Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Summary

We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Abstract

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)–accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

Introduction

Cholinergic modulation of circuit excitability contributes to fundamental aspects of cognition, and altered cholinergic modulation is a feature of neurodegenerative and neuropsychiatric disorders including Alzheimer’s disease, Parkinson’s disease, schizophrenia and addiction^1-4. An established mechanism of cholinergic facilitation of synaptic transmission in the CNS is via direct activation of nAChRs localized at pre-synaptic sites. Activation of these pre-synaptic receptors leads to increased intracellular Ca²⁺ ([Ca²⁺]_i) in pre-synaptic terminals – both directly, due to the relatively high calcium conductance of certain nAChR subtypes, and indirectly, via intracellular signaling cascades⁵, thereby enhancing neurotransmitter release. In fact, the activation of pre-synaptic nAChRs has been linked with changes in release of a wide variety of neurotransmitters including glutamate, GABA, ACh, and dopamine^6-10. Although this process has been studied indirectly using electrophysiological methods at various synapses, optical reporters of [Ca²⁺]_i and synaptic vesicle recycling allow more direct and temporally precise measurement of pre-synaptic phenomena.

Pre-synaptic localization of nAChRs has been demonstrated convincingly with direct immuno-gold labeling of nAChRs at the electron microscopic (EM) level^11,12. Several other techniques have also been used to address the nAChR localization indirectly, including detecting locations of nAChRs subunit- fluorescent protein chimeras in cultured neurons^13,14, electrophysiological recording of nAChR currents in synaptic terminals^15,16, monitoring nicotine induced changes in [Ca²⁺]_i in synaptic nerve terminals by live cell imaging¹⁷, and indirect monitoring of neurotransmitter release at the synaptic terminal by live cell imaging techniques with fluorescent indicators, including exocytosis of synaptic vesicles viewed by styryl amphipathic FM dyes (FM1-43 and FM4-64) and/or synapto-pHluorin and by specific fluorescent neurotransmitter reporters, such as CNiFERs for ACh and iGluSnFr for glutamate^18-20. Overall, these current approaches for identifying pre-synaptic localization of nAChRs are complicated, and require special systems and techniques to allow reliable identification and physiological monitoring of pre-synaptic activity.

Here we describe protocols and equipment for an in vitro co-culture system of a ventral hippocampal (vHipp) – nucleus accumbens (nAcc) circuit that provides direct access to identify and analyze both pre- and post-synaptic components of synaptic transmission. We show examples of pre-synaptic localization of nAChRs and the live cell imaging of nAChR mediated Ca²⁺ signaling and neurotransmitter release along vHipp axons. A natural (and straightforward) extension of the protocol presented here is the preparation of pre- and post-synaptic contacts comprised of neurons from different genotypes. In this manner the contribution of a particular gene product to the pre- and/or post-synaptic mechanisms of modulation can be assessed directly.

Protocol

All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 2012) and studies were approved by Institutional Animal Care and Use for Research Committees at Stony Brook University (#1618 and #1792).

1. vHipp-nAcc Synaptic Co-cultures

1. Sacrifice mice (postnatal day 0 - 3, from wild-type (WT) or α7 nAChRs transgenic mouse line) with CO₂. Decapitate each pup and save the tail in a 1.5 ml centrifuge tube for post facto genotyping. Perform the following steps in a sterilized hood.
2. Remove the top of the skull with scissors and forceps to expose the whole brain. Starting at the juncture where the cerebral cortices part from each other caudally, obtain a coronal section that contains the posterior cortices including the vHipp^5,21 under a dissecting microscope.
3. Dissect vHipp tissue strips containing the CA1-subiculum region out according to a developing mouse brain atlas²² (Figure 1A). Transfer to a 35 mm culture dish containing cold culture media (4 °C), cut into small thin slices (around 100 µm × 100 µm microslices).
4. Pre-incubate 12 mm poly-D-lysine/laminin-coated glass coverslips inside 24 well tissue culture plates with culture media (~ 500 μl) for at least 30 min in the incubator at 37 °C. Remove media before plating microslices.
5. Plate with a fire-polished Pasteur pipette at the center of the coverslip with minimal amount of media (~ 50 μl, containing ~ 20 pieces of microslices) to facilitate attachment of the explants. Plate vHipp microslices originating from a single animal (either the +/+ or the -/- genotype of the transgenic α7 line) on each coverslip.
6. After the explants settle onto the coverslip, gently add additional media (~ 100 μl) by the wall so that the level of the media is high enough to completely cover the explants on the periphery, but not those at the center of the coverslip.
7. After an O/N incubation in 37 °C, 5% CO₂ incubator, disperse nAcc neurons from embryonic days 18 to postnatal day 1 WT mice and add to the explants.
   1. Dissect out nAcc tissues according to a developing mouse brain atlas²⁰ (Figure 1B).
   2. Cut into small pieces (around 500 µm × 500 µm microslices) and transfer to a 15 ml tube.
   3. Treat with 0.25% trypsin (2 ml) for 15 min at 37 °C. Wash tissue chunks three times with cold washing media (5 ml, 4 °C) followed by one wash in cold culture media (5 ml, 4 °C). Allow suspension to rest for 5 min in each wash step, and then pour off supernatant carefully.
   4. Dissociate cells by gentle trituration in 2 ml of culture media with a lightly fire-polished Pasteur pipette.
   5. Transfer cell suspension to another 15 ml tube and centrifuge at 2,000 x rpm for 5 min. Remove the supernatant and resuspend the cells in culture media at 1 ml per 6 pups. Disperse cells by gently pipetting in media several times with a fire-polished Pasteur pipette.
   6. Add 0.25 ml of dispersed nAcc cells to each coverslip with vHipp explants.
   7. Maintain the cultures in a humidified 37 °C, 5% CO₂ incubator. Place culture plates on dampened sterile gauze pads to mechanically stabilize and maintain humidity, facilitating synapse formation.

