Top

Published in:

Open Access 01-04-2017 | Original Article

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

Authors: Arnauld Belmer, Paul M. Klenowski, Omkar L. Patkar, Selena E. Bartlett

Published in: Brain Structure and Function | Issue 3/2017

Abstract

Serotonin neurons arise from the brainstem raphe nuclei and send their projections throughout the brain to release 5-HT which acts as a modulator of several neuronal populations. Previous electron microscopy studies in rats have morphologically determined the distribution of 5-HT release sites (boutons) in certain brain regions and have shown that 5-HT containing boutons form synaptic contacts that are either symmetric or asymmetric. In addition, 5-HT boutons can form synaptic triads with the pre- and postsynaptic specializations of either symmetrical or asymmetrical synapses. However, due to the labor intensive processing of serial sections required by electron microscopy, little is known about the neurochemical properties or the quantitative distribution of 5-HT triads within whole brain or discrete subregions. Therefore, we used a semi-automated approach that combines immunohistochemistry and high-resolution confocal microscopy to label serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions. We also used antibodies against key pre- (synaptophysin) and postsynaptic components of excitatory (PSD95) or inhibitory (gephyrin) synapses to (1) identify putative 5-HTergic boutons within SERT immunoreactive axons and, (2) quantify their close apposition to neurochemical excitatory or inhibitory synapses. We provide a 5-HTergic axon density map and have determined the ratio of synaptic triads consisting of a 5-HT bouton in close proximity to either neurochemical excitatory or inhibitory synapses within different limbic brain areas. The ability to model and map changes in 5-HTergic axonal density and the formation of triadic connectivity within whole brain regions using this rapid and quantitative approach offers new possibilities for studying neuroplastic changes in the 5-HTergic pathway.

Available only for authorised users

Aghajanian GK, Marek GJ (1997) Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells. Neuropharmacology 36:589–599CrossRefPubMed

Audet MA, Descarries L, Doucet G (1989) Quantified regional and laminar distribution of the serotonin innervation in the anterior half of adult rat cerebral cortex. J Chem Neuroanat 2:29–44PubMed

Azmitia EC (1999) Serotonin neurons, neuroplasticity, and homeostasis of neural tissue. Neuropsychopharmacology 21:33S–45S. doi:10.1016/S0893-133X(99)00022-6 CrossRefPubMed

Bankson MG, Yamamoto BK (2004) Serotonin-GABA interactions modulate MDMA-induced mesolimbic dopamine release. J Neurochem 91:852–859. doi:10.1111/j.1471-4159.2004.02763.x CrossRefPubMed

Belmer A, Patkar OL, Pitman KM, Bartlett SE (2016) Serotonergic plasticity in alcohol addiction. Brain Plast 1:177–206. doi:10.3233/BPL-150022 CrossRef

Brown P, Molliver ME (2000) Dual serotonin (5-HT) projections to the nucleus accumbens core and shell: relation of the 5-HT transporter to amphetamine-induced neurotoxicity. J Neurosci Off J Soc Neurosci 20:1952–1963

Bunin MA, Wightman RM (1998) Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission. J Neurosci Off J Soc Neurosci 18:4854–4860

Busse B, Smith S (2013) Automated analysis of a diverse synapse population. PLoS Comput Biol 9:e1002976. doi:10.1371/journal.pcbi.1002976 CrossRefPubMedPubMedCentral

Cameron DL, Williams JT (1994) Cocaine inhibits GABA release in the VTA through endogenous 5-HT. J Neurosci Off J Soc Neurosci 14:6763–6767

Ciocchi S, Herry C, Grenier F et al (2010) Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 468:277–282. doi:10.1038/nature09559 CrossRefPubMed

Ciranna L (2006) Serotonin as a modulator of glutamate- and GABA-mediated neurotransmission: implications in physiological functions and in pathology. Curr Neuropharmacol 4:101–114CrossRefPubMedPubMedCentral

