Analysis of single neurons by perforated patch clamp recordings and

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Analysis of single neurons by perforated patch clamp recordings and MALDI-TOF mass spectrometry Susanne Neupert, Debora Fusca, Peter Kloppenburg, and Reinhard Predel ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00163 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018

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ACS Chemical Neuroscience

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Analysis of single neurons by perforated patch clamp recordings and MALDI-TOF mass spectrometry

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Susanne Neupert1†, Debora Fusca1,2, Peter Kloppenburg1,2, Reinhard Predel1†

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All authors contributed equally to this work.

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University of Cologne, Department of Biology, Institute for Zoology, Zülpicher Strasse 47b, 50674 Cologne,

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Germany 2

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University

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of Cologne, Zülpicher Str. 47 b, D-50674 Cologne, Germany

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†

Correspondence to: [email protected]; University of Cologne, Department for Biology, Institute for

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Zoology, 50674 Cologne, Germany

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or

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[email protected]; University of Cologne, Department for Biology, Institute for Zoology, 50674 Cologne,

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Germany

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Running title: Probing on single cell level

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KEYWORDS: single cell analysis, perforated patch recording, neuropeptides, acetylcholine, insect

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olfactory system, MALDI-TOF MS

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Author contributions

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SN: Conceptualization, Experiments, Analysis, Writing, Funding acquisition

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DF: Conceptualization, Experiments, Analysis, Writing

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PK: Conceptualization, Writing, Funding acquisition

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RP: Conceptualization, Writing, Funding acquisition

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ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Single cell mass spectrometry has become an established technique to study specific molecular

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properties such as the neuropeptide complement of identified neurons. Here, we describe a strategy to

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characterize, by MALDI-TOF mass spectrometry, neurochemical properties of neurons that were

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identified by their electrophysiological and neuroanatomical properties. The workflow for the first

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time combined perforated patch clamp recordings with dye loading by electroporation for

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electrophysiological and neuroanatomical characterization as well as chemical profiling of somata by

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MALDI-TOF mass spectrometry with subsequent immunocytochemistry. To develop our protocol, we

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used identified central olfactory neurons from the American cockroach Periplaneta americana. First,

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the combined approach was optimized using a relative homogenous, well-characterized neuron

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population of uniglomerular projection neurons, which show acetylcholine esterase immunoreactivity.

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The general applicability of this combined approach was verified on local interneurons, which are a

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diverse neuron population expressing highly differentiated neuropeptidomes. Thus, this study shows

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that the newly established protocol is suitable to comprehensively analyze electrophysiological,

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neuroanatomical, and molecular properties of single neurons. We consider this approach an important

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step to foster single cell analysis in a wide variety of neuron types.

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Introduction

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The functional and computational properties of neuronal networks are largely determined by

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the properties of the neurons that form these networks. Thus, to better understand brain function it is

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crucial to define the functional and morphological phenotypes of the brain’s component neurons.

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Ideally this goal should be addressed by combining several different methods such as cell labeling,

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immunocytochemistry, transcriptomics, mass spectrometry and electrophysiological recordings with

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single cell resolution. Previous studies have elegantly and very successfully used various combinations

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of these methods to analyze single neurons [1-4]. To physiologically and neurochemically characterize

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single neurons, combinations of cellular electrophysiology and mass spectrometry (MS) are highly

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desirable. Previous studies combined whole-cell patch clamp recordings with single cell capillary

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electrophoresis-MS [5]. In this study, our goal was to establish a combination of perforated patch

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clamp recordings, dye loading, and matrix-assisted laser desorption/ionization MALDI-TOF MS to

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detect potential neurotransmitters or neuromodulators in single neurons from intact brain samples.

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Perforated patch-clamp recordings allow long lasting current and voltage clamp recordings from small

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vertebrate and invertebrate neurons in vitro and in vivo with minimal impact on the intracellular

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pathways [e.g. 6,7,8,9]. Single cell MALDI-TOF mass spectrometry (SCMS) has been established as a

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rapid and robust technique for studying specific molecular properties such as the neuropeptide and

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metabolites complement of identified neurons. First performed on giant neurons of molluscs [10],

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SCMS is meanwhile commonly used in neurobiology [5, 11-15]. Here, we illustrate a combination of

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these approaches on identified neurons of the insect olfactory system.

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In insects, the antennal lobe (AL) is the first synaptic processing station in the olfactory

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pathway, and is considered the functional analog of the vertebrate olfactory bulb [16-20]. The antennal

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olfactory receptor neurons, each expressing a single functional receptor gene, direct their axons to the

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AL, where they collate by receptor type and congregate into specific glomeruli. In the glomeruli they

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provide synaptic input to local interneurons (LNs) and projection neurons (PN). The LNs are a

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heterogeneous neuron population with different physiological and morphological phenotypes

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mediating complex inhibitory and excitatory interactions between glomerular pathways to structure

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the olfactory representation in the AL. Projection neurons, the analog of the mammalian mitral and

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tufted cells [21], transfer the integrated olfactory information to higher order processing centers in the

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protocerebrum.

