The physiological functions of glycine receptors (GlyRs) depend on their subcellular locations. In axonal terminals of the central neurons, GlyRs trigger a slow facilitation of presynaptic transmitter release; however, their spatial relationship to the release sites is not known. In this study, we examined the distribution of GlyRs in the rat glutamatergic calyx of Held nerve terminal using high-resolution pre-embedding immunoelectron microscopy. We performed a quantitative analysis of GlyR-associated immunogold (IG) labeling in 3D reconstructed calyceal segments. A variable density of IG particles and their putative accumulations, inferred from the frequency distribution of inter-IG distances, indicated a non-uniform distribution of the receptors in the calyx. Subsequently, increased densities of IG particles were found in calyceal swellings, structures characterized by extensive exocytosis of glutamate. In swellings as well as in larger calyceal stalks, IG particles did not tend to accumulate near the glutamate releasing zones. On the other hand, GlyRs in swellings (but not in stalks) preferentially occupied membrane regions, unconnected to postsynaptic cells and presumably accessible by ambient glycine. Furthermore, the sites with increased GlyR concentrations were found in swellings tightly juxtaposed with GABA/glycinergic nerve endings. Thus, the results support the concept of an indirect mechanism underlying the modulatory effects of calyceal GlyRs, activated by glycine spillover. We also suggest the existence of an activity-dependent mechanism regulating the surface distribution of α homomeric GlyRs in axonal terminals of central neurons.

Department of Auditory Neuroscience Laboratory of Synaptic Transmission Institute of Experimental Medicine Academy of Sciences of the Czech Republic Prague Czech Republic

Department of Biomathematics Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

Department of Physiology 2 University of Freiburg Freiburg Germany ; BIOSS Centre for Biological Signalling Studies University of Freiburg Freiburg Germany

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Alle H., Geiger J. R. (2007). GABAergic spill-over transmission onto hippocampal mossy fiber boutons. J. Neurosci. 27, 942–950 10.1523/jneurosci.4996-06.2007 PubMed DOI PMC

Awatramani G. B., Price G. D., Trussell L. O. (2005). Modulation of transmitter release by presynaptic resting potential and background calcium levels. Neuron 48, 109–121 10.1016/j.neuron.2005.08.038 PubMed DOI

Barbour B., Hausser M. (1997). Intersynaptic diffusion of neurotransmitter. Trends Neurosci. 20, 377–384 PubMed

Belenky M. A., Sagiv N., Fritschy J. M., Yarom Y. (2003). Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus. Neuroscience 118, 909–923 10.1016/s0306-4522(03)00062-9 PubMed DOI

Bernard V., Somogyi P., Bolam J. P. (1997). Cellular, subcellular and subsynaptic distribution of AMPA-type glutamate receptor subunits in the neostriatum of the rat. J. Neurosci. 17, 819–833 PubMed PMC

Billups B. (2005). Colocalization of vesicular glutamate transporters in the rat superior olivary complex. Neurosci. Lett. 382, 66–70 10.1016/j.neulet.2005.02.071 PubMed DOI

Boehm S., Kubista H. (2002). Fine tuning of sympathetic transmitter release via ionotropic and metabotropic presynaptic receptors. Pharmacol. Rev. 54, 43–99 10.1124/pr.54.1.43 PubMed DOI

Brickley S. G., Mody I. (2012). Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease. Neuron 73, 23–34 10.1016/j.neuron.2011.12.012 PubMed DOI PMC

Caruncho H. J., Guidotti A., Lindstrom J., Costa E., Pesold C. (1997). Subcellular localization of the alpha 7 nicotinic receptor in rat cerebellar granule cell layer. Neuroreport 8, 1431–1433 10.1097/00001756-199704140-00021 PubMed DOI

Chu Y., Fioravante D., Thanawala M., Leitges M., Regehr W. G. (2012). Calcium-dependent isoforms of protein kinase C mediate glycine-induced synaptic enhancement at the calyx of held. J. Neurosci. 32, 13796–13804 10.1523/JNEUROSCI.2158-12.2012 PubMed DOI PMC

Danbolt N. C. (2001). Glutamate uptake. Prog. Neurobiol. 65, 1–105 10.1016/S0301-0082(00)00067-8 PubMed DOI

Darstein M., Petralia R. S., Swanson G. T., Wenthold R. J., Heinemann S. F. (2003). Distribution of kainate receptor subunits at hippocampal mossy fiber synapses. J. Neurosci. 23, 8013–8019 PubMed PMC