Danielson E, Lee SH (2014) SynPAnal: software for rapid quantification of the density and intensity of protein puncta from fluorescence microscopy images of neurons. PLoS One. doi:10.1371/journal.pone.0115298 PubMedPubMedCentral

de Strauss CVA, Vicente MA, Zangrossi H (2013) Activation of 5-HT1A receptors in the rat basolateral amygdala induces both anxiolytic and antipanic-like effects. Behav Brain Res 246:103–110. doi:10.1016/j.bbr.2013.03.005 CrossRefPubMed

De-Miguel FF, Trueta C (2005) Synaptic and extrasynaptic secretion of serotonin. Cell Mol Neurobiol 25:297–312CrossRefPubMed

Descarries L, Riad M, Parent M (2010) Ultrastructure of the serotonin innervation in the mammalian central nervous system. In: Jacobs BL, Müller CP (eds) Handbook of behavioral neuroscience. Elsevier, Amsterdam, pp 65–101

Dumitriu D, Berger SI, Hamo C et al (2012) Vamping: stereology-based automated quantification of fluorescent puncta size and density. J Neurosci Methods 209:97–105. doi:10.1016/j.jneumeth.2012.05.031 CrossRefPubMedPubMedCentral

Fogarty MJ, Hammond LA, Kanjhan R et al (2013) A method for the three-dimensional reconstruction of Neurobiotin^TM-filled neurons and the location of their synaptic inputs. Front Neural Circuits 7:153. doi:10.3389/fncir.2013.00153 CrossRefPubMedPubMedCentral

Gilpin NW, Herman MA, Roberto M (2015) The central amygdala as an integrative hub for anxiety and alcohol use disorders. Biol Psychiatry 77:859–869. doi:10.1016/j.biopsych.2014.09.008 CrossRefPubMed

González-Maeso J, Ang RL, Yuen T et al (2008) Identification of a serotonin/glutamate receptor complex implicated in psychosis. Nature 452:93–97. doi:10.1038/nature06612 CrossRefPubMedPubMedCentral

Hatzidimitriou G, McCann UD, Ricaurte GA (1999) Altered serotonin innervation patterns in the forebrain of monkeys treated with (±)3,4-methylenedioxymethamphetamine 7 years previously: factors influencing abnormal recovery. J Neurosci Off J Soc Neurosci 19:5096–5107

Hervé D, Pickel VM, Joh TH, Beaudet A (1987) Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons. Brain Res 435:71–83CrossRefPubMed

Ippolito DM, Eroglu C (2010) Quantifying synapses: an immunocytochemistry-based assay to quantify synapse number. J Vis Exp JoVE. doi:10.3791/2270 PubMed

Jiang X, Xing G, Yang C et al (2008) Stress impairs 5-HT2A receptor-mediated serotonergic facilitation of GABA release in juvenile rat basolateral amygdala. Neuropsychopharmacology 34:410–423. doi:10.1038/npp.2008.71 CrossRefPubMed

Jiang H, Fang D, Kong L-Y et al (2014) Sensitization of neurons in the central nucleus of the amygdala via the decreased GABAergic inhibition contributes to the development of neuropathic pain-related anxiety-like behaviors in rats. Mol Brain 7:72. doi:10.1186/s13041-014-0072-z CrossRefPubMedPubMedCentral

Johnson SW, Mercuri NB, North RA (1992) 5-hydroxytryptamine1B receptors block the GABAB synaptic potential in rat dopamine neurons. J Neurosci Off J Soc Neurosci 12:2000–2006

Kalin NH, Shelton SE, Davidson RJ (2004) The role of the central nucleus of the amygdala in mediating fear and anxiety in the primate. J Neurosci Off J Soc Neurosci 24:5506–5515. doi:10.1523/JNEUROSCI.0292-04.2004 CrossRef

Kanazawa H, Ieda M, Kimura K et al (2010) Heart failure causes cholinergic transdifferentiation of cardiac sympathetic nerves via gp130-signaling cytokines in rodents. J Clin Invest 120:408–421. doi:10.1172/JCI39778 CrossRefPubMedPubMedCentral

