HLLIL

#Puncta Punota size Total puncta # Puncta Puncta size Total pjneta

Excitatory synapses inhibitory synapses

Figure 9.3. Spinal Neurons Deficient in y-Pcdhs Make Fewer Synapses in Vitro. (A-C) Spinal neuron cultures from control and homozygous Pcdh-y truncation embryos (tr/tr) cultured for 9 days and immunostained for the somato-dendritic protein MAP2 (A) and markers of excitatory (VGluts, vesicular glutamate transporters; B) or inhibitory (GAD, glutamic acid decarboxylase; C) presynaptic terminals. Despite normal neuronal differentiation in mutant cultures (see ref. 9 for further data), the numbers of both excitatory and inhibitory synapses are significantly reduced. Quantification confirms this reduction and further demonstrates a significant reduction in the size of excitatory terminals (D; *p < 0.001, mean ± SEM of data from four cultures per genotype). Bar = 30 ^m. The micrographs in A-C show the same fields within each genotype.

missing cytoplasmic region is not strictly required for potential y-Pcdh functions in the promotion of spinal interneuron survival.

To further substantiate the synaptic defects observed in Pcdh-y mutant mice, as well as to establish a model system in which the mechanism of these defects could be elucidated, spinal cord neurons were cultured from Pcdh-y truncation mutant embryos9. Mutant cultures exhibited normal neuronal differentiation with no evidence of excessive apoptosis, as expected from our analysis of the intact spinal cord. Immunostaining of mature cultures (>8 days in vitro) for synaptic markers, however, revealed that Pcdh-y mutant neurons made 40-50% fewer synapses than did control neurons. The number of both excitatory (vesicular glutamate transporter-positive) and inhibitory (glutamic acid decarboxylase-positive) terminals were significantly reduced, as was the size of excitatory terminals (Figure 9.3). Patch-clamp recordings of individual neurons in these cultures revealed that Pcdh-ymutant neurons exhibited normal resting membrane potentials, and were able to conduct action potentials at normal depolarization thresholds. However, the amplitude of both excitatory and inhibitory spontaneous synaptic currents was reduced by ~50% in mutant neurons9. The reduced current amplitudes in mutant neurons, along with the decrease in the size of some synaptic terminals, suggest that y-Pcdhs may be important for the maturation of synaptic contacts into fully functioning synapses. The significant reduction in the number of immunostained synaptic puncta could indicate a role for y-Pcdh function in either initial synaptogenesis or in synaptic maintenance.

Together, our data provide the first genetic analysis of any of the clustered protocadherins, demonstrating that the y-Pcdhs are critically required for both synapse development and neuronal survival in the spinal cord. While confirming that these molecules are indispensable for neural development is an important first step, much remains unknown about the mechanisms by which protocadherins exert their functions. The following section highlights some of the critical issues to be addressed and discusses progress made by several recent studies.

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