embryonic stages and P21, and their expression domains become largely distinct. Comparison of the distribution of En-expressing Purkinje cells relative to early- and late-onset Purkinje cell M-L stripe proteins revealed that although En1- and En2-expressing Purkinje cell domains do not strictly align with those of ZEBRINII at P21, a clear pattern exists that is most evident at E17.5 by an inverse correlation between the level of En2 expression and PLCß4 and EPHA4.

Development of axon-target specificity of ponto-cerebellar afferents.

The function of neuronal networks relies on selective assembly of synaptic connections during development. We examined how synaptic specificity emerges in the pontocerebellar projection. Analysis of axon-target interactions with correlated light-electron microscopy revealed that developing pontine mossy fibers elaborate extensive cell-cell contacts and synaptic connections with Purkinje cells, an inappropriate target. Subsequently, mossy fiber-Purkinje cell connections are eliminated resulting in granule cell-specific mossy fiber connectivity as observed in mature cerebellar circuits. Formation of mossy fiber-Purkinje cell contacts is negatively regulated by Purkinje cell-derived BMP4. BMP4 limits mossy fiber growth in vitro and Purkinje cell-specific ablation of BMP4 in mice results in exuberant mossy fiber-Purkinje cell interactions. These findings demonstrate that synaptic specificity in the pontocerebellar projection is achieved through a stepwise mechanism that entails transient innervation of Purkinje cells, followed by synapse elimination. Moreover, this work establishes BMP4 as a retrograde signal that regulates the axon-target interactions during development.

Transcriptional control of synaptic differentiation by retrograde signals.

Synaptic differentiation during development is a multi-step process, which requires reciprocal communication between pre- and postsynaptic cells. Cell surface interactions can induce the assembly of synaptic specializations but maintenance and growth of synapses depend on transcriptional regulation. Transcriptional responses associated with synaptic differentiation are observed in central and peripheral neurons and depend on retrograde signals coming from the target region. Although the identity of most of the retrograde signaling pathways remains to be identified, the TGFbeta family of growth factors have emerged as one crucial signal at the neuromuscular junction. Here, we discuss evidence for transcriptional control during synaptic differentiation and the signaling pathways mediating retrograde TGFbeta signaling.