Kasper JM, Booth RG, Peris J (2015) Serotonin-2C receptor agonists decrease potassium-stimulated GABA release in the nucleus accumbens. Synap N Y N 69:78–85. doi:10.1002/syn.21790 CrossRef

Kishimoto K, Koyama S, Akaike N (2000) Presynaptic modulation of synaptic gamma-aminobutyric acid transmission by tandospirone in rat basolateral amygdala. Eur J Pharmacol 407:257–265CrossRefPubMed

Kiss JP (2008) Theory of active antidepressants: a nonsynaptic approach to the treatment of depression. Neurochem Int 52:34–39. doi:10.1016/j.neuint.2007.04.006 CrossRefPubMed

Klenowski PM, Fogarty MJ, Belmer A et al (2015) Structural and functional characterization of dendritic arbors and GABAergic synaptic inputs on interneurons and principal cells in the rat basolateral amygdala. J Neurophysiol 114:942–957. doi:10.1152/jn.00824.2014 CrossRefPubMedPubMedCentral

Koyama S, Kubo C, Rhee JS, Akaike N (1999) Presynaptic serotonergic inhibition of GABAergic synaptic transmission in mechanically dissociated rat basolateral amygdala neurons. J Physiol 518(Pt 2):525–538CrossRefPubMedPubMedCentral

Koyama S, Matsumoto N, Kubo C, Akaike N (2000) Presynaptic 5-HT3 receptor-mediated modulation of synaptic GABA release in the mechanically dissociated rat amygdala neurons. J Physiol 529(Pt 2):373–383CrossRefPubMedPubMedCentral

Koyama S, Matsumoto N, Murakami N et al (2002) Role of presynaptic 5-HT1A and 5-HT3 receptors in modulation of synaptic GABA transmission in dissociated rat basolateral amygdala neurons. Life Sci 72:375–387CrossRefPubMed

Kuramochi M, Nakamura S (2009) Effects of postnatal isolation rearing and antidepressant treatment on the density of serotonergic and noradrenergic axons and depressive behavior in rats. Neuroscience 163:448–455. doi:10.1016/j.neuroscience.2009.06.017 CrossRefPubMed

Lang EJ, Paré D (1997) Synaptic and synaptically activated intrinsic conductances underlie inhibitory potentials in cat lateral amygdaloid projection neurons in vivo. J Neurophysiol 77:353–363PubMed

Lang EJ, Paré D (1998) Synaptic responsiveness of interneurons of the cat lateral amygdaloid nucleus. Neuroscience 83:877–889CrossRefPubMed

Lebrand C, Cases O, Wehrlé R et al (1998) Transient developmental expression of monoamine transporters in the rodent forebrain. J Comp Neurol 401:506–524CrossRefPubMed

LeDoux J (2007) The amygdala. Curr Biol 17:R868–R874. doi:10.1016/j.cub.2007.08.005 CrossRefPubMed

Marek GJ, Aghajanian GK (1998) The electrophysiology of prefrontal serotonin systems: therapeutic implications for mood and psychosis. Biol Psychiatry 44:1118–1127CrossRefPubMed

McMahon HT, Bolshakov VY, Janz R et al (1996) Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release. Proc Natl Acad Sci U S A 93:4760–4764CrossRefPubMedPubMedCentral

Miner LH, Schroeter S, Blakely RD, Sesack SR (2000) Ultrastructural localization of the serotonin transporter in superficial and deep layers of the rat prelimbic prefrontal cortex and its spatial relationship to dopamine terminals. J Comp Neurol 427:220–234CrossRefPubMed

Miyagawa K, Tsuji M, Fujimori K et al (2011) Prenatal stress induces anxiety-like behavior together with the disruption of central serotonin neurons in mice. Neurosci Res 70:111–117. doi:10.1016/j.neures.2011.02.002 CrossRefPubMed

