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

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-016-1278-x) contains supplementary material, which is available to authorized users.

Excitatory/inhibitory balance of serotonergic axon connectivity in the brain

“…This suggests a potential role of cortical 5-HT in the regulation of neurotransmitter release. In line with this, we showed that 5-HT boutons essentially form synaptic triads with excitatory synapses in the upper layer of the mouse prefrontal cortex and were located closer to the presynaptic than the postsynaptic specializations, also suggesting a role of 5-HT in the regulation of excitatory neurotransmitter (glutamate) release 13 . The potential role of 5-HT in the regulation of excitatory transmission has been demonstrated in electrophysiology studies showing a 5-HT-mediated increase in glutamate release and amplitude of glutamatergic EPSCs at the apical dendrites of pyramidal neurons 19 .…”

“…Therefore, we have recently developed a semi-quantitative method combining high-resolution confocal microscopy and 3D reconstruction of 5-HT transporter SERT-positive axons to map the distribution of 5-HT boutons apposed to either excitatory or inhibitory neurochemical synapses in the mouse limbic brain 13 . Briefly, we labeled serotonin transporter (SERT) immunoreactive axons and reconstruct in 3D their distribution within limbic brain regions.…”

“…In parallel, we used the same masking function to isolate the synaptophysin puncta outside SERT fibers (SYNout) and their engagement into excitatory (SYNout/PSD95 spot pairs) or inhibitory (SYNout/GEPH spot pairs) neurochemical synapses. Finally, we quantified the density of 5-HT boutons forming "triads" with either excitatory or inhibitory neurochemical synapses in various limbic regions of the mouse brain 13 .…”

Acute Ethanol Administration Upregulates Synaptic α4-Subunit of Neuronal Nicotinic Acetylcholine Receptors within the Nucleus Accumbens and Amygdala

“…Images were deconvolved using Huygens Professional v16.10 (Scientific Volume Imaging) with 100 iterations, quality threshold at 0.001, signal to noise ratio at 15 for the three channels. Images were subsequently analysed in Imaris 8.2.1 (Bitplane), as previously described ( Belmer et al, 2017 ). Total YFP punctate fluorescence (α4-YFP) ( Figure 1A ) was quantified using the spot detection function of Imaris, with a spot diameter of 0.4 μm ( Figure 1B ).…”

Neural connection supporting endogenous 5-hydroxytryptamine influence on autonomic activity in medial prefrontal cortex

“…Since Syn, the molecular marker for presynaptic vesicle membrane, can be used for quantitative estimation of synaptic formation (Belmer et al, 2016), the finding that about 16% of the 5-HTergic varicosities densely distributed in layer V of mPFC expressed Syn suggests an equivalent fraction of 5-HT swellings may possibly engage in putative synaptic construct in the same region. This ratio is comparable with that of an EM study showing that about 19% of the total 5-HT varicosities in laminae V-VI of ventral mPFC formed synapses (Miner et al, 2000).…”