This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.
Research Article: New Research | Neuronal Excitability
Axonal Type III Nrg1 Controls Glutamate Synapse Formation and GluA2
Trafficking in Hippocampal-Accumbens Connections
Nrg1/ErbB signaling modulate glutamatergic transmission
Chongbo Zhong1, Wendy Akmentin1, Chuang Du2, Lorna W. Role1 and David A Talmage3
1
Department of Neurobiology and Behavior, Center for Nervous System Disorder, Stony Brook University, Stony
Brook, NY s11794
2
Department of Neuroscience, Tufts University School of Medicine, Boston, MA
3
Department of Pharmacological Science, Center for Nervous System Disorder, Stony Brook University, Stony
Brook, NY 11794
DOI: 10.1523/ENEURO.0232-16.2017
Received: 5 August 2016
Revised: 23 January 2017
Accepted: 6 February 2017
Published: 15 February 2017
Author Contributions: CZ, LWR and DAT Designed Research; CZ, WA and CD Performed Research; CZ,
LWR and DAT Analyzed data and Wrote the paper.
Funding: NIH
NS022061
Funding: NIH
MH087473
Conflict of Interest: Authors report no conflict of interest.
This research was supported by the National Institutes of Health Grants NS022061, and MH087473 to L.W.R.
and D.A.T.
Correspondence should be addressed to either Chongbo Zhong, Department of Neurobiology
and Behavior; Center for Nervous System Disorder; Stony Brook University, Stony Brook, NY 11794;
[email protected] or David A Talmage, Department of Pharmacological Science; Center for
Nervous System Disorder; Stony Brook University, Stony Brook, NY 11794; [email protected]
Cite as: eNeuro 2017; 10.1523/ENEURO.0232-16.2017
Alerts: Sign up at eneuro.org/alerts to receive customized email alerts when the fully formatted version of this
article is published.
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading
process.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any
medium provided that the original work is properly attributed.
Copyright © 2017 the authors
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
1. Manuscript Title
Axonal type III Nrg1 controls glutamate synapse formation and GluA2 trafficking in hippocampalaccumbens connections
2. Abbreviated Title (50 character maximum)
Nrg1/ErbB signaling modulate glutamatergic transmission
3. List all Author Names and Affiliations in order as they would appear in the published article
Chongbo Zhong, Department of Neurobiology and Behavior; Center for Nervous System Disorder; Stony
Brook University, Stony Brook, NY 11794;
Wendy Akmentin, Department of Neurobiology and Behavior; Center for Nervous System Disorder; Stony
Brook University, Stony Brook, NY 11794;
Chuang Du, Department of Neuroscience, Tufts University School of Medicine, Boston, MA
Lorna W. Role, Department of Neurobiology and Behavior; Center for Nervous System Disorder;
Neuroscience Institute; Stony Brook University, Stony Brook, NY 11794;
David A Talmage, Department of Pharmacological Science; Center for Nervous System Disorder; Stony
Brook University, Stony Brook, NY 11794;
4. Author Contributions:
CZ, LWR and DAT Designed Research; CZ, WA and CD Performed Research; CZ, LWR and DAT Analyzed
data and Wrote the paper
5. Correspondence should be addressed to (include email address)
Chongbo Zhong, Department of Neurobiology and Behavior; Center for Nervous System Disorder; Stony
Brook University, Stony Brook, NY 11794; [email protected]
David A Talmage, Department of Pharmacological Science; Center for Nervous System Disorder; Stony
Brook University, Stony Brook, NY 11794; [email protected]
6. Number of Figures: 5
7. Number of Tables: 1
8. Number of Multimedia: 0
9. Number of words for Abstract: 131
10. Number of words for Significance Statement: 87
11. Number of words for Introduction: 472
12. Number of words for Discussion: 971
13. Acknowledgements
We thank technical support from Mallory Myers and Johnathan Beck.
14. Conflict of Interest
The authors declare that there is no conflict of interests regarding the publication of this paper.
15. Funding sources
This research was supported by the National Institutes of Health Grants NS022061, and MH087473 to
L.W.R. and D.A.T.
1
47
Abstract
48
Altered neuregulin 1 (Nrg1) / ErbB signaling and glutamatergic hypofunction have been
49
implicated in the pathophysiology of schizophrenia. Here, we employed gene chimeric
50
ventral
51
electrophysiology, immunocytochemistry, FM1-43 vesicle fusion and electron microscopy
52
techniques to examine the pre- and post-synaptic mechanisms of genetic deficits in
53
Nrg1/ErbB signaling induced glutamatergic dysfunctions. Reduced presynaptic type III
54
Nrg1 expression along vHipp axons decreases the number of glutamate synapses and
55
impairs GluA2 trafficking in the postsynaptic nAcc neurons, resulting in decreased
56
frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs).