Deleuze C., Runquist M., Orcel H., Rabié A., Dayanithi G., Alonso G., et al. (2005). Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system. Neuroscience 135, 475–483 10.1016/j.neuroscience.2005.05.024 PubMed DOI

Dodson P. D., Billups B., Rusznák Z., Szûcs G., Barker M. C., Forsythe I. D. (2003). Presynaptic rat Kv1.2 channels suppress synaptic terminal hyperexcitability following action potential invasion. J. Physiol. 550, 27–33 10.1113/jphysiol.2003.046250 PubMed DOI PMC

Duerstock B. S., Bajaj C. L., Borgens R. B. (2003). A comparative study of the quantitative accuracy of three-dimensional reconstructions of spinal cord from serial histological sections. J. Microsc. 210, 138–148 10.1046/j.1365-2818.2003.01130.x PubMed DOI

Elezgarai I., Díez J., Puente N., Azkue J. J., Benítez R., Bilbao A., et al. (2003). Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body. Neuroscience 118, 889–898 10.1016/s0306-4522(03)00068-x PubMed DOI

Engelman H. S., MacDermott A. B. (2004). Presynaptic ionotropic receptors and control of transmitter release. Nat. Rev. Neurosci. 5, 135–145 10.1038/nrn1297 PubMed DOI

Faber D. S., Korn H. (1988). Synergism at central synapses due to lateral diffusion of transmitter. Proc. Natl. Acad. Sci. U S A 85, 8708–8712 10.1073/pnas.85.22.8708 PubMed DOI PMC

Farrant M., Nusser Z. (2005). Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat. Rev. Neurosci. 6, 215–229 10.1038/nrn1625 PubMed DOI

Fiala J. C. (2005). Reconstruct: a free editor for serial section microscopy. J. Microsc. 218, 52–61 10.1111/j.1365-2818.2005.01466.x PubMed DOI

Friauf E., Aragón C., Löhrke S., Westenfelder B., Zafra F. (1999). Developmental expression of the glycine transporter GLYT2 in the auditory system of rats suggests involvement in synapse maturation. J. Comp. Neurol. 412, 17–37 10.1002/(sici)1096-9861(19990913)412:1<17::aid-cne2>3.3.co;2-5 PubMed DOI

Geiman E. J., Zheng W., Fritschy J. M., Alvarez F. J. (2002). Glycine and GABA(A) receptor subunits on Renshaw cells: relationship with presynaptic neurotransmitters and postsynaptic gephyrin clusters. J. Comp. Neurol. 444, 275–289 10.1002/cne.10148 PubMed DOI

Gladding C. M., Raymond L. A. (2011). Mechanisms underlying NMDA receptor synaptic/extrasynaptic distribution and function. Mol. Cell. Neurosci. 48, 308–320 10.1016/j.mcn.2011.05.001 PubMed DOI

Hardingham G. E., Bading H. (2010). Synaptic versus extrasynaptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat. Rev. Neurosci. 11, 682–696 10.1038/nrn2911 PubMed DOI PMC

Hermida D., Mateos J. M., Elezgarai I., Puente N., Bilbao A., Bueno-López J. L., et al. (2010). Spatial compartmentalization of AMPA glutamate receptor subunits at the calyx of held synapse. J. Comp. Neurol. 518, 163–174 10.1002/cne.22189 PubMed DOI

Hruskova B., Trojanova J., Kulik A., Kralikova M., Pysanenko K., Bures Z., et al. (2012). Differential distribution of glycine receptor subtypes at the rat calyx of held synapse. J. Neurosci. 32, 17012–17024 10.1523/JNEUROSCI.1547-12.2012 PubMed DOI PMC

Huang H., Trussell L. O. (2008). Control of presynaptic function by a persistent Na(+) current. Neuron 60, 975–979 10.1016/j.neuron.2008.10.052 PubMed DOI PMC

Jaskolski F., Coussen F., Mulle C. (2005). Subcellular localization and trafficking of kainate receptors. Trends Pharmacol. Sci. 26, 20–26 10.1016/j.tips.2004.11.008 PubMed DOI

Jeong H. J., Jang I. S., Moorhouse A. J., Akaike N. (2003). Activation of presynaptic glycine receptors facilitates glycine release from presynaptic terminals synapsing onto rat spinal sacral dorsal commissural nucleus neurons. J. Physiol. 550, 373–383 10.1113/jphysiol.2003.041053 PubMed DOI PMC