2. Immunocytochemistry

1. Fix cultures (5 - 7 days in vitro) in 4% paraformaldehyde/4% sucrose/PBS (~500 μl per coverslip, 20 min, RT).
2. Permeabilize cultures with 0.25% Triton X-100/ PBS (~500 μl per coverslip, 5 min, RT).
3. Block cultures with 10% normal donkey serum in PBS (~500 μl per coverslip, 30 min, RT) prior to antibody staining. Incubate with primary antibodies O/N at 4 °C. Use the following primary antibodies: anti- nAChRs (α₄-ECD 1:500; anti-α₅ subunits, 1:500), anti-vesicular glutamate transporter 1 (1:250), anti-GAD65 (1:100).
4. Wash primary antibodies out with PBS (3 times/~500 μl per coverslip, 5 min) and then incubate cultures in secondary antibodies conjugated to Alexa 488 (~500 μl per coverslip, 1:500) or Alexa 594 (~500 μl per coverslip, 1:500) for 1 hr at 37 °C.
5. Incubate cultures in αBgTx conjugated to Alexa 594 (~500 μl per coverslip, 1:1,000 in culture media) for 45 min at 37 °C prior to fixation for labeling surface α7*nAChRs.
6. Mount and seal coverslips.
7. Capture images with a fluorescence microscope equipped with Plan-Apochromat objectives (20X with 0.8 NA or 63X oil with 1.4 NA) and a CCD camera.

3. FM1-43 Based Vesicular Fusion

1. Maintain cultures (5 - 7 days in vitro) in an imaging chamber, mount the chamber on spinning disk confocal microscope. Continuously perfuse (1 ml/min) with HBS cocktail at RT.
2. Stop perfusion and load cultures with 10 µM FM1-43 in 56 mM K⁺ ACSF (~500 μl) for 90 sec (staining), wash external dye away for 15 min with Ca²⁺ free HBS containing ADVASEP-7 (0.1 mM) to scavenge membrane-bound FM1-43.
3. Stimulate the cultures with 56 mM K⁺ ACSF (~500 μl) without FM1-43 for 120 sec (destaining), reload cultures with FM1-43 using the same conditions, and wash again with Ca²⁺ free HBS containing ADVASEP-7 for 15 min.
4. Acquire FM1-43 fluorescence images with spinning disk confocal microscope equipped with a Plan-Apochromat objective (60X water with 1.4 NA, excitation 488 nm, emission 530 nm) and capture images with a CCD camera.
5. Collect baseline FM1-43 fluorescence images (every 2 s for 1 min) as pre-nicotine control; apply nicotine (1 µM) by rapid perfusion (2 ml/min) for 1 min, and then wash nicotine out with HBS cocktail. Keep capture time-lapse images before, during, and after nicotine application every 2 sec for 5 min.
6. Quantify FM1-43 fluorescence intensity before (F_staining) and after (F_destaining) nicotine application at each synaptic bouton.
7. Calculate total amount of releasable FM1-43 fluorescence along vHipp axons by quantifying the difference of FM1-43 fluorescence intensity before and after nicotine application (ΔF = F_staining-F_destaining). Analyze fraction of fluorescence intensity decrease after nicotine with the following equation: F_decrease% = ΔF/F_staining.

4. Calcium Imaging

1. Rinse cultures (5 - 7 days in vitro) quickly with normal HBS, then load cultures with 5 µM Fluo-4 Ca²⁺ indicator (AM ester) and 0.02% Pluronic F-127 in HBS (2 ml) for 30 min at 37 °C and 5% CO₂.
2. Wash out Fluo-4 solution with HBS (3 times/5 min). Return cultures to incubator (37 °C / 5% CO₂) for at least 30 min.
3. Maintain cultures in the same imaging chamber and same conditions as described in FM1-43 based vesicular fusion section (see step 3.1).
4. Collect Fluo-4 fluorescence images of axonal projections from the vHipp micro-slices with a Plan-Apochromat objective (60X water with 1.4 NA, excitation 488 nm, emission 530 nm) and a CCD camera.
5. Collect baseline Fluo-4 fluorescence images (every 10 sec for 2 min) as pre-nicotine control, apply nicotine (1 µM) by rapid perfusion (2 ml/min) for 1 min, and then wash nicotine out with HBS cocktail. Keep capture time-lapse images before, during, and after nicotine application every 10 sec for 30 min.
6. Save all frames of the raw fluo-4 fluorescence images, export as a series of TIFF format images for further analyzing.
7. Collect the integrated intensity of fluo-4 fluorescence along vHipp axons before and after nicotine application.
8. Normalize integrated fluorescence intensity with the following equation: ΔF/F₀ =  (F-F₀)/F₀, where F₀ is the background-corrected pre-nicotine integrated fluorescence intensity and F is the integrated fluorescence intensity along vHipp axons at each time point after nicotine application. Analyze and plot normalized integrated fluorescence intensity.

Results

The preparation employed consists of gene chimeric co-cultures of vHipp–nAcc circuits in vitro. Projections emanating from vHipp microslices, as pre-synaptic axonal input, can make synaptic contacts with post-synaptic targets, the dispersed neurons from nAcc. Nicotine induced a sustained (≥ 30 min) facilitation of glutamatergic transmission from nAcc neurons innervated by vHipp axons²¹ and prolonged calcium signaling along vHipp axons⁵ via pre-synaptic α7*nAChRs.

Discussion

The co-culture preparation described re-capitulates ventral hippocampal–accumbens circuits in vitro. This preparation permits relatively straightforward and reliable examination of the spatial and temporal profiles by which activation of pre-synaptic nAChRs elicit enhanced glutamatergic transmission^{5, 21}.

Co-cultures are defined as the growth of different specific cell types in one dish which can provide physiological conditions in vitro to demonstrate in...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1. Culture Media (50 ml)
Neurobasal	GIBCO	10888022	48 ml
B-27 Supplements	GIBCO	0080085-SA	1 ml
Penicillin-Streptomycin	GIBCO	10908-010	0.5 ml
GlutaMAX Supplement	GIBCO	35050-061	0.5 ml
Brain-derived neurotrophic factor (BDNF)	GIBCO	15140-122	20 ng/ml
2. washing media (HBSS, 100 ml)
HBSS, no calcium, no magnesium, no phenol red	GIBCO	14175-095	99 ml
HEPES ( 1M)	GIBCO	15630-130	1 ml
3. HEPES buffered saline  (HBS)   pH=7.3
NaCl	Sigma	S9888	135 mM
KCl	Sigma	P9333	5 mM
MgCl2	Sigma	M8266	1 mM
CaCl2,	Sigma	C1016	2 mM
HEPES	Sigma	H3375	10 mM
Glucose	Sigma	G0350500	10 mM
4. HBS Cocktail for live imaging pH=7.3
NaCl	Sigma	S9888	135 mM
KCl	Sigma	P9333	5 mM
MgCl2	Sigma	M8266	1 mM
CaCl2,	Sigma	C1016	2 mM
HEPES	Sigma	H3375	10 mM
Glucose	Sigma	G0350500	10 mM
tetrodotoxin	Tocris	1078	2 µM
bicuculline	Tocris	131	10 µM
D-AP-5	Tocris	105	50 µM
CNQX	Tocris	1045	20 µM
LY341495	Tocris	1209	10 µM
5. Calcium-free  HBS   pH=7.3
NaCl	Sigma	S9888	135 mM
KCl	Sigma	P9333	5 mM
MgCl2	Sigma	M8266	1 mM
HEPES	Sigma	H3375	10 mM
Glucose	Sigma	G0350500	10 mM
6. 56 mM Potassium ACSF pH=7.4
NaCl	Sigma	S9888	119 mM
KCl	Sigma	P9333	56 mM
MgSO4.7H	Sigma	M1880	1.3 mM
CaCl2	Sigma	C1016	2.5 mM
NaH2PO4	Sigma	S8282	1 mM
NaHCO3	Sigma	S5761	26.2 mM
Glucose	Sigma	G0350500	10 mM