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In the first part of this study we illustrate the performance and workflow of combined

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perforated patch clamp recordings and SCMS on uniglomerular PNs (uPNs). The relative

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homogeneity of the uPN population makes them suitable not only to show the feasibility of our

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combined approach, but also to demonstrate its reliability and reproducibility. This analysis is the first

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proving that the uPNs are cholinergic, which was previously hypothesized based on indirect evidence

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from immunostainings against the biosynthetic enzyme choline acetyl transferase [2,22,23],

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acetylcholine esterase [24-27] and acetylcholine receptors [25,27]. In the second part, to show the 3



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method’s general versatility and applicability, we analyzed the peptide profiles of single

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physiologically and morphologically identified LNs in the antennal lobe. Previous studies using

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immunocytochemistry and MALDI-TOF MS have already revealed a striking diversity of

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neuropeptides in different LN types [2,23,28].

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Combining perforated patch clamp recordings and MALDI-TOF MS allows detailed,

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unambiguous mass spectrometric analysis of putative neurotransmitters and neuromodulators in

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electrophysiologically and morphologically identified neurons. Since perforated patch clamp

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recordings are suitable for long lasting electrophysiological recordings without disrupting cytosolic

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signaling, we consider this approach an important step to foster single cell analysis in a wide variety of

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neuron types.

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Results and Discussion

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The main goal of this study was to establish an experimental protocol to assess

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electrophysiological, morphological and biochemical parameters of single neurons by combining

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perforated patch clamp recordings and MALDI-TOF MS. The results are presented in two parts. First,

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we illustrate in detail the strategy with its consecutive processing steps on uPNs of P. americana

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(Fig. 1). We chose these neurons since the homogeneity of the uPN population makes them particular

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suitable to optimize, validate and show the performance, reliability and reproducibility of this

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approach. Second, we show the performance and general applicability of our workflow on different

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LN types of the insect AL.

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Step 1: Dye loading by electroporation and perforated patch clamp recordings

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Uniglomerular projection neurons were pre-identified by the position and size of their somata,

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which are mostly located in the ventral portion of the ventrolateral somata group [36] (Fig. 1A, B).

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This pre-identification has a high success rate (90%), and was verified in each case by a physiological

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characterization (e.g. spiking versus non-spiking) during and a morphological classification after the

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recording. In initial experiments we performed whole-cell patch-clamp recordings and biocytin tracing

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of individual neurons [23]. This approach is often used to analyze the electrophysiological and

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morphological properties of single neurons. However, in our experiments we observed a distinct

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decrease of transmitters and/or neuropeptides over time due to the exchange of the cytosol with the

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pipette solution which diminished or even abolished the respective ion signals in subsequent mass

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spectrometric analyses. While 86% of the experiments yielded sufficient ion signal intensities when

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recordings lasted less than 5 minutes prior to dissection for mass spectrometry (n = 7), success rate

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decreased to 35% when whole cell recordings were extended to 20 - 40 minutes (n = 17). To overcome

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this issue, we switched to perforated patch-clamp configuration with gramicidin as ionophore, which

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preserved the integrity of intracellular components and kept them available for subsequent mass

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spectrometric analyses. 4


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Since the perforated patch-clamp method hinders diffusion of dyes or intracellular markers

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into the cells, these markers need to be ‘actively moved’ across the membrane by electroporation.

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Therefore, the patch pipette was filled with internal solution containing the intracellular marker

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biocytin as well as gramicidin. Immediately after the patch pipette formed a tight seal on the

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membrane of the target soma of >1 GΩ and before the gramicidin-mediated perforation process

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started, 5-10 500 ms trains of short voltage pulses at 200 Hz (-1 V, duration 1 ms, interpulse interval

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5 ms) were applied to electroporate biocytin across the membrane into the neuron (Fig. 1C-E). During

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the following perforation process with the ionophore, the access resistance (Ra) was constantly

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monitored and electrophysiological experiments were started after Ra and the action potential

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amplitude were stable (after about 15 – 30 min). All recorded uPNs generated Na+-driven action

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potentials upon stimulation with depolarizing current injection, had a membrane potential of -64.6 ±

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8.4 mV (n = 11), and cell input resistance of 97.9 ± 36.9 MΩ, which is in line with previous whole-cell

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recordings [29]. We did not observe differences in resting membrane potential between perforated

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patch recordings with or without initial electroporation for dye loading. Evidently, the electroporation

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protocol did not cause significant amounts of hydrophobic ionophore to cross the membrane. Single-

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cell labeling confirmed that all analyzed neurons were uPNs. Each of these neurons was characterized

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by arborizations in a single glomerulus only and sent its axon via the medial antennal lobe tract

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(mALT) to the protocerebrum (see [37]), innervating the mushroom body calyces and the lateral horn

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(Fig. 1F).