Mo B, Feng N, Renner K, Forster G (2008) Restraint stress increases serotonin release in the central nucleus of the amygdala via activation of corticotropin-releasing factor receptors. Brain Res Bull 76:493–498. doi:10.1016/j.brainresbull.2008.02.011 CrossRefPubMedPubMedCentral

Muller JF, Mascagni F, McDonald AJ (2007) Serotonin-immunoreactive axon terminals innervate pyramidal cells and interneurons in the rat basolateral amygdala. J Comp Neurol 505:314–335. doi:10.1002/cne.21486 CrossRefPubMed

Muramatsu M, Lapiz MDS, Tanaka E, Grenhoff J (1998) Serotonin inhibits synaptic glutamate currents in rat nucleus accumbens neurons via presynaptic 5-HT1B receptors. Eur J Neurosci 10:2371–2379. doi:10.1046/j.1460-9568.1998.00248.x CrossRefPubMed

Narboux-Nême N, Pavone LM, Avallone L et al (2008) Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). Neuropharmacology 55:994–1005. doi:10.1016/j.neuropharm.2008.08.020 CrossRefPubMed

Nielsen K, Brask D, Knudsen GM, Aznar S (2006) Immunodetection of the serotonin transporter protein is a more valid marker for serotonergic fibers than serotonin. Synap N Y N 59:270–276. doi:10.1002/syn.20240 CrossRef

Nunez-Parra A, Maurer RK, Krahe K, Smith RS, Araneda RC (2013) Disruption of centrifugal inhibition to olfactory bulb granule cells impairs olfactory discrimination. Proc Natl Acad Sci U S A 110(36):14777–14782CrossRefPubMedPubMedCentral

O’Dell LE, Parsons LH (2004) Serotonin1B receptors in the ventral tegmental area modulate cocaine-induced increases in nucleus accumbens dopamine levels. J Pharmacol Exp Ther 311:711–719. doi:10.1124/jpet.104.069278 CrossRefPubMed

Ohta K-I, Miki T, Warita K et al (2014) Prolonged maternal separation disturbs the serotonergic system during early brain development. Int J Dev Neurosci Off J Int Soc Dev Neurosci 33:15–21. doi:10.1016/j.ijdevneu.2013.10.007 CrossRef

Oleskevich S, Descarries L, Watkins KC et al (1991) Ultrastructural features of the serotonin innervation in adult rat hippocampus: an immunocytochemical description in single and serial thin sections. Neuroscience 42:777–791CrossRefPubMed

Parsons LH, Koob GF, Weiss F (1999) RU 24969, a 5-HT1B/1A receptor agonist, potentiates cocaine-induced increases in nucleus accumbens dopamine. Synap N Y N 32:132–135. doi:10.1002/(SICI)1098-2396(199905)32:2<132:AID-SYN6>3.0.CO;2-V CrossRef

Pickel VM, Chan J (1999) Ultrastructural localization of the serotonin transporter in limbic and motor compartments of the nucleus accumbens. J Neurosci Off J Soc Neurosci 19:7356–7366

Rainnie DG (1999) Serotonergic modulation of neurotransmission in the rat basolateral amygdala. J Neurophysiol 82:69–85PubMed

Rainnie DG, Asprodini EK, Shinnick-Gallagher P (1991) Inhibitory transmission in the basolateral amygdala. J Neurophysiol 66:999–1009PubMed

Roberto M, Gilpin NW, Siggins GR (2012) The central amygdala and alcohol: role of -aminobutyric acid, glutamate, and neuropeptides. Cold Spring Harb Perspect Med 2:a012195–a012195. doi:10.1101/cshperspect.a012195 CrossRefPubMedPubMedCentral

Sanders J, Singh A, Sterne G et al (2015) Learning-guided automatic three dimensional synapse quantification for drosophila neurons. BMC Bioinformatics. doi:10.1186/s12859-015-0616-y PubMedPubMedCentral