Jones I. W., Wonnacott S. (2004). Precise localization of alpha7 nicotinic acetylcholine receptors on glutamatergic axon terminals in the rat ventral tegmental area. J. Neurosci. 24, 11244–11252 10.1523/jneurosci.3009-04.2004 PubMed DOI PMC

Jourdain P., Bergersen L. H., Bhaukaurally K., Bezzi P., Santello M., Domercq M., et al. (2007). Glutamate exocytosis from astrocytes controls synaptic strength. Nat. Neurosci. 10, 331–339 10.1038/nn1849 PubMed DOI

Kharazia V. N., Weinberg R. J. (1997). Tangential synaptic distribution of NMDA and AMPA receptors in rat neocortex. Neurosci. Lett. 238, 41–44 10.1016/s0304-3940(97)00846-x PubMed DOI

Kieval J. Z., Hubert G. W., Charara A., Paré J. F., Smith Y. (2001). Subcellular and subsynaptic localization of presynaptic and postsynaptic kainate receptor subunits in the monkey striatum. J. Neurosci. 21, 8746–8757 PubMed PMC

Kneussel M., Betz H. (2000). Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model. Trends Neurosci. 23, 429–435 10.1016/s0166-2236(00)01627-1 PubMed DOI

Kopach O., Voitenko N. (2013). Extrasynaptic AMPA receptors in the dorsal horn: evidence and functional significance. Brain Res. Bull. 93, 47–56 10.1016/j.brainresbull.2012.11.004 PubMed DOI

Kopp-Scheinpflug C., Dehmel S., Tolnai S., Dietz B., Milenkovic I., Rübsamen R. (2008). Glycine-mediated changes of onset reliability at a mammalian central synapse. Neuroscience 157, 432–445 10.1016/j.neuroscience.2008.08.068 PubMed DOI

Kubota H., Alle H., Betz H., Geiger J. R. (2010). Presynaptic glycine receptors on hippocampal mossy fibers. Biochem. Biophys. Res. Commun. 393, 587–591 10.1016/j.bbrc.2010.02.019 PubMed DOI

Kullmann D. M. (2001). Presynaptic kainate receptors in the hippocampus: slowly emerging from obscurity. Neuron 32, 561–564 10.1016/s0896-6273(01)00507-4 PubMed DOI

Kusumi A., Fujiwara T. K., Morone N., Yoshida K. J., Chadda R., Xie M., et al. (2012). Membrane mechanisms for signal transduction: the coupling of the meso-scale raft domains to membrane-skeleton-induced compartments and dynamic protein complexes. Semin. Cell Dev. Biol. 23, 126–144 10.1016/j.semcdb.2012.01.018 PubMed DOI

Leão R. M., Kushmerick C., Pinaud R., Renden R., Li G. L., Taschenberger H., et al. (2005). Presynaptic Na+ channels: locus, development and recovery from inactivation at a high-fidelity synapse. J. Neurosci. 25, 3724–3738 10.1523/jneurosci.3983-04.2005 PubMed DOI PMC

Lester H. A., Dibas M. I., Dahan D. S., Leite J. F., Dougherty D. A. (2004). Cys-loop receptors: new twists and turns. Trends Neurosci. 27, 329–336 10.1016/s0166-2236(04)00109-2 PubMed DOI

Lévi S., Schweizer C., Bannai H., Pascual O., Charrier C., Triller A. (2008). Homeostatic regulation of synaptic GlyR numbers driven by lateral diffusion. Neuron 59, 261–273 10.1016/j.neuron.2008.05.030 PubMed DOI

Masugi-Tokita M., Tarusawa E., Watanabe M., Molnár E., Fujimoto K., Shigemoto R. (2007). Number and density of AMPA receptors in individual synapses in the rat cerebellum as revealed by SDS-digested freeze-fracture replica labeling. J. Neurosci. 27, 2135–2144 10.1523/jneurosci.2861-06.2007 PubMed DOI PMC

Meier J., Vannier C., Sergé A., Triller A., Choquet D. (2001). Fast and reversible trapping of surface glycine receptors by gephyrin. Nat. Neurosci. 4, 253–260 10.1038/85099 PubMed DOI

Morkve S. H., Hartveit E. (2009). Properties of glycine receptors underlying synaptic currents in presynaptic axon terminals of rod bipolar cells in the rat retina. J. Physiol. 587, 3813–3830 10.1113/jphysiol.2009.173583 PubMed DOI PMC