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Step 2: Soma dissection for mass spectrometry

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Following the electrophysiological characterization, the patch pipette was detached from the

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recorded uPN by applying gentle positive pressure and then carefully retracted (Fig. 1G). To collect

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the soma, a collecting pipette with a larger tip diameter (~ 2/3 of the soma) was positioned at the cell

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body using a second micromanipulator (Fig. 1 H, I). After applying gentle negative pressure to

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capture this soma, it could be separated and removed from the brain. The soma was then sucked into

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the tip of the collecting pipette (Fig. 1J). Finally, the outer tip of the pipette containing the soma was

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crushed directly on the sample plate for MALDI-TOF mass spectrometry and air-dried. This procedure

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yielded the most reproducible mass spectra with sufficient ion intensity. In our initial experiments, we

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released the captured soma directly on the sample plate by applying gentle positive pressure but this

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procedure resulted in a clear decrease of neuropeptide ion signals and an increase in false positive ion

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signals. These problems likely resulted from contaminations due to excessive deposition of saline from

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the pipette.

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Step 3: Single cell MALDI-TOF mass spectrometry

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Most reports about SCMS in insects focused on neurosecretory neurons producing peptide

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hormones [e.g. 11,15,38-41]. Such cells are regulated by the transcription factors dimmed and 5



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crytocephal [42] which results in a large number of peptide-containing dense core vesicles and, hence,

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also a high concentration of neuropeptides in the somata. In peptidergic interneurons such as AL

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neurons, the detection of neuropeptides is more difficult (see [28]). This can primarily be attributed to

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a much lower peptide concentration in these cells compared to neurosecretory dimmed-cells. Because

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of that, we had to optimize some established parameters for sample preparation (see [43]) to analyze

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interneurons such as uPNs by SCMS. First of all, cell preparations (see step 2) were not rinsed with

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water prior to matrix application on the sample plate. Secondly, the crushed outer tip of the capillary

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containing the cell body was covered with only 10-20 nl saturated CHCA solution diluted 1:2 with

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methanol/water (50/50). Settings for detection of transmitters (m/z 100 - 300) and putative

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neuropeptides (m/z 600 - 4000) were optimized separately using a laser power with minimal matrix

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ablation.

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First, we analyzed uPN somata in the mass range of m/z 100 – 300 to verify the hypothesis

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that acetylcholine (ACh) is a potential neurotransmitter in uPNs, which was based on indirect evidence

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from previous immunostainings against the biosynthetic enzymes choline acetyltransferase and

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acetylcholine esterase as well as acetylcholine receptors [22-27]. In contrast to other transmitters such

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as the biogenic amines octopamine and tyramine, the detection of ACh does not require a pre-

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extraction, specific sample purification or derivatization of samples for analysis by MALDI-TOF MS

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[44]. Resulting mass spectra of individual dissected uPNs (N=14) shows a distinct ion signal at m/z

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146.12 (Fig. 1K), which was confirmed unambiguously as ACh in all preparations by tandem MS

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experiments. (Fig 1 L).

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Next, we analyzed all uPN sample preparations in the mass range of m/z 600-4000 for

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neuropeptide profiling. Resulting mass spectra from 2 of 14 isolated uPN somata revealed ion signals

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typical of neuropeptides from the allatostatin-A (ast-A) gene (Fig. 1M). These results confirmed for

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the first time the presence of neuropeptides in uPNs of insects. In fact, the detection of AST-A

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peptides corresponds with the positive anti-AST-A immunostaining in the mALT [28] as well as in the

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lateral horn and the mushroom body calyx where the axonal endings of uPNs are located. Since AST-

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A immunoreactivity was almost exclusively detected in axons forming the mALT but not in uPN

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somata, these immunostainings may also explain why AST-A peptides were only sporadically

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detected in mass spectra from uPN somata. It is conceivable that uPNs express ast-A only occasionally

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and the somata are therefore devoid of AST-A peptides most of the time. The presence of

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neuropeptides in axons but not in somata is unusual for insect neurons.

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Step 4: Immunostaining as a control to verify the distribution of neuropeptides, which have been

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detected by mass spectrometry

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As described above (step 3), AST-A signals in mass spectra from uPNs were usually missing

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or represented with very low signal intensity. The mass spectrum shown in Fig. 1L, represented a rare

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case of an uPN preparation with distinct AST-A ion signals. Therefore, co-labeling of biocytin and 6


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anti-AST-A serum has been performed. With this approach, we confirmed not only the presence of

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AST-A peptides in this specific uPN but provided evidence that many other uPN somata of that

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specimen contained anti AST-A immunoreactive material as well (Fig. 1N-Q).

