Functional Characterization of Individual Pre- and Postsynaptic Partners In the Drosophila Larval Central Nervous System Using CaMPARI

Summary

This protocol assesses functional synaptic activity between defined neuronal partners in vivo in the central nervous system of Drosophila melanogaster larvae using genetically encoded tools. CsChrimson-mediated optogenetic stimulation of presynaptic cIVda sensory neurons induces calcium-dependent photoconversion of CaMPARI in postsynaptic Basin-4 interneurons.

Abstract

Connectome studies have greatly expanded understanding of synaptic connectivity in the nervous systems of various species. While characterization of synaptic partners using electron microscopy (EM) provides detailed anatomical information, functional aspects of neuronal networks require complementary approaches. Recent studies in Drosophila have revealed not only the complete connectome of the larval, as well as male and female adult central nervous system, but also the cellular components of neuronal networks that regulate specific behaviors, such as the larval nociceptive network. By the third instar larval stage, class IV dendritic arborization (cIVda) multidendritic sensory neurons (nociceptors) establish most synaptic contacts with Basin-4 interneurons. To assess the functional relevance of these anatomical connections, the genetically encoded calcium indicator CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) was employed in live, undissected third-instar larvae as an activity-dependent reporter to evaluate synaptic connectivity between cIVda neurons and Basin-4 interneurons. CaMPARI is a ratiometric fluorescent indicator whose emission spectrum changes in response to elevated intracellular calcium levels. Under baseline conditions, CaMPARI fluoresces green; in the presence of high calcium concentrations and upon exposure to photoconversion light (~400 nm), it irreversibly switches to red fluorescence. Because calcium influx into postsynaptic neurons is a hallmark of synaptic activation, CaMPARI photoconversion provides a readout of functional synaptic signaling. A step-by-step method is presented to immobilize third-instar larvae on a microscope slide for optogenetic activation of cIVda nociceptors using the red-shifted channelrhodopsin CsChrimson, combined with simultaneous CaMPARI photoconversion in Basin-4 neurons. Calcium-dependent photoconversion in Basin-4 neurons, despite internal movements and changes in focal plane, provides functional evidence of synaptic connectivity between these cells. This serves as proof-of-principle for the use of CaMPARI in combination with presynaptic optogenetic stimulation in intact, undissected Drosophila larvae.

Introduction

Identifying precise synaptic partners in the central nervous system (CNS) enables tracking of the formation and refinement of synaptic connections during nervous system development. Accordingly, efforts have focused on the use of volumetric electron microscopy (EM) to reconstruct not only complete connectomes in powerful genetic model organisms such as Caenorhabditis elegans^1,2 and Drosophila melanogaster^3,4,5, but also millimeter-scale EM volumes of complex mammalian brains, including the mouse prim....

Protocol

All experimental procedures involving Drosophila melanogaster were conducted in accordance with institutional guidelines. The genotype w; Basin4-LexA/CyO-TwGFP; ppk-GAL4/ppk-GAL4 (obtained from recombining RRID:BDSC_54899¹² and RRID:BDSC_32079¹⁶) was used to drive optogenetic and CaMPARI expression (using the line w^-, UAS-IVS-CsChrimson.mVenus, LexAOp-CaMPARI2; Sp/Cyo obtained from recombining RRID:BDSC_81085 with second chromosome markers) in pre- and postsynaptic partners, respectively. The research tools used in this protocol are listed in the Table of Materials.

Results

Following this protocol, functional connectivity between cIVda neurons and Basin-4 interneurons was assessed in live, undissected D. melanogaster third instar larvae using CaMPARI (Figure 2). Prior to any stimulation with photoconversion (PC) light or optogenetic activation, Basin-4 interneurons displayed green fluorescence due to cytosolic CaMPARI expression (Figure 2A). No red fluorescence was detected under baseline conditions (F.......

Discussion

The protocol described here enables qualitative and quantitative assessment of synaptic connectivity between defined synaptic partners in intact D. melanogaster larvae. This approach leverages the combination of optogenetic stimulation of cIVda sensory neurons with CaMPARI-based visualization of activated postsynaptic Basin-4 interneurons to monitor synaptic activity in the central nervous system (CNS) of undissected larvae. Previous applications of CaMPARI in D. melanogaster focused primarily on adult .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1 M Phosphate Buffered Saline (PBS)	Sigma Aldrich	P5368-10PAK	Chemical used in solution to hydrate and mount larvae
All-trans-retinal (ATR)	Sigma Aldrich	R2500-500mg	Chemical added to food to activate optogenetic tools
Confocal Microscope	ZEISS	LSM900 Confocal	Confocal microscope, Objective lens (40×/1.2 NA), Light/laser source (405, 488, 561 nm)
Coverslips (18x18mm)	VWR	48366-205	Used to mount animals for imaging
Ethanol (95%)	Sigma Aldrich	E7023	Used as solvent for powder ATR
Fiji (ImageJ)	Open source	RRID:SCR_002285	Used for imaging analysis
Prism version 10.4.1	GraphPad Software Inc.	RRID:SCR_002798	Used for statistical analysis
Microscope slides	VWR	48300-041	Used to mount animals for imaging
Modeling clay	Flinn scientific	FB0600	Used to hold cover glass on microscope slide with larvae in between
Paintbrush	Genesee scientific	59-204	Used to transfer larvae between locations
Standard cornmeal agar medium	Genesee scientific	66-123	Food can also be prepared using similar standard recipes and ingredients
Standard Drosophila plastic vials (25mm x 95mm Drosophila narrow vials)	Genesee scientific	32-109	Large vials or bottles can also be used
Three-well micro spot plate	Electron Microscopy Sciences	71561-01	Plate used to separate and clean individual larva
Drosophila stock (genotype: w; Basin4-LexA/CyO-TwGFP; ppk-GAL4/ppk-GAL4)	Bloomington Drosophila Stock Center	RRID:BDSC_54899 & RRID:BDSC_32079	Obtained from recombining RRID:BDSC_54899 and RRID:BDSC_32079
Drosophila stock (genotype: w, UAS-IVS-CsChrimson.mVenus, LexAOp-CaMPARI2; Sp/CyO)	Bloomington Drosophila Stock Center	RRID:BDSC_81085	Obtained from recombining RRID:BDSC_81085 with second chromosome markers