Muller E., Le-Corronc H., Legendre P. (2008). Extrasynaptic and postsynaptic receptors in glycinergic and GABAergic neurotransmission: a division of labor? Front. Mol. Neurosci. 1:3 10.3389/neuro.02.003.2008 PubMed DOI PMC

Newpher T. M., Ehlers M. D. (2008). Glutamate receptor dynamics in dendritic microdomains. Neuron 58, 472–497 10.1016/j.neuron.2008.04.030 PubMed DOI PMC

Nusser Z., Sieghart W., Somogyi P. (1998). Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J. Neurosci. 18, 1693–1703 PubMed PMC

Nyíri G., Cserép C., Szabadits E., Mackie K., Freund T. F. (2005). CB1 cannabinoid receptors are enriched in the perisynaptic annulus and on preterminal segments of hippocampal GABAergic axons. Neuroscience 136, 811–822 10.1016/j.neuroscience.2005.01.026 PubMed DOI

Paspalas C. D., Goldman-Rakic P. S. (2005). Presynaptic D1 dopamine receptors in primate prefrontal cortex: target-specific expression in the glutamatergic synapse. J. Neurosci. 25, 1260–1267 10.1523/jneurosci.3436-04.2005 PubMed DOI PMC

Perkins G. A., Tjong J., Brown J. M., Poquiz P. H., Scott R. T., Kolson D. R., et al. (2010). The micro-architecture of mitochondria at active zones: electron tomography reveals novel anchoring scaffolds and cristae structured for high-rate metabolism. J. Neurosci. 30, 1015–1026 10.1523/JNEUROSCI.1517-09.2010 PubMed DOI PMC

Petralia R. S. (2012). Distribution of extrasynaptic NMDA receptors on neurons. ScientificWorldJournal 2012:267120 10.1100/2012/267120 PubMed DOI PMC

Pinheiro P. S., Mulle C. (2008). Presynaptic glutamate receptors: physiological functions and mechanisms of action. Nat. Rev. Neurosci. 9, 423–436 10.1038/nrn2379 PubMed DOI

Price G. D., Trussell L. O. (2006). Estimate of the chloride concentration in a central glutamatergic terminal: a gramicidin perforated-patch study on the calyx of held. J. Neurosci. 26, 11432–11436 10.1523/jneurosci.1660-06.2006 PubMed DOI PMC

Reyes-Haro D., Müller J., Boresch M., Pivneva T., Benedetti B., Scheller A., et al. (2010). Neuron-astrocyte interactions in the medial nucleus of the trapezoid body. J. Gen. Physiol. 135, 583–594 10.1085/jgp.200910354 PubMed DOI PMC

Rowland K. C., Irby N. K., Spirou G. A. (2000). Specialized synapse-associated structures within the calyx of held. J. Neurosci. 20, 9135–9144 PubMed PMC

Rubio M. E., Soto F. (2001). Distinct localization of P2X receptors at excitatory postsynaptic specializations. J. Neurosci. 21, 641–653 PubMed PMC

Ruiz A., Fabian-Fine R., Scott R., Walker M. C., Rusakov D. A., Kullmann D. M. (2003). GABAA receptors at hippocampal mossy fibers. Neuron 39, 961–973 10.1016/s0896-6273(03)00559-2 PubMed DOI PMC

Ruiz A. J., Kullmann D. M. (2013). Ionotropic receptors at hippocampal mossy fibers: roles in axonal excitability, synaptic transmission and plasticity. Front. Neural Circuits 6:112 10.3389/fncir.2012.00112 PubMed DOI PMC

Rusakov D. A., Kullmann D. M., Stewart M. G. (1999). Hippocampal synapses: do they talk to their neighbours? Trends Neurosci. 22, 382–388 10.1016/s0166-2236(99)01425-3 PubMed DOI

Sätzler K., Söhl L. F., Bollmann J. H., Borst J. G., Frotscher M., Sakmann B., et al. (2002). Three-dimensional reconstruction of a calyx of held and its postsynaptic principal neuron in the medial nucleus of the trapezoid body. J. Neurosci. 22, 10567–10579 PubMed PMC

Sheets E. D., Simson R., Jacobson K. (1995). New insights into membrane dynamics from the analysis of cell surface interactions by physical methods. Curr. Opin. Cell Biol. 7, 707–714 10.1016/0955-0674(95)80113-8 PubMed DOI