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General applicability

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To demonstrate the performance and general applicability of this approach also for profiling of

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other neurons, we used LNs of the insect AL. The LNs form a diverse neuron population with

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different electrophysiological and morphological phenotypes including spiking and non-spiking

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neurons [29,35]. In contrast to uPNs they have processes in many or all glomeruli. Since the different

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LN types can release various transmitters and neuromodulators, they can mediate complex inhibitory

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and excitatory interactions between glomerular pathways to structure the olfactory representation in

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the AL, which ultimately shapes the tuning profile of the PNs [45-50].

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Type I LNs generate Na+ driven action potentials, express GABA immunoreactivity, and are

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inhibitory [29, 51]. In this study we observed variabilities between the neuropeptidomes of individual

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type I LNs (Fig. 2). The three analyzed type I LNs always contained allatotropin (AT) and two of

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these cells showed co-localization of AT either with tachykinin-related peptides (TKs) or short

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neuropeptide F (sNPF). These findings are in agreement with previous studies where these

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neuropeptides were always abundant in mass spectra of dissected type I LN soma clusters [28], which

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were also immunoreactive to AT and TK antisera [2].

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Type II LNs do not generate Na+ driven action potentials and represent a heterogeneous group

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of neurons that are divided in different subpopulations depending on electrophysiological properties

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and glomerular innervation pattern [2,52]. In each of our analyses (N=17), current injection evoked

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depolarizations in type II LNs but no Na+ driven action potentials (see Fig. 3 A, B). This response

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made it possible to conclusively distinguish type II LNs from uPNs and type I LNs, which respond

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with Na+-driven action potentials. Resulting mass spectra of isolated type II LNs revealed distinct ion

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signals of TKs as well as sNPF as potential neuromodulators. Furthermore, in 4 out of 17 type II LNs

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we detected, by mass spectrometry, the presence of ACh (Fig. 3A). These cells likely belong to type

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IIa1 LNs, which were previously shown to express ChAT immunoreactivity [2]. We also analysed type

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II LNs, which did not contain ACh (Fig 3B). The neuropeptide complement, however, clearly varied

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among these type II LNs and covered TKRPs, sNPF, extended FMRFamides, AT, allatostatin-C (Ast-

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C) and peptides derived from the neuropeptide-like precursor-1 precursor. This variability of the

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peptidome of type II LNs was already suggested previously by mass spectrometric analyses of few

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type II LN soma clusters [28].

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Taken together we have shown that perforated patch clamp recordings, single cell labeling,

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MALDI-TOF MS and immunocytochemistry, which are all utmost powerful methods in neuroscience

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research, can be successfully combined on the single cell level. This approach allows detailed,

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unambiguous mass spectrometric analysis of putative neurotransmitters and neuromodulators that are 7



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synthesized in electrophysiologically and morphologically identified neurons. Since perforated patch

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clamp recordings are suitable for long lasting current and voltage clamp recordings of various cell

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types with minimal impact on the integrity of the cytoplasmic pathways, we consider this approach an

238

important step to foster single neuron analysis.

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Methods

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Animals

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Cockroaches (P. americana) were reared in crowded colonies at a constant temperature of 27°C under

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a 13:11 light:dark photoperiod on a diet of dry rodent food, oatmeal and water. Experiments were

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performed with adult males.

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Brain preparation and cell identification

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The intact brain preparation was based on an approach described previously [29-31], in which the

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entire olfactory network is left intact. Insects were anesthetized by CO2, placed in a custom build

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holder and the head with antennae was immobilized with tape (Tesa ExtraPower Gewebeband, Tesa,

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Hamburg, Germany). Subsequently, the head capsule was opened under visual control (stereo

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microscope M5-63302, Wild Heerbrugg, Switzerland) by cutting a window between the two

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compound eyes at the bases of the antennae. The brain was dissected in insect saline of the following

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composition (in mM): 185 NaCl, 4 KCl, 6 CaCl2, 2 MgCl2, 10 HEPES, 35 D-glucose; adjusted to pH

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7.2 with NaOH and to 430 mOsm with glucose. The brain was then transferred to a Sylgard-coated

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(Dow Corning Corp., Midland, Michigan, USA) recording chamber (~3 ml volume), positioned for the

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recording and fixed with tungsten pins (diameter: 0.05 mm). To gain better access to the olfactory

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neurons of the antennal lobe (AL), the AL was carefully desheathed with fine forceps. To improve

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access to the recording sites, the brain was enzymatically treated at room temperature (24°C) by a

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mixture of papain (0.3 mg·ml-1, P4762, Sigma-Aldrich, Darmstadt, Germany) and Fluka L-cysteine (1

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mg·ml-1, 30090, Sigma-Aldrich) dissolved in insect saline. Enzyme treatment was stopped after 3 min

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by constantly rinsing the brain with 10 to 20 ml of ice-cold insect saline using a 2.5 ml plastic Pasteur

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pipette. During the recording experiment, the brain was superfused with insect saline at a flow rate of

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~2 ml·min-1. Somata of the AL neurons were visualized with a fixed stage upright microscope

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(BX51WI, Olympus, Hamburg, Germany) using a 40x water-immersion objective (UMPLFL, x40, 0.8

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numerical aperture, 3.3 mm working distance, Olympus, Hamburg, Germany) and infrared differential

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interference contrast optics.