Schätzle P, Wuttke R, Ziegler U, Sonderegger P (2012) Automated quantification of synapses by fluorescence microscopy. J Neurosci Methods 204:144–149. doi:10.1016/j.jneumeth.2011.11.010 CrossRefPubMed

Schrader M, Hell SW, Van der Voort HTM (1996) Potential of confocal microscopes to resolve in the 50–100 nm range. Appl Phys Lett 69:3644–3646CrossRef

Séguéla P, Watkins KC, Descarries L (1989) Ultrastructural relationships of serotonin axon terminals in the cerebral cortex of the adult rat. J Comp Neurol 289:129–142. doi:10.1002/cne.902890111 CrossRefPubMed

Sheng M, Pak DT (2000) Ligand-gated ion channel interactions with cytoskeletal and signaling proteins. Annu Rev Physiol 62:755–778. doi:10.1146/annurev.physiol.62.1.755 CrossRefPubMed

Sheng M, Sala C (2001) PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24:1–29. doi:10.1146/annurev.neuro.24.1.1 CrossRefPubMed

Sigal YM, Speer CM, Babcock HP, Zhuang X (2015) Mapping synaptic input fields of neurons with super-resolution imaging. Cell 163:493–505. doi:10.1016/j.cell.2015.08.033 CrossRefPubMedPubMedCentral

Smiley JF, Goldman-Rakic PS (1996) Serotonergic axons in monkey prefrontal cerebral cortex synapse predominantly on interneurons as demonstrated by serial section electron microscopy. J Comp Neurol 367:431–443. doi:10.1002/(SICI)1096-9861(19960408)367:3<431:AID-CNE8>3.0.CO;2-6 CrossRefPubMed

Snyder-Keller AM, Keller RW (1993) Prenatal cocaine increases striatal serotonin innervation without altering the patch/matrix organization of intrinsic cell types. Brain Res Dev Brain Res 74:261–267CrossRefPubMed

Soiza-Reilly M, Commons KG (2014) Unraveling the architecture of the dorsal raphe synaptic neuropil using high-resolution neuroanatomy. Front Neural Circuits. doi:10.3389/fncir.2014.00105 PubMedPubMedCentral

Soiza-Reilly M, Anderson WB, Vaughan CW, Commons KG (2013) Presynaptic gating of excitation in the dorsal raphe nucleus by GABA. Proc Natl Acad Sci U S A 110:15800–15805. doi:10.1073/pnas.1304505110 CrossRefPubMedPubMedCentral

Sun N, Cassell MD (1993) Intrinsic GABAergic neurons in the rat central extended amygdala. J Comp Neurol 330:381–404. doi:10.1002/cne.903300308 CrossRefPubMed

Tabuchi K, Blundell J, Etherton MR, Hammer RE, Liu X, Powell CM et al (2007) A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318(5847):71–76CrossRefPubMedPubMedCentral

Tallini YN, Shui B, Greene KS et al (2006) BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons. Physiol Genomics 27:391–397. doi:10.1152/physiolgenomics.00092.2006 CrossRefPubMed

Theile JW, Morikawa H, Gonzales RA, Morrisett RA (2009) Role of 5-hydroxytryptamine2C receptors in Ca₂ ⁺ -dependent ethanol potentiation of GABA release onto ventral tegmental area dopamine neurons. J Pharmacol Exp Ther 329:625–633. doi:10.1124/jpet.108.147793 CrossRefPubMedPubMedCentral

Tomer R, Ye L, Hsueh B, Deisseroth K (2014) Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9(7):1682–1697CrossRefPubMedPubMedCentral

Tyagarajan SK, Fritschy J-M (2014) Gephyrin: a master regulator of neuronal function? Nat Rev Neurosci 15:141–156. doi:10.1038/nrn3670 CrossRefPubMed

