Classic hallucinogens such as lysergic acid diethylamide are thought to elicit their psychotropic actions via serotonin receptors of the 5-hydroxytryptamine 2A subtype (5-HT 2AR). One likely site for these effects is the prefrontal cortex (PFC). Previous studies have shown that activation of 5-HT2ARs in this region results in a robust increase in spontaneous glutamatergic synaptic activity, and these results have led to the widely held idea that hallucinogens elicit their effect by modulating synaptic transmission within the PFC. Here, we combine cellular and molecular biological approaches, including single-cell 5-HT 2ARs inactivation and 5-HT2AR rescue over a 5-HT 2AR knockout genetic background, to distinguish between competing hypotheses accounting for these effects. The results from these experiments do not support the idea that 5-HT 2ARs elicit the release of an excitatory retrograde messenger nor that they activate thalamocortical afferents, the two dominant hypotheses. Rather, they suggest that 5-HT 2ARs facilitate intrinsic networks within the PFC. Consistent with this idea, we locate a discrete subpopulation of pyramidal cells that is strongly excited by 5-HT 2AR activation.gene gun ͉ in vitro electrophysiology ͉ organotypic slices ͉ serotonin ͉ hallucinogen T he idea that classic hallucinogens such as lysergic acid diethylamide and psylocibin act by interfering with serotonergic neurotransmission can be traced to the middle of the 20th century (1). It was, however, not until the 1980s that serotonin receptors of the 5-hydroxytryptamine 2A subtype (5-HT 2A R) were identified as the molecular target for these agents (refs. 2, 3; reviewed in refs. 4, 5). Subsequent brain imaging studies in human subjects have extended these findings to identify the prefrontal cortex (PFC), which is highly enriched in these receptors, as a key brain region in mediating the effects of hallucinogens (6, 7). These findings have led to the now widely accepted view that activation of 5-HT 2A R in the prefrontal is a key biological step leading to the psychological effects of hallucinogens (5, 8).Our understanding of the mechanisms by which 5-HT 2A R activation elicits the sensory and behavioral manifestation of hallucinogens would be enriched by a precise understanding of how these receptors modulate cellular and network excitability in the PFC. To that effect, a number of studies have addressed the electrophysiological effects signaled by 5-HT 2A Rs in this region. There is general concordance that the most robust cellular effect observed in pyramidal cell of the PFC on stimulation of 5-HT 2A Rs involves an increase in both the frequency and amplitude of glutamatergic spontaneous excitatory postsynaptic potentials/spontaneous excitatory postsynaptic currents (sEPSCs) (9)(10)(11)(12)(13)(14). This observation thus points to 5-HT 2A Rs as powerful modulators of the excitability of PFC networks and reconciles evidence implicating both glutamatergic and serotonergic systems in the actions of hallucinogens (15).Although multiple ...

The synaptic vesicle-associated cysteine string protein (CSP) is critical for neurotransmitter release at the neuromuscular junction (NMJ) of Drosophila, where the approximately 4% of mutant flies lacking CSP that survive to adulthood exhibit spastic jumping and shaking, temperature-sensitive paralysis, and premature death. Previously, it has been shown that CSP is also required for nerve terminal growth and the prevention of neurodegeneration in Drosophila and mice. At larval csp null mutant NMJs of Drosophila, intracellular recordings from the muscle showed that evoked release is significantly reduced at room temperature. However, it remained unclear whether the reduction in evoked release might be due to a loss of synaptic boutons, loss of synapses, and alterations in trafficking of vesicles to synapses. To resolve these issues, we have examined synaptic structure and function of csp null mutant NMJs at the level of single boutons. csp null mutations proportionally reduce the number of synaptic boutons of both motor neurons (1s and 1b) innervating larval muscles 6 and 7, while the number of synapses per bouton remains normal. However, focal recordings from individual synaptic boutons show that nerve-evoked neurotransmitter release is also impaired in both 1s and 1b boutons. Further, our ultrastructural analyses show that the reduction in evoked release at low stimulation frequencies is not due to a loss of synapses or to alterations in docked vesicles at synapses. Together, these data suggest that CSP promotes synaptic growth and evoked neurotransmitter release by mechanistically independent signaling pathways.

The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins.

We genetically characterized the synaptic role of the Drosophila homologue of human DCAF12, a putative cofactor of Cullin4 (Cul4) ubiquitin ligase complexes. Deletion of Drosophila DCAF12 impairs larval locomotion and arrests development. At larval neuromuscular junctions (NMJs), DCAF12 is expressed presynaptically in synaptic boutons, axons, and nuclei of motor neurons. Postsynaptically, DCAF12 is expressed in muscle nuclei and facilitates Cul4-dependent ubiquitination. Genetic experiments identified several mechanistically independent functions of DCAF12 at larval NMJs. First, presynaptic DCAF12 promotes evoked neurotransmitter release. Second, postsynaptic DCAF12 negatively controls the synaptic levels of the glutamate receptor subunits GluRIIA, GluRIIC, and GluRIID. The down-regulation of synaptic GluRIIA subunits by nuclear DCAF12 requires Cul4. Third, presynaptic DCAF12 is required for the expression of synaptic homeostatic potentiation. We suggest that DCAF12 and Cul4 are critical for normal synaptic function and plasticity at larval NMJs.

Accumulating evidence illustrates the significance of chaperone systems for the regulation and maintenance of neuronal and synaptic function. The significance of synaptic chaperones is best illustrated by cysteine-string protein (CSP), a member of the DnaJ/Hsp40 family of Hsp70/Hsc70 co-chaperones. CSP recruits the ubiquitous chaperone Hsc70 to synaptic vesicles forming a chaperone complex that maintains synaptic function and prevents neurodegeneration. Here, we summarize studies that demonstrate CSP's neuroprotective role for synaptic function and discuss insights into its possible clientele (the proteins whose function it facilitates). General IntroductionSynaptic transmission occurs at highly specialized cell-cell contact sites, termed synapses. In the presynaptic axon terminal, quantal packages of neurotransmitter are stored in synaptic vesicles. Regulated neurotransmitter release is initiated by a depolarization-dependent Ca 2+ influx through voltage-gated Ca 2+ channels that triggers the fusion of synaptic vesicles with the presynaptic membrane releasing neurotransmitter into the synaptic cleft. In turn, activation of specific neurotransmitter receptors in the postsynaptic cell membrane induces a postsynaptic response in less than 100 ms after the presynaptic trigger. After exocytosis, synaptic vesicle membranes and proteins are rapidly recaptured and locally recycled ensuring that synapses can faithfully sustain transmitter release during prolonged periods of high nerve activity.Presynaptic terminals resemble specialized secretory machines that exhibit an unparalleled autonomy and durability. Both Ca 2+ signaling and synaptic vesicle fusion are driven by a series of protein-protein and protein-lipid interactions that involve a complex interplay among a specialized set of synaptic proteins and the