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Whole cell and perforated patch recordings

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Whole cell recordings were performed as described previously [32]. The patch electrode was filled

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with internal (referred as pipette-) solution containing (in mM) 190 K-aspartate, 10 NaCl, 1 CaCl2, 2

8


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MgCl2, 10 HEPES, and 10 EGTA adjusted to pH 7.2 with KOH, resulting in an osmolarity of ∼ 415

272

mOsm.

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Perforated patch recordings were performed using protocols modified from [6,7]. The tip of the patch

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pipette was filled with pipette solution (see above) and back filled with 25–75 µg·ml-1 gramicidin-

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containing pipette solution to achieve perforated patch recordings. Gramicidin (G5002; Sigma,

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Steinheim, Germany) was dissolved in dimethyl sulfoxide (DMSO; D8418, Sigma) in a final

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concentration of 0.25 – 0.75 % as described previously [33,34] and was added to the pipette solution

278

shortly before use. DMSO had no obvious effect on the investigated neurons. During the perforation

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process access resistance (Ra) was constantly monitored and experiments were started after Ra had

280

reached steady state (~15 – 30 min) and the action potential amplitude was stable. While we aimed for

281

reproducible recording conditions, Ra had a certain variability. Typically Ra was between 30-40 MΩ.

282

To assess intrinsic electrophysiological parameters in current clamp Ra’s above 60MΩ were excluded.

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With Ras below 60 MΩ the action potential amplitudes and waveforms were similar as recorded in the

284

whole cell patch clamp configuration [29, 35]. In principle recordings with higher Ras (up to 100 MΩ)

285

might still be used to monitor action potential frequency.

286 287

Electrophysiology

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Electrodes with tip resistances between 3 and 5 MΩ were fashioned from borosilicate glass (0.86 mm

289

inner diameter; 1.5 mm outer diameter; GB150-8P; Science Products GmbH, Hofheim, Germany)

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with a vertical pipette puller (PP-830; Narishige, London, UK). Recordings were performed with an

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EPC10 patch-clamp amplifier (HEKA, Lambrecht, Germany) controlled by the program PatchMaster

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(version 2.32; HEKA) running under Windows. Data were sampled at 10 kHz and low-pass filtered at

293

2 kHz with a four-pole Bessel filter. Whole-cell capacitance was determined by using the capacitance

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compensation (C-slow) of the EPC10. Cell input resistances (RM) were calculated from voltage

295

responses to small hyperpolarizing current pulses. The calculated liquid junction potential (with

296

Patcher's Power Tools plug-in from http://www.mpibpc.gwdg.de/abteilungen/140/software/index.html

297

for Igor Pro 6 (Wavemetrics, Lake Oswego, OR, USA)) between internal and external solutions was

298

compensated. Hyperpolarizing and depolarizing current steps were applied to monitor passive and

299

active membrane properties.

300 301

Dye loading of neurons

302

For cell labeling, 1% biocytin (B4261, Sigma-Aldrich) was added to the pipette solution. When

303

recorded in whole cell patch clamp configuration, biocytin was loaded into the cell by application of a

304

hyperpolarizing current of 0.2 - 0.6 nA for 20 - 40 min. In the perforated patch configuration, the

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neurons were juxtasomal filled with biocytin by electroporation via the patch pipette. When a seal

306

resistance of ≥ 1 GΩ was reached, a sequence of 5-10 200 Hz trains (500 ms) of 1 ms high voltage

9



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square pulses (-1V) with an interstimulus interval of 5 ms was applied. Biocytin was then allowed to

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diffuse into the neurites for at least 20 min.

309 310

Sample preparation for single cell MALDI-TOF mass spectrometry

311

Immediately after electrophysiological recording and dye loading, somata were isolated and prepared

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for direct transmitter and neuropeptide profiling by matrix-assisted laser desorption/ionization

313

(MALDI) time-of-flight (TOF) mass spectrometry. First, the soma, while still fixed by the patch

314

pipette, was carefully moved for- and backward to separate the soma from attached neurons.