Van Bockstaele EJ, Pickel VM (1993) Ultrastructure of serotonin-immunoreactive terminals in the core and shell of the rat nucleus accumbens: cellular substrates for interactions with catecholamine afferents. J Comp Neurol 334:603–617. doi:10.1002/cne.903340408 CrossRefPubMed

Van Bockstaele EJ, Chan J, Pickel VM (1996) Pre- and postsynaptic sites for serotonin modulation of GABA-containing neurons in the shell region of the rat nucleus accumbens. J Comp Neurol 371:116–128. doi:10.1002/(SICI)1096-9861(19960715)371:1<116:AID-CNE7>3.0.CO;2-6 CrossRefPubMed

Veinante P, Freund-Mercier MJ (1998) Intrinsic and extrinsic connections of the rat central extended amygdala: an in vivo electrophysiological study of the central amygdaloid nucleus. Brain Res 794:188–198CrossRefPubMed

Vicente MA, Zangrossi H (2014) Involvement of 5-HT2C and 5-HT1A receptors of the basolateral nucleus of the amygdala in the anxiolytic effect of chronic antidepressant treatment. Neuropharmacology 79:127–135. doi:10.1016/j.neuropharm.2013.11.007 CrossRefPubMed

Wang G, Smith SJ (2012) Sub-diffraction limit localization of proteins in volumetric space using bayesian restoration of fluorescence images from ultrathin specimens. PLoS Comput Biol. doi:10.1371/journal.pcbi.1002671

Washburn MS, Moises HC (1992) Inhibitory responses of rat basolateral amygdaloid neurons recorded in vitro. Neuroscience 50:811–830CrossRefPubMed

Xue X, Shao S, Li M et al (2013) Maternal separation induces alterations of serotonergic system in different aged rats. Brain Res Bull 95:15–20. doi:10.1016/j.brainresbull.2013.03.003 CrossRefPubMed

Yan Q-S, Yan S-E (2001) Serotonin-1B receptor-mediated inhibition of [3H]GABA release from rat ventral tegmental area slices. J Neurochem 79:914–922. doi:10.1046/j.1471-4159.2001.00643.x CrossRefPubMed

Zhou FC, Xu Y, Bledsoe S, Lin R, Kelley MR (1996) Serotonin transporter antibodies: production, characterization, and localization in the brain. Brain Res Mol Brain Res 43(1–2):267–278CrossRefPubMed

Zhou L, Huang K-X, Kecojevic A et al (2006) Evidence that serotonin reuptake modulators increase the density of serotonin innervation in the forebrain. J Neurochem 96:396–406. doi:10.1111/j.1471-4159.2005.03562.x CrossRefPubMed

Zhou W, Lee YM, Guy VC, Freed CR (2009) Embryonic stem cells with GFP knocked into the dopamine transporter yield purified dopamine neurons in vitro and from knock-in mice. Stem Cells 27:2952–2961. doi:10.1002/stem.216 CrossRefPubMed

Title: Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain
Authors: Arnauld Belmer
Paul M. Klenowski
Omkar L. Patkar
Selena E. Bartlett
Publication date: 01-04-2017
Publisher: Springer Berlin Heidelberg
Published in: Brain Structure and Function / Issue 3/2017
Print ISSN: 1863-2653
Electronic ISSN: 1863-2661
DOI: https://doi.org/10.1007/s00429-016-1278-x

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Mapping the connectivity of serotonin transporter immunoreactive axons to excitatory and inhibitory neurochemical synapses in the mouse limbic brain

Abstract

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 3/2017

Axon topography of layer 6 spiny cells to orientation map in the primary visual cortex of the cat (area 18)

A cognitive fMRI study in non-manifesting LRRK2 and GBA carriers

Multiparametric characterization of neuronal subpopulations in the ventrolateral preoptic nucleus

A seed-based cross-modal comparison of brain connectivity measures

Functional and neurochemical interactions within the amygdala–medial prefrontal cortex circuit and their relevance to emotional processing

Principles of ipsilateral and contralateral cortico-cortical connectivity in the mouse