Parcellation of cerebellins 1, 2, and 4 among different subpopulations of dorsal horn neurons in mouse spinal cord.

The cerebellins (Cblns) are a family of secreted proteins that are widely expressed throughout the nervous system, but whose functions have been studied only in the cerebellum and striatum. Two members of the family, Cbln1 and Cbln2, bind to neurexins on presynaptic terminals and to GluRδs postsynaptically, forming trans-synaptic triads that promote synapse formation. Cbln1 has a higher binding affinity for GluRδs and exhibits greater synaptogenic activity than Cbln2. In contrast, Cbln4 does not form such triads and its function is unknown. The different properties of the three Cblns suggest that each plays a distinct role in synapse formation. To begin to elucidate Cbln function in other neuronal systems, we used in situ hybridization to examine Cbln expression in the mouse spinal cord. We find that neurons expressing Cblns 1, 2, and 4 tend to occupy different laminar positions within the dorsal spinal cord, and that Cbln expression is limited almost exclusively to excitatory neurons. Combined in situ hybridization and immunofluorescent staining shows that Cblns 1, 2, and 4 are expressed by largely distinct neuronal subpopulations, defined in part by sensory input, although there is some overlap and some individual neurons coexpress two Cblns. Our results suggest that differences in connectivity between subpopulations of dorsal spinal cord neurons may be influenced by which Cbln each subpopulation contains. Competitive interactions between axon terminals may determine the number of synapses each forms in any given region, and thereby contribute to the development of precise patterns of connectivity in the dorsal gray matter.

Localization of cerebellin-2 in late embryonic chicken brain: implications for a role in synapse formation and for brain evolution.

Cerebellin-1 (Cbln1), the most studied member of the cerebellin family of secreted proteins, is necessary for the formation and maintenance of parallel fiber-Purkinje cell synapses. However, the roles of the other Cblns have received little attention. We previously identified the chicken homolog of Cbln2 and examined its expression in dorsal root ganglia and spinal cord (Yang et al. [2010] J Comp Neurol 518:2818-2840). Interestingly, Cbln2 is expressed by mechanoreceptive and proprioceptive neurons and in regions of the spinal cord where those afferents terminate, as well as by preganglionic sympathetic neurons and their sympathetic ganglia targets. These findings suggest that Cbln2 may demonstrate a tendency to be expressed by synaptically connected neuronal populations. To further assess this possibility, we examined Cbln2 expression in chick brain. We indeed found that Cbln2 is frequently expressed by synaptically connected neurons, although there are exceptions, and we discuss the implications of these findings for Cbln2 function. Cbln2 expression tends to be more common in primary sensory neurons and in second-order sensory regions than it is in motor areas of the brain. Moreover, we found that the level of Cbln2 expression for many regions of the chicken brain is very similar to that of the mammalian homologs, consistent with the view that the expression patterns of molecules playing fundamental roles in processes such as neuronal communication are evolutionarily conserved. There are, however, large differences in the pattern of Cbln2 expression in avian as compared to mammalian telencephalon and in other regions that show the most divergence between the two lineages.

Identification of cerebellin2 in chick and its preferential expression by subsets of developing sensory neurons and their targets in the dorsal horn.

The cerebellins are a family of four secreted proteins, two of which, Cbln1 and Cbln3, play an important role in the formation and maintenance of parallel fiber-Purkinje cell synapses. We have identified the chicken homologue of Cbln2 and, through the use of in situ hybridization, shown that it is expressed by specific subsets of neurons in the dorsal root ganglia (DRGs) and spinal cord starting shortly after those neurons are generated. In the developing spinal cord, Cbln2 is highly expressed by dI1, dI3, dI5, and dILB dorsal interneurons and to a lesser extent by dI2, dI4, dI6, and dILA dorsal interneurons, but not by ventral (v0-v3) interneurons. After the spinal cord has matured and neurons have migrated to their final destinations, Cbln2 is abundant in the dorsal horn. In the DRGs, Cbln2 is expressed by TrkB+ and TrkC+ sensory neurons, but not by TrkA+ sensory neurons. Interestingly, regions of the spinal cord where TrkB+ and TrkC+ afferents terminate (i.e., laminae II, III, IV, and VI) exhibit the highest levels of Cbln2 expression. Cbln2 is also expressed by preganglionic sympathetic neurons and their targets in the sympathetic chain ganglia. Thus, the results show that Cbln2 is frequently expressed by synaptically connected neuronal populations. This, in turn, raises the possibility that if Cbln2, like Cbln1, plays a role in the formation and maintenance of synapses, it may somehow mediate bi-directional communication between discrete populations of neurons and their appropriate neuronal targets.