Spirou G. A., Chirila F. V., von Gersdorff H., Manis P. B. (2008). Heterogeneous Ca2+ influx along the adult calyx of held: a structural and computational study. Neuroscience 154, 171–185 10.1016/j.neuroscience.2008.04.002 PubMed DOI PMC

Tamaru Y., Nomura S., Mizuno N., Shigemoto R. (2001). Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: differential location relative to pre- and postsynaptic sites. Neuroscience 106, 481–503 10.1016/s0306-4522(01)00305-0 PubMed DOI

Taschenberger H., Leão R. M., Rowland K. C., Spirou G. A., von Gersdorff H. (2002). Optimizing synaptic architecture and efficiency for high-frequency transmission. Neuron 36, 1127–1143 10.1016/s0896-6273(02)01137-6 PubMed DOI

Trigo F. F., Bouhours B., Rostaing P., Papageorgiou G., Corrie J. E., Triller A., et al. (2010). Presynaptic miniature GABAergic currents in developing interneurons. Neuron 66, 235–247 10.1016/j.neuron.2010.03.030 PubMed DOI

Trigo F. F., Marty A., Stell B. M. (2008). Axonal GABAA receptors. Eur. J. Neurosci. 28, 841–848 10.1111/j.1460-9568.2008.06404.x PubMed DOI

Triller A., Choquet D. (2005). Surface trafficking of receptors between synaptic and extrasynaptic membranes: and yet they do move! Trends Neurosci. 28, 133–139 10.1016/j.tins.2005.01.001 PubMed DOI

Turecek R., Trussell L. O. (2001). Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse. Nature 411, 587–590 10.1038/35079084 PubMed DOI

Turecek R., Trussell L. O. (2002). Reciprocal developmental regulation of presynaptic ionotropic receptors. Proc. Natl. Acad. Sci. U S A 99, 13884–13889 10.1073/pnas.212419699 PubMed DOI PMC

Uwechue N. M., Marx M. C., Chevy Q., Billups B. (2012). Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes. J. Physiol. 590, 2317–2331 10.1113/jphysiol.2011.226605 PubMed DOI PMC

Verhoog M. B., Mansvelder H. D. (2011). Presynaptic ionotropic receptors controlling and modulating the rules for spike timing-dependent plasticity. Neural Plast. 2011:870763 10.1155/2011/870763 PubMed DOI PMC

Vizi E. S., Fekete A., Karoly R., Mike A. (2010). Non-synaptic receptors and transporters involved in brain functions and targets of drug treatment. Br. J. Pharmacol. 160, 785–809 10.1111/j.1476-5381.2009.00624.x PubMed DOI PMC

Walmsley B., Alvarez F. J., Fyffe R. E. (1998). Diversity of structure and function at mammalian central synapses. Trends Neurosci. 21, 81–88 10.1016/s0166-2236(97)01170-3 PubMed DOI

Wei W., Zhang N., Peng Z., Houser C. R., Mody I. (2003). Perisynaptic localization of delta subunit-containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus. J. Neurosci. 23, 10650–10661 PubMed PMC

Wimmer V. C., Horstmann H., Groh A., Kuner T. (2006). Donut-like topology of synaptic vesicles with a central cluster of mitochondria wrapped into membrane protrusions: a novel structure-function module of the adult calyx of held. J. Neurosci. 26, 109–116 10.1523/jneurosci.3268-05.2006 PubMed DOI PMC

Xiong W., Chen S. R., He L., Cheng K., Zhao Y. L., Chen H., et al. (2014). Presynaptic glycine receptors as a potential therapeutic target for hyperekplexia disease. Nat. Neurosci. 17, 232–239 10.1038/nn.3615 PubMed DOI PMC

Zafra F., Aragón C., Olivares L., Danbolt N. C., Giménez C., Storm-Mathisen J. (1995). Glycine transporters are differentially expressed among CNS cells. J. Neurosci. 15, 3952–3969 PubMed PMC

Zarei M. M., Radcliffe K. A., Chen D., Patrick J. W., Dani J. A. (1999). Distributions of nicotinic acetylcholine receptor alpha7 and beta2 subunits on cultured hippocampal neurons. Neuroscience 88, 755–764 10.1016/s0306-4522(98)00246-2 PubMed DOI

Zeilhofer H. U., Studler B., Arabadzisz D., Schweizer C., Ahmadi S., Layh B., et al. (2005). Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice. J. Comp. Neurol. 482, 123–141 10.1002/cne.20349 PubMed DOI

The role of GABAB receptors in the subcortical pathways of the mammalian auditory system