315

Subsequently, the soma was collected using a collecting pipette with an inner diameter of about 2/3 of

316

the cell body width. The intact soma was then partially absorbed in the pipette tip and slowly pulled

317

out of the cell cluster. During this procedure, the soma was entirely absorbed into the tip of the

318

collecting pipette, creating a plug at the end of the pipette tip. The complete dissection was

319

documented using a two photon laser scanning system installed on the Olympus microscope. Only

320

somata without any visible contaminations were used for further analysis. The collecting pipette was

321

completely removed from the patch-clamp setup and connected to a tube coupled with a 0.1-10 µl

322

pipette tip (Eppendorf, Germany) to control the transfer of saline on the sample plate. Finally, the

323

collecting pipette tip with the dissected cell was smashed onto the sample plate for MALDI-TOF mass

324

spectrometry. About 10–20 nl of saturated α-cyano-4-hydroxycinnamic acid (CHCA) (Sigma-Aldrich)

325

dissolved in 60% methanol (0.1% TFA) and finally diluted 1:2 with 50 % methanol was loaded onto

326

the dried samples over a period of a few seconds using a glass capillary (Hilgenberg GmbH, Malsfeld,

327

Germany). Each spot was air-dried and then covered for a few seconds with purified water to reduce

328

salt contamination.

329 330

MALDI-TOF/TOF mass spectrometry

331

MALDI-TOF mass spectra were acquired in positive ion mode on a 4800 Proteomics Analyzer with

332

TOF/TOF optics (AB Sciex Germany GmbH, Darmstadt). All acquisitions were taken in manual

333

mode. Initially the instrument was operated in reflectron mode using a delayed extraction time of 100

334

ns, 75 % grid voltage, 0.02-0.06 % guide wire voltage, and an accelerating voltage of 20 kV. Laser

335

strength was adjusted to provide the optimal signal-to-noise ratio. The higher mass range of m/z 600 -

336

4000 was used to detect neuropeptide signals and settings optimized for the lower mass range of m/z

337

100 - 300 were used to detect the neurotransmitter ACh. An external mass spectrum calibration was

338

performed using synthetic peptides from an AB Sciex peptide standard kit or a synthetic standard

339

mixture of 1M acetylcholine, 1M γ-aminobutyric acid (GABA) and 1M threonine. The data obtained

340

in these experiments were handled using the Data explorer 4.3 software package. Tandem mass

341

spectrometry (MS/MS) was performed with and/or without collision-induced dissociation. The

342

number of laser shots used to obtain a spectrum varied from 2000–5000, depending on ion signal

343

intensity. The identities of the ion signals described in this study have been verified using MS/MS 10


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344

fragmentation of the molecules. Fragment patterns of peptides with masses corresponding to

345

theoretical masses of Periplaneta peptides were compared with the respective theoretical fragments

346

obtained from ProteinProspector (http://prospector.ucsf.edu) and fragment pattern of ACh was

347

compared with that provided at MELTIN: Metabolite and Tandem MS Database provided by the

348

Scripps Center for Metabolomics (http://meltin.scripps.edu). Principal Component Analysis (PCA)

349

was performed on the raw data by calculating the first three components of a compiled binary feature

350

list representing the detected ion signals in a mass range of m/z 100-300 (small molecules).

351 352

Visualization of Biocytin

353

After dissection of the soma, the brain was fixed in Roti-Histofix (P0873, Carl Roth, Karlsruhe,

354

Germany) for about 12 h at 4 °C and then rinsed in 0.1 M phosphate buffered saline (PBS) (pH 7.2,

355

three times for 20 min). To facilitate streptavidin penetration, these preparations were treated with a

356

mixture of collagenase/dispase (1 mg ml-1, 10 269 638 001, Roche Diagnostics, Mannheim, Germany)

357

and hyaluronidase (1 mg ml-1, H3506, Sigma-Aldrich) in PBS for 20 min at 37 °C; rinsed in PBS

358

(three times for 10 minutes, 4 °C) and preincubated for 40 min in PBS containing 1 % Triton X-100

359

(SERVA, Heidelberg, Germany) and 10 % normal goat serum (S-1000, Vector Labs, Burlingame,

360

CA). Afterwards, the preparations were incubated in Alexa Fluor 633 conjugated streptavidin (Kat. S-

361

21375; Molecular Probes, Eugene, Oregon) dissolved (1:400) in PBS containing 10 % normal goat

362

serum for 1-2 days at 4 °C. After that, samples were rinsed in PBS (3 x 10 min, 4 °C), dehydrated in

363

an ascending ethanol series (50 %, 70 %, 90 %, 2 x 100 %; 10 min each), cleared and mounted in

364

methylsalicylate (M6752, Sigma-Aldrich, Germany).

365 366

Immunocytochemistry

367

Brains were preincubated for 30 min in 5 % normal goat serum dissolved in PBS and then incubated in

368

anti-Diploptera punctata allatostatin A-7 serum (1:2; 5F10 kindly provided by B. Stay) diluted in

369

PBS, 1 % Triton X-100 and 10 % normal goat serum for 3-4 days at 4°C on a laboratory shaker.