57
Reduced expression of axonal type III Nrg1 along vHipp projections also decreases
58
functional synaptic vesicle clustering and vesicular trafficking to presynaptic vHipp axonal
59
terminals.
60
transmission via both pre- and post-synaptic mechanisms.
Hippocampus
(vHipp)
–
accumbens
(nAcc)
co-culture
from
mouse,
These findings suggest that Nrg1/ErbB signaling modulate glutamatergic
61
62
63
64
65
66
Key words: neuregulin 1; glutamatergic transmission; presynaptic maturation; synaptic
67
vesicles; neurotransmitter release; electron microscopy; FM1-43
2
68
Significance Statement
69
Presynaptic Nrg1 to post-synaptic ErbB signaling contributes to excitatory synapse
70
formation and plasticity, and disturbances in this signaling are thought to contribute to a
71
number of structural and functional endophenotypes associated with schizophrenia. In
72
this study we uncover a new role for axonal Nrg1 signaling in the formation and functional
73
maturation of glutamatergic, pre-synaptic specializations. Axonal signaling by Nrg1 adds
74
an additional layer of complexity to the role of these molecules in neuronal development
75
and might provide additional insight into their contribution to the etiology of schizophrenia.
76
77
78
79
80
81
82
83
84
85
86
3
87
Introduction
88
Schizophrenia is a highly heritable neuropsychiatric illness affecting about 1% of the
89
world’s population. Alterations of glutamate-mediated synaptic neurotransmission, in
90
particular glutamatergic hypofunction, are implicated in the pathophysiology of
91
schizophrenia, although there is no consensus on either the origin of these dysfunctions or
92
to what extent glutamatergic dysfunctions are causative for the disorder (Paz et al., 2008;
93
Gaspar et al., 2009; Lin et al., 2012; Coyle et al., 2012). The major mechanisms leading to
94
altered glutamatergic synaptic strength include modulating neurotransmitter (glutamate)
95
release at presynaptic terminals and altering the glutamate receptor responses at
96
postsynaptic terminals (González-Forero et al., 2012; Allam et al., 2015).
97
Studies of candidate schizophrenia risk genes and post-mortem analyses have
98
implicated changes in neuregulin 1 (Nrg1) signaling as a contributor to disease associated
99
endophenotypes (Stefansson et al., 2002; Banerjee et al., 2010). The Nrg1 gene encodes
100
a family of ligands for ErbB receptor tyrosine kinases (Hobbs et al., 2002; Grossmann et
101
al., 2009). Nrg1/ErbB signaling plays a complex role in regulating glutamatergic synaptic
102
transmission. For example, Nrg1-ErbB signaling affects excitatory synaptogenesis on
103
cortical interneurons (Ting et al., 2011), interferes with LTP at hippocampal synapses
104
(Kwon et al., 2005), and is necessary for plasticity at cortical-amygdala synapses (Jiang et
105
al., 2013). A critical question that emerges from these studies is whether altered Nrg1
106
signaling contributes to the glutamatergic hypofunction seen in patients with schizophrenia
107
and/or psychoses, and if so, how?
4
108
Mechanistically, Nrg1 signaling has been shown to alter NMDA (Gu et al., 2005;
109
Hahn et al., 2006; Pitcher et al., 2011) and AMPA receptor levels and trafficking (Abe et al.,
110
2011; Fenster et al., 2012), GABA-A (Okada and Corfas 2004; Fazzari et al., 2010),
111
nicotinic acetylcholine receptor (nAChRs) (Liu et al., 2001; Kim et al., 2013) expression,
112
and presynaptic targeting of nAChRs (Hancock et al., 2008). These recent studies
113
converge on the idea that critical balance of Nrg1/ErbB signaling is required for
114
maintaining optimal glutamatergic synaptic plasticity.
