Synaptic contacts of gamma-aminobutyric acid (GABA) -immunoreactive neurons in honeybee mushroom bodies were studied by using electron microscopic immunocytochemistry. In the lip region of the calyx neuropil, GABA-immunoreactive profiles formed synapses onto both small postsynaptic profiles (76%) and large immunonegative boutons (4%), which were likely to belong to the intrinsic and extrinsic mushroom body neurons, respectively. Three morphologic types of the large immunonegative boutons were distinguished: "light," "dark," and "dense core"; all of them received synaptic inputs from the GABA-immunoreactive profiles. A significant proportion of the synapses formed by the GABA-immunoreactive neurons in the lip region (20%) were input synapses from immunonegative neurons. Analysis of thin serial sections showed that the output and input synapses formed microcircuits in which both large immunonegative boutons and small postsynaptic profiles were involved. We interpret these findings to show that negative feedforward and feedback loops exist within the microcircuits of the lip region.

Axospinous synapses are traditionally divided according to postsynaptic density (PSD) configuration into a perforated subtype characterized by a complex-shaped PSD and nonperforated subtype exhibiting a simple-shaped, disc-like PSD. It has been hypothesized that perforated synapses are especially important for synaptic plasticity because they have a higher efficacy of impulse transmission. The aim of the present study was to test this hypothesis. The number of postsynaptic AMPA receptors (AMPARs) is widely regarded as the major determinant of synaptic efficacy. Therefore, the expression of AMPARs was evaluated in the two synaptic subtypes and compared with that of NMDA receptors (NMDARs). Postembedding immunogold electron microscopy was used to quantify the immunoreactivity following single labeling of AMPARs or NMDARs in serial sections through the CA1 stratum radiatum of adult rats. The results showed that all perforated synapses examined were immunopositive for AMPARs. In contrast, only a proportion of nonperforated synapses (64% on average) contained immunogold particles for AMPARs. The number of immunogold particles for AMPARs was markedly and significantly higher in perforated synapses than in immunopositive nonperforated synapses. Although all synapses of both subtypes were NMDAR immunopositive perforated synapses contained significantly more immunogold particles for NMDARs than nonperforated ones. Multivariate analysis of variance revealed that the mode of AMPAR and NMDAR expression is related to the complexity of PSD configuration, not only to PSD size. These findings support the notion that perforated synapses may evoke larger postsynaptic responses relative to nonperforated synapses and, hence, contribute to an enhancement of synaptic transmission associated with some forms of synaptic plasticity.

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Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31–246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate α- and β-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3′ region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3′ splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.