370

Following overnight washing in PBS-1 % Triton X-100, pH 7.6, a Cy3-tagged goat anti-mouse

371

secondary antibody were incubated on a laboratory shaker at a concentration of 1:300 for 3 days at 4

372

°C. The preparations were washed again overnight in PBS 1 % Triton X-100, pH 7.6. Finally, the

373

buffer was replaced by purified water. For clearing of tissues, brain preparations were dehydrated in

374

ethanol, cleared in methylsalycylate (Sigma, Steinheim, Germany) and mounted in Entellan (Merck,

375

Darmstadt, Germany).

376 377

Image processing

378

The labeled preparations (immunostainings and dye loaded cell projections) were examined with a

379

confocal laser scanning microscope (ZEISS LSM 510 Meta system; Jena, Germany), equipped with C-

380

Apochromat 10x/0.45W (NA 0.45), Plan-Apochromat 20x/0.75W (NA 0.75), and Plan-Apochromat 11



Page 12 of 23

381

63x/1.4W (Oil, NA=1.4)) objectives using the multi-track mode. For that, streptavidin-Alexa 633 was

382

excited with at 633 nm and emission was collected via a 650 nm LP filter and Cy3 was excited at 543

383

nm and emission collected via a 560 - 615 nm BP filter. Serial optical sections were analyzed with

384

optical sections from 0.3 to 0.8 µm and assembled into combined images using the Zeiss LSM 5 image

385

browser version 3. Scaling, contrast enhancement and z-projections were performed with ImageJ

386

v1.44o and the WCIF plug-in bundle (www.uhnresearch.ca/facilities/wcif/). The final figures were

387

exported and processed to adjust brightness and contrast with Adobe Photoshop 7.0 software (Adobe

388

Systems, San Jose, CA) and Adobe Illustrator CS5 (Adobe Systems, San Jose, CA).

389 390

Acknowledgment

391

We thank Tobias Lamkemayer (University of Cologne) for support during mass spectrometric

392

analysis, and Axel Kersting and Helmut Wratil (University of Cologne) for technical support. Funding

393

was provided by the Deutsche Forschungsgemeinschaft (German Research foundation) (SN: NE911/3-

394

1; RP: PR766/9-1; PR595/10-1; PK: KL762/5-1, KL762/6-1)

395

12


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References

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MS-based peptidomics of individual paraformaldehyde-fixed and immunolabeled neurons. Chem.

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Biol. 19, 1010-9.

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2. Fusca, D., Schachtner, J., Kloppenburg, P. (2015) Colocalization of allatotropin and tachykinin-

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physiologically different local interneuron types of the antennal lobe. J. Neurosci. 29, 716-726.

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responses to female sex pheromone in the male sphinx moth Manduca sexta. J. Neurosci. 19, 8172–

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changes in intracellular chloride concentration. J. Neurosci. Methods 57, 27-35.

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homologous neurons express sequence-related neuropeptides that originate from different genes. J.

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identified neurons demonstrates a highly differentiated expression pattern of FXPRLamides in the

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42. Gauthier, S.A., Hewes, R.S. (2006) Transcriptional regulation of neuropeptide and peptide

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43. Neupert, S., Predel, R. (2010) Peptidomic analysis of single identified neurons. Methods Mol. 615,

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45. Wilson, R.I., Turner, G.C., and Laurent, G. (2004) Transformation of olfactory representations in

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the Drosophila antennal lobe. Science 303, 366–370.

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46. Stopfer, M. (2005) Olfactory coding: inhibition reshapes odor responses. Curr. Biol. 15, 996–

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R998.

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the Drosophila antennal lobe: anything goes? J. Neurosci. 28, 13075–13087.

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48. Shang, Y., Claridge-Chang, A., Sjulson, L., Pypaert, M., and Miesenbock, G. (2007) Excitatory

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49. Olsen, S.R., Bhandawat, V., Wilson, R.I. (2007) Excitatory interactions between olfactory

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processing channels in the Drosophila antennal lobe. Neuron 54, 89–103.

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50. Olsen, S.R., and Wilson, R.I. (2008). Lateral presynaptic inhibition mediates gain control in an

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olfactory circuit. Nature 452, 956–960.

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51. Warren, B., and Kloppenburg, P. (2014). Rapid and Slow Chemical Synaptic Interactions of

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Cholinergic Projection Neurons and GABAergic Local Interneurons in the Insect Antennal Lobe. J.

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Neurosci. 34, 13039–13046.

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52. Husch, A., Paehler, M., Fusca, D., Paeger, L., Kloppenburg, P. (2009) Distinct

521

electrophysiological properties in subtypes of nonspiking olfactory local interneurons correlate with

522

their cell type-specific Ca2+ current profiles. J. Neurophysiol. 102, 2834-2845.