115
Glutamatergic projections from the ventral subiculum of the hippocampus innervate
116
the GABAergic medium spiny neurons in the ventral striatum / nucleus accumbens. Type
117
III Nrg1 is expressed in the ventral subiculum of the hippocampus but not in ventral
118
striatum; in contrast the Nrg1 receptor, ErbB4 is widely expressed in the early post-natal
119
ventral striatum (Chen et al., 2008). Recently, it has been demonstrated that bi-directional
120
signaling by the Type III isoforms of Nrg1 is essential for establishing and maintaining
121
functional levels of presynaptic α7 containing nAChRs on ventral subicular and cortical
122
glutamatergic projections (Zhong et al., 2008; Jiang et al., 2013). As a result, Type III
123
Nrg1 heterozygotes have pronounced deficits in α7 nAChR-dependent presynaptic
124
modulation of glutamate release and compromised glutamatergic synaptic plasticity. In
125
this report we investigated a more general role for ventral hippocampal presynaptic Type
126
III Nrg1 signaling in establishing glutamatergic synapses on nucleus accumbens medium
127
spiny neurons (MSN).
128
129
5
130
Materials and Methods
131
vHipp micro-slices culture and vHipp-nAcc synaptic co-culture
132
All animal experiments were carried out in accordance with the National Institutes of
133
Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23,
134
revised 2012). Animals used in these studies were derived from crosses of heterozygous
135
male and female Nrg1tm1.1Lwr mice in which the exon unique to Type III Nrg1 isoform has
136
been disrupted (Wolpowitz et al., 2000) (on a C57Bl6/J background).
137
For vHipp micro-slices culture, ventral CA1 and subiculum area (vHipp) from WT or
138
Nrg1tm1.1Lwr +/- mice (postnatal day 0-3, P0-P3) were dissected, sliced into 150×150 μm
139
pieces, and then plated onto poly-D-lysine/laminin-coated glass coverslips (BD Sciences,
140
Bedford, MA) in a minimal volume (50 μl) of culture media (Neurobasal, 2% B-27 (GIBCO,
141
Grand Island, NY) and 20 ng/ml brain-derived neurotrophic factor (R&D Systems,
142
Minneapolis, MN)). After the microslices attached to the substrate, 100 μl of additional
143
culture media were added. Prior studies using a similar vHipp microsclice preparation in
144
the absence of post-synaptic targets, demonstrated that nicotine regulated calcium
145
signaling and FM1-43 vesicle cycling in glutamatergic axons from the vHipp micro-slices
146
(Zhong et al., 2008; Zhong et al., 2013; Zhong et al., 2015). Here, we use vHipp-nAcc
147
synaptic co-cultures to examine the effects of Nrg1 – ErbB4 signaling on functional
148
glutamatergic synapse formation (Figure 1), and we use vHipp micro-slices cultured alone
149
to focus on axonal presynaptic-like specializations (Figures 2 – 5).
150
For vHipp-nAcc synaptic co-cultures, nucleus accumbens (nAcc) (ED18 – P1) from WT
151
mice (C57BL/6J) were dissected out and dispersed with 0.25% trypsin (GIBCO, Grand
6
152
Island, NY) for 15 min at 37oC, followed by gentle trituration in culture media. Dispersed
153
nAcc neurons were added to the vHipp micro-slices plated the prior day at 0.25
154
ml/coverslip. All cultures were maintained in a humidified 37oC, 5% CO2 incubator.
155
Electrophysiological Recordings
156
Synaptic currents were recorded from WT nAcc neurons contacted with vHipp axons
157
from WT or Nrg1+/- mice by whole-cell configuration of the patch clamp technique. Briefly,
158
after 5-7 days in vitro, vHipp-nAcc co-cultures in a recording chamber were continuously
159
perfused with extracellular solution (in mM): 145 NaCl, 3 KCl, 2.5 CaCl2, 10 HEPES, and
160
10 glucose, pH = 7.4. The intracellular solution included (in mM): 3 NaCl, 150 KCl, 1 MgCl2,
161
1 EGTA, 10 HEPES, 5 MgATP, and 0.3 NaGTP, pH = 7.2. Bicuculline (Tocris, Ellisville,
162
MO), and TTX (Sigma, St. Louis, MO) were included in the perfusion as noted. Synaptic
163
currents were filtered at 10 KHz with a 8-pole Bessel filter (DC Amplifier/Filter, Warner
164
Instruments, Hamden, CT) prior to acquisition and digitization through a DigiData 1200B
165
A/D interface with pCLAMP 8 (Axon Instruments, Foster City, CA). The amplitudes and
166
frequency of TTX resistant, spontaneous synaptic currents were measured with
167
MiniAnalysis (RRID: SCR_014441 Synaptosoft).
