Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871 bp designer eukaryotic chromosome, synIII, which is based on the 316,617 bp native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, tRNAs, transposons and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in “a mater” derivatives resulting from loss of the MATα allele on synIII. The total synthesis of synIII represents the first complete design and synthesis of a eukaryotic chromosome, establishing S. cerevisiae as the basis for designer eukaryotic genome biology.

Summary Older concepts of a hard-wired adult brain have been overturned in recent years by in vivo imaging studies revealing synaptic remodeling, now thought to mediate rearrangements in microcircuit connectivity. Using three-color labeling and spectrally resolved two-photon microscopy, we monitor in parallel the daily structural dynamics (assembly or removal) of excitatory and inhibitory postsynaptic sites on the same neurons in mouse visual cortex in vivo. We find that dynamic inhibitory synapses often disappear and reappear again in the same location. The starkest contrast between excitatory and inhibitory synapse dynamics is on dually innervated spines, where inhibitory synapses frequently recur while excitatory synapses are stable. Monocular deprivation, a model of sensory input-dependent plasticity, shortens inhibitory synapse lifetimes and lengthens intervals to recurrence, resulting in a new dynamic state with reduced inhibitory synaptic presence. Reversible structural dynamics indicates a fundamentally new role for inhibitory synaptic remodeling – flexible, input-specific modulation of stable excitatory connections.

Dendrites of cortical pyramidal neurons contain intermingled excitatory and inhibitory synapses. We studied the local mechanisms that regulate the formation and distribution of synapses. We found that local γ-aminobutyric acid (GABA) release on dendrites of mouse cortical layer 2/3 pyramidal neurons could induce gephyrin puncta and dendritic spine formation via GABA type A receptor activation and voltage-gated calcium channels during early postnatal development. Furthermore, the newly formed inhibitory and excitatory synaptic structures rapidly gained functions. Bidirectional manipulation of GABA release from somatostatin-positive interneurons increased and decreased the number of gephyrin puncta and dendritic spines, respectively. These results highlight a noncanonical function of GABA as a local synaptogenic element shaping the early establishment of neuronal circuitry in mouse cortex.

SUMMARY Competition between synapses contributes to activity-dependent refinement of the nervous system during development. Does local competition between neighboring synapses drive circuit remodeling during experience-dependent plasticity in the cerebral cortex? Here, we examined the role of activity-mediated competitive interactions in regulating dendritic spine structure and function on hippocampal CA1 neurons. We found that high-frequency glutamatergic stimulation at individual spines, which leads to input-specific synaptic potentiation, induces shrinkage and weakening of nearby unstimulated synapses. This heterosynaptic plasticity requires potentiation of multiple neighboring spines, suggesting that a local threshold of neural activity exists beyond which inactive synapses are punished. Notably, inhibition of calcineurin, IP3Rs, or group I mGluRs blocked heterosynaptic shrinkage without blocking structural potentiation, and inhibition of CaMKII blocked structural potentiation without blocking heterosynaptic shrinkage. Our results support a model in which activity-induced shrinkage signal, and not competition for limited structural resources, drives heterosynaptic structural and functional depression during neural circuit refinement.

Refinement of neural circuits in the mammalian cerebral cortex shapes brain function during development and in the adult. However, the signaling mechanisms underlying the synapse-specific shrinkage and loss of spiny synapses when neural circuits are remodeled remain poorly defined. Here, we show that low-frequency glutamatergic activity at individual dendritic spines leads to synapse-specific synaptic weakening and spine shrinkage on CA1 neurons in the hippocampus. We found that shrinkage of individual spines in response to lowfrequency glutamate uncaging is saturable, reversible, and requires NMDA receptor activation. Notably, shrinkage of large spines additionally requires signaling through metabotropic glutamate receptors (mGluRs) and inositol 1,4,5-trisphosphate receptors (IP 3 Rs), supported by higher levels of mGluR signaling activity in large spines. Our results support a model in which signaling through both NMDA receptors and mGluRs is required to drive activity-dependent synaptic weakening and spine shrinkage at large, mature dendritic spines when neural circuits undergo experience-dependent modification.two-photon microscopy | long-term depression | synaptic plasticity | spine dynamics S tructural plasticity of neurons, such as the growth and retraction of dendritic spines, is thought to contribute to the experience-dependent changes in brain circuitry that mediate learning and memory (1, 2). In particular, the destabilization and loss of spiny synapses plays a critical role in the refinement of neural circuits during development and during learning. Indeed, spine elimination occurs more frequently than does spine formation in young rodents between the first and the third month of age (3-5), a period when experience-dependent refinement of neural circuitry is in its peak. Furthermore, several in vivo studies demonstrate that manipulations leading to experience-dependent circuit plasticity also increase the rate of spine shrinkage and loss (6-9). However, it remains unclear how neural activity drives the selective shrinkage and loss of individual dendritic spines in response to sensory experience.Dendritic spines occur in a wide variety of shapes and sizes (10, 11), and their stability is strongly correlated with spine size (3, 12, 13). Small spines are in general more motile and thought to serve as substrates for plasticity, or "learning" spines; large spines are more stable and thought to serve as components of functioning neural circuits, or "memory" spines (12,14). It is the selective shrinkage and loss of individual circuit-incorporated, persistent spines that underlies experience-dependent circuit refinement (6, 9, 15). Thus, experience-dependent circuit remodeling requires an activity-dependent mechanism that selectively induces shrinkage and retraction of those specific individual dendritic spines that are no longer useful for circuit function.Previous studies have established that low-frequency glutamatergic stimulation (LFS), which causes long-term depression (LTD) of synaptic transmission in...