Cristofari et al. VAChT and VGLUT3 distribution in CINs 1 Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities

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Cristofari et al. VAChT and VGLUT3 distribution in CINs

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities

suggests colocalization and segregation of the two transporters in synaptic vesicles

Paola Cristofari 1†, Mazarine Desplanque 1†, Odile Poirel 1, Alison Hébert 1, Sylvie Dumas 2,

Etienne Herzog 3, Lydia Danglot 4,5,6, David Geny 5, Jean-François Gilles 7,

Audrey Geeverding8, Susanne Bolte 7, Alexis Canette 8, Michaël Trichet 7, Véronique Fabre 1,

Stéphanie Daumas 1, Nicolas Pietrancosta 1,9, Salah El Mestikawy 1,10 and

Véronique Bernard 1,11*

†Both authors contributed equally to this work

1 Sorbonne Université - CNRS UMR 8246 - INSERM U1130 - Neuroscience Paris Seine -

Institut de Biologie Paris Seine (NPS - IBPS), F-75005 Paris, France.

2 Oramacel, F-75006 Paris, France.

3 Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, F-

33000 Bordeaux, France.

4 Université de Paris, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM

U1266, Membrane Traffic in Healthy & Diseased Brain, F-75014 Paris, France.

5 Université de Paris, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM

U1266, NeurImag Imaging facility, F-75014 Paris, France.

6 GHU PARIS Psychiatrie & Neurosciences, F-75014 Paris, France.

7 Imaging facility of the Institut de Biologie Paris‐Seine (IBPS) - Sorbonne Université, F-

75005 Paris, France.

8 Sorbonne Université, CNRS, Institut de Biologie Paris-Seine (IBPS), Service de

microscopie électronique (IBPS-SME), F-75005, Paris.

9 Sorbonne Université - CNRS UMR 7203 – Laboratoire des BioMolécules, F-75005 Paris,

France.

10 Douglas Mental Health University Institute, Department of Psychiatry, McGill University,

Montreal, Canada.

11 Lead contact

*Correspondence: Véronique Bernard (veronique.bernard@inserm.fr)

Cristofari et al. VAChT and VGLUT3 distribution in CINs

Keywords:

Striatum, striatal cholinergic interneurons, acetylcholine-glutamate cotransmission, vesicular

acetylcholine transporter, VAChT, Type 3 vesicular glutamate transporter, VGLUT3,

scanning electron microscopy (SEM), STimulated Emission Depletion microscopy (STED).

Cristofari et al. VAChT and VGLUT3 distribution in CINs

Abstract

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to

regulate the striatal network since they express vesicular transporters for ACh (VAChT) and

Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or

independently from cholinergic varicosities is an open question. The answer to that question

requires the multichannel detection of vesicular transporters at the level of single synaptic

vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy

(STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs.

Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots

revealed that 34 % of CINs SVs contain both VAChT and VGLUT3. In addition, 40 % of

SVs expressed only VAChT while 26 % of SVs contain only VGLUT3.

These results suggest that SVs from CINs have the potential to store simultaneously or

independently ACh and/or Glut. Overall, these morphological findings support the notion that

CINs varicosities can signal with either ACh or Glut or both with an unexpected level of

complexity.

Cristofari et al. VAChT and VGLUT3 distribution in CINs

Introduction

The striatum regulates reward- and habit-guided behaviors as well as locomotor activity

(Graybiel, 2008; Palmiter, 2008) and is involved in a wide range of neurological and

psychiatric disorders (Crittenden et al., 2014; Florio et al., 2018). Striatal GABAergic

medium spiny output neurons are modulated by dopaminergic inputs as well by striatal

cholinergic interneurons (CINs) (Witten et al., 2010; Lim et al., 2014). CINs represent 1% of

striatal neurons but form an extensively ramified network (Morris et al., 2004; Cragg, 2006;

Goldberg and Reynolds, 2011). CINs express vesicular transporters for acetylcholine

(VAChT) and for glutamate (VGLUT3) and consequently regulate the striatal network with

both acetylcholine (ACh) and glutamate (Glut) (Gras et al., 2002, 2008; Higley et al., 2011).

In the striatum, VGLUT3 enhances ACh accumulation and cholinergic transmission (Gras et

al., 2008). Similarly, recently, it was found that Zn2+ facilitate Glut vesicular accumulation

through the presence of two transporters ZnT3 and VGLUT1 on the same vesicles (Upmanyu

et al., 2022). This presynaptic mechanism named “vesicular synergy” has also been reported

in serotonergic, dopaminergic and GABAergic terminals (Hnasko et al., 2010; El Mestikawy

et al., 2011; Amilhon et al., 2010; Frahm et al., 2015; Trudeau and El Mestikawy, 2018;

Zander et al., 2010). The current mechanistic explanation of vesicular synergy is based on a

VGLUT-dependent acidification of SVs due to the intralumenal accumulation of Glut (Gras

et al., 2008; Amilhon et al., 2010; Hnasko et al., 2010; El Mestikawy et al., 2011; Frahm et

al., 2015). This model and the fact that immuno-isolated VGLUT3-positive SVs from rat

striatum adsorbed VGLUT3- and VAChT-positive vesicles suggest (but does not prove) that

VGLUT3 and VAChT are expressed on the same population of SVs. However, several recent

findings and indirect evidence conflict with this hypothesis. For example, cholinergic neurons

from the medial habenula, projecting to the interpeduncular nucleus, corelease ACh and Glut

(Ren et al., 2011; Frahm et al., 2015; Souter et al., 2022). Optogenetic single pulse stimulation

Cristofari et al. VAChT and VGLUT3 distribution in CINs

of interpeduncular cholinergic terminals triggers glutamatergic currents whereas their tetanic

stimulation elicit cholinergic currents (Ren et al., 2011). Therefore, these cholinergic

terminals have the capacity to independently release ACh or Glut depending on their firing

frequency. This finding suggests that in the IPN, ACh and Glut are stored, at least partially, in

independent pools of SVs. In addition, a recent study demonstrated, using single-vesicle

imaging techniques, that only a minority of SVs from total brain extract contains two

transporters (Upmanyu et al., 2022). Therefore, whether a single SV from CINs can express

VAChT or / and VGLUT3 and consequently store independently or co-store ACh or Glut

remains to be established.

One way to test our hypotheses is to visualize vesicular transporters to determine their

distribution at the level of single SVs. Here, we used nanoscale imaging with STimulated

Emission Depletion microscopy (STED) to assess the relative distribution of VAChT and

VGLUT3 in CINs SVs. We show that striatal cholinergic varicosities contain three

subpopulations of SVs expressing either VAChT or VGLUT3, or both. These findings

provide morphological evidence that open the way to a deeper understanding of mechanisms

underlying ACh/Glut co-transmission by CINs.

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Cristofarietal.VAChTandVGLUT3distributioninCINs1NanoscopicdistributionofVAChTandVGLUT3instriatalcholinergicvaricositiessuggestscolocalizationandsegregationofthetwotransportersinsynapticvesiclesPaolaCristofari1†,MazarineDesplanque1†,OdilePoirel1,AlisonHébert1,SylvieDumas2,EtienneHerzog3,LydiaDanglot4...

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Cristofari et al. VAChT and VGLUT3 distribution in CINs 1 Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities

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