Nerve growth factors, substrate and cell adhesion molecules, and protein synthesis are considered necessary for most developmental programs, including cell proliferation, migration, differentiation, axogenesis, pathfinding, and synaptic plasticity. Their direct involvement in synapse formation, however, has not yet been fully determined. The neurite outgrowth that precedes synaptogenesis is contingent on protein synthesis, the availability of externally supplied growth factors, and substrate adhesion molecules. It is therefore difficult to ascertain whether these factors are also needed for synapse formation. To examine this issue directly we reconstructed synapses between the cell somata of identified Lymnaea neurons. We show that when paired in the presence of brain conditioned medium (CM), mutual inhibitory chemical synapses between neurons right pedal dorsal 1 (RPeD1) and visceral dorsal 4 (VD4) formed in a soma-soma configuration (86%; n ϭ 50). These synapses were reliable and target cell specific and were similar to those seen in the intact brain. To test whether synapse formation between RPeD1 and VD4 required de novo protein synthesis, the cells were paired in the presence of anisomycin (a nonspecific protein synthesis blocker). Chronic anisomycin treatment (18 hr) after cell pairing completely blocked synaptogenesis between RPeD1 and VD4 (n ϭ 24); however, it did not affect neuronal excitability or responsiveness to exogenously applied transmitters (n ϭ 7), nor did chronic anisomycin treatment affect synaptic transmission between pairs of cells that had formed synapses (n ϭ 5). To test the growth and substrate dependence of synapse formation, RPeD1 and VD4 were paired in the absence of CM [defined medium; (n ϭ 22)] on either plain plastic culture dishes (n ϭ 10) or glass coverslips (n ϭ 10). Neither CM nor any exogenous substrate was required for synapse formation. In summary, our data provide direct evidence that synaptogenesis in this system requires specific, cell contact-induced, de novo protein synthesis but does not depend on extrinsic growth factors or substrate adhesion molecules.Key words: synapse formation; in vitro; growth factors; Lymnaea; soma-soma synapses; mollusks To f unction properly, the adult brain relies heavily on neuronal connectivity patterns that are orchestrated during early embryonic development (McMahan, 1990;Nelson et al., 1990;Goodman and Shatz, 1993;Hall and Sanes, 1993; Jessel and Kandel, 1993;Goodman, 1994Goodman, , 1996Grantyn et al., 1995;Katz and Shatz, 1996;Wu et al., 1996;Spencer et al., 1997). Yet, the cellular and molecular mechanisms (intrinsic and /or extrinsic) that determine the specificity of synaptic connections in the nervous system remain poorly understood. This gap in our f undamental knowledge regarding nervous system development (and also regeneration) owes its existence to the complexity of the mammalian brain: synapse formation between defined pre-and postsynaptic neurons can be studied only rarely in the intact nervous system. Various in vivo and in...

The cellular basis of long-term memory (LTM) storage is not completely known. We have developed a preparation where we are able to specify that a single identified neuron, Right Pedal Dorsal 1 (RPeD1), is a site of LTM formation of associative learning in the pond snail, Lymnaea stagnalis. We demonstrated this by ablating the soma of the neuron but leaving behind its functional primary neurite, as evidenced by electrophysiological and behavioral analyses. The soma-less RPeD1 neurite continues to be a necessary participant in the mediation of aerial respiratory behavior, associative learning, and intermediate-term memory (ITM); however, LTM cannot be formed. However, if RPeD1's soma is ablated after LTM consolidation has occurred, LTM can still be accessed. Thus the soma of RPeD1 is a site of LTM formation.

The human face is a complex assemblage of highly variable yet clearly heritable anatomic structures that together make each of us unique, distinguishable, and recognizable. Relatively little is known about the genetic underpinnings of normal human facial variation. To address this, we carried out a large genomewide association study and two independent replication studies of Bantu African children and adolescents from Mwanza, Tanzania, a region that is both genetically and environmentally relatively homogeneous. We tested for genetic association of facial shape and size phenotypes derived from 3D imaging and automated landmarking of standard facial morphometric points. SNPs within genes SCHIP1 and PDE8A were associated with measures of facial size in both the GWAS and replication cohorts and passed a stringent genomewide significance threshold adjusted for multiple testing of 34 correlated traits. For both SCHIP1 and PDE8A, we demonstrated clear expression in the developing mouse face by both whole-mount in situ hybridization and RNA-seq, supporting their involvement in facial morphogenesis. Ten additional loci demonstrated suggestive association with various measures of facial shape. Our findings, which differ from those in previous studies of European-derived whites, augment understanding of the genetic basis of normal facial development, and provide insights relevant to both human disease and forensics.

Reconsolidation of a long-term memory (LTM) in the snail Lymnaea stagnalis can be disrupted by cooling, an RNA synthesis blocker (actinomycin D), and by specifically ablating the soma of a cell we know is a site of LTM consolidation (right pedal dorsal 1, RPeD1). Aerial respiratory behavior was conditioned operantly by applying a gentle tactile stimulus to the pneumostome area (the respiratory orifice) every time the snail began to open its pneumostome to perform aerial respiration. This resulted in a reduction of this behavior while leaving cutaneous respiration intact. One week after training one-half of the animals received a memory reactivation session, which was similar to the original training (i.e., animals received reinforcement). All animals then received 1 hr of cooling, an injection of actinomycin D or saline, or the soma ablation procedure. This was followed by a test for savings 4 hr or 4 d later, which was also similar to the original training. Only those animals that received both the memory reactivation session and the treatment showed memory impairment during the test for savings. That is, the impairment was contingent on memory reactivation. These data indicate that reconsolidation requires both new RNA and protein synthesis to stabilize a reactivated memory, and it demonstrates that the soma of RPeD1, a cell that we have shown previously to be required in the consolidation of an LTM, is necessary for reconsolidation. These data suggest that the critical molecular processes occurring during both consolidation and reconsolidation transpire in the same cell in Lymnaea.

Aerial respiration of the pond snail, Lymnaea stagnalis, can be operantly conditioned; however, the parameters necessary to produce long-term (LTM) or intermediate term memory (ITM) have not previously been investigated. We conducted training using procedures that varied in the duration of the training session, the number of training sessions per day or the amount of time between subsequent training sessions (SI). We found that by varying the duration and frequency of the training session learning could be differentially produced. Furthermore, the ability to form LTM was dependent not only on the duration of the training session was also the interval between training sessions, the SI. Thus it was possible to produce ITM, which persists for up to 3 hr, and not form LTM, which persists at least 18 hr. Learning, ITM, and LTM can be differentially produced by altering the SI, the duration of the training session, or the number of training sessions per day. These findings may allow us to begin to elucidate the underlying neural mechanisms of learning, ITM, and LTM.