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Page 18 of 23

Legends

524 525

Fig. 1. Workflow developed for the analysis and differentiation of AL neurons, performed on a single

526

uniglomerular projection neuron (uPN) from the cockroach brain. A) Schematic overview of the

527

cockroach brain with antennal lobe (AL), mushroom body (MB), lateral horn (LH), and a single uPN.

528

B) Setup for electrophysiology with recording chamber, recording pipette and collecting pipette. C)

529

Pre-identification of the uPN soma by position and size. Scale bar: 10 µm D) Dye (biocytin) loading

530

into the uPN by electroporation E) Perforated patch-clamp recording of Na+ driven action potentials

531

typical for uPNs upon depolarizing current injections. F) Visualization of biocytin distribution using

532

conjugated streptavidin. As expected, the uPN arborized in a single glomerulus and the axon projected

533

via the mALT to the mushroom body calyces and the lateral horn. Scale bar: 100 µm G-J) Soma

534

dissection for mass spectrometry. First, the recording pipette (G) was retracted and replaced by a

535

collecting pipette (H) with a larger tip diameter. The uPN soma was then separated from the

536

neighboring cells (I) and completely absorbed into the tip of the collecting pipette (J) Scale bar: 10

537

µm. The tip of the capillary containing the cell soma was finally directly crushed onto a sample plate

538

for MALDI-TOF mass spectrometry. K-M) MALDI-TOF mass spectra from a single uPN preparation.

539

Ion signal of acetylcholine (ACh) was obtained in the mass range at m/z 100-300 (K) and substance

540

confirmed (L) whereas AST- A peptides were detected under experimental conditions optimized for

541

the mass range of m/z 600-4000 (M). N-Q) Anti-AST-A immunostaining (green) confirmed the

542

presence of AST-A peptides in uPNs. Double labeling with biocytin (magenta) and anti-Ast-A serum

543

shows anti-AST-A staining in the complete uPN cluster (N), double labeling in the single glomerulus

544

entered by the analyzed uPN (N, O; scale bar: 50 µm) as well as the axonal endings in the mushroom

545

body calyx (P; Scale bar: 20 µm). The morphology of the analyzed uPN is reconstructed in Q. Insets

546

marked with asterisks depict the immunostained regions of the brain which are shown in N, O, and P).

547

MB, mushroom body; LH, lateral horn; AL, antennal lobe; uPN, uniglomerular projection neuron;

548

mALT, medial antennal lobe tract; Glom, glomerulus; ACh, acetylcholin; AST-A, allatostatin-A; OL,

549

optic lobe.

550 551

Fig. 2. Analysis of a type I LN. A) Electrophysiological recording of type I LN with Na+-driven action

552

potentials on stimulation with depolarizing current injection, B) Morphology, revealed by biocytin/

553

streptavidin labeling which is characterized by the innervation of multiple glomeruli. The glomeruli

554

show characteristic differences in neurite densities.. (C) Mass spectrum, which revealed co-

555

localization of short neuropeptide F (sNPF) and allatotropin (AT). D) Ion signals marked with

556

asterisks represent CHCA matrix ion signals and were detected in few preparations (gray shaded in C).

557

Scale bar: 50µm. CHCA, α-cyano-4-hydroxycinnamic acid; LN, local interneuron; sNPF, short

558

neuropeptide F; AT, allatotropin; PP, precursor peptide. MGC, macrorglomerulus; sNPF, short

559

neuropeptide F; AT, allatotropin. 18


Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


560 561

Fig. 3. Analysis of type II LNs with electrophysiological recording, intracellular staining, and direct

562

SCMS. The latter provides information about neuropeptides (left image) and presence/absence of the

563

transmitter acetylcholin (ACh) (right image). Both types of type II LNs are characterized by non-linear

564

depolarizations but no generation of Na+-driven action potentials upon depolarizing current injections

565

and homogeneous innervation pattern in all glomeruli. Insets in left image panels: Top, voltage

566

responses to hyper- and depolarizing current steps; bottom, morphology, revealed by biocytin/

567

streptavidin labeling. Scale bars: 100 µm. A) Type IIa LN with detection of ACh using lower mass

568

range settings and detection of co-localized tachykinin-related peptides (TKs) and short neuropeptide

569

F (sNPF) using higher mass range settings. Based on the presence of ACh, we suggest that this neuron

570

belongs to the type IIa1 LN subpopulation. B) Type II LN with absence of ACh and co-localized TKs,

571

extended FMRFamides (FMRFa), and allatotropin (AT), CHCA, α-cyano-4-hydroxycinnamic acid .

19



234x414mm (300 x 300 DPI)


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135x200mm (600 x 600 DPI)



159x140mm (300 x 300 DPI)


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Graphical abstract 39x32mm (300 x 300 DPI)


Analysis of single neurons by perforated patch clamp recordings and

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