168
Immunocytochemistry and reagents
169
After 5-7 days in vitro, vHipp-nAcc co-cultures were fixed in 4% paraformaldehyde/4%
170
sucrose /PBS (20 min, room temperature), permeabilized with 0.25% Triton X-100/ PBS (5
171
min, RT), blocked with 10% normal donkey serum in PBS (30 min, RT), and then
172
incubated in primary antibodies overnight at 4°C. The following primary antibodies were
173
used: anti-PSD95 (1:500, Cat# P246 RRID: AB_260911 Sigma-Aldrich), anti-vesicular
7
174
glutamate transporter 1 (1:250, Cat# 135 302 Synaptic Systems), anti-pan-axonal
175
neurofilament marker (1:1000, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), anti-
176
GluA2, extracellular, clone 6C4 (1:500, Cat# MAB397 EMD Millipore), anti-GluA2 + GluA3,
177
C-terminal (1:500, Cat# PA1-4660 Thermo Fisher Scientific). Cultures were washed and
178
incubated in secondary antibodies conjugated to Alexa 488 (1:500; Cat# A-21206 RRID:
179
AB_141708 Molecular Probes) or Alexa 594 (1:500; Cat# A-21203 RRID: AB_141633
180
Molecular Probes) for 1 h at RT. Slips were mounted using VectaShield (with DAPI, Cat#
181
H-1200 RRID: AB_2336790 Vector Laboratories), and images were captured using a
182
microscope (Axio Imager A1; Carl Zeiss, Inc.) equipped with Plan-Apochromat objectives
183
(63× oil with 1.4 NA), a CCD camera (Hamamatsu), and Metamorph software (Version 7.1,
184
MetaMorph Microscopy Automation and Image Analysis Software, RRID:SCR_002368
185
Molecular Devices).
186
To label surface GluA2 (sGluA2), vHipp-nAcc co-cultures were incubated in anti-GluA2
187
antibody, extracellular, clone 6C4 (1:500) for 45 min at 37°C prior to fixation. Then, total
188
GluA2 was visualized with anti-GluA2 + GluA3 antibody, C-terminal (1:500) with the
189
normal staining procedures.
190
Morphological synaptic contacts were identified as puncta of presynaptic (vGluT1) and
191
postsynaptic (PSD95, sGluA2) marker proteins that are formed during synapse formation
192
and maturation (Ippolito et al., 2010). First, the number of vGluT1 and PSD95 clusters
193
were measured separately along nAcc neurites contacted by vGluT1-positive vHipp axons
194
using Metamorph software. The number of PSD95 positive vGluT1 clusters (co-
195
localization) along nAcc neurites were counted as synaptic contacts. We considered
196
vGluT1 and PSD95 (or sGluA2) puncta to be co-localized if markers directly overlapped
8
197
(yellow puncta) or were closely apposed to each other (Chao et al., 2007). The lengths of
198
vHipp axonal projections and nAcc neurites were also measured in Metamorph. The
199
overall ratio of surface GluA2 to total GluA2 from all neurites was also quantified.
200
FM1-43 based imaging and analysis
201
After 5-7 days in vitro, vHipp micro-slices cultures were maintained in an imaging
202
chamber (Live Imaging Services, Olten Switzerland; containing 1 ml fresh normal HBS)
203
mounted on a Olympus IX81 DSU (spinning disk confocal) microscope (Olympus America
204
Inc., Center Valley, PA) under continuous perfusion (1 ml/min) with HBS containing 2 μM
205
tetrodotoxin (TTX, Tocris), 10 μM bicuculline (Tocris), 50 μM D-AP-5 (Tocris), 20 μM
206
CNQX (Tocris). vHipp microslices were loaded with 10 μM FM1-43 (Molecular Probes,
207
Eugene, OR) in 56 mM K+ ACSF for 90 s, external dye was washed away in Ca2+ free
208
HEPES buffered saline (HBS, 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10
209
mM glucose, pH 7.4) containing ADVASEP-7 (0.1 mM, Sigma) to scavenge membrane-
210
bound FM1-43 for 15 min. Fluorescence images of vHipp axons were collected by a Plan-
211
Apochromat objective (60× oil with 1.4 NA, excitation 488 nm, emission 530 nm) and
212
captured with a CCD camera (Hamamatsu) every 1.5 s for 5 min. Image acquisition was
213
performed using Slidebook software (Version 5, Olympus). After 1 minute of baseline data
214
collection, 56 mM K+ ACSF without FM1-43 was applied for 120 s to confirm that high K+
215
depolarization can induce destaining of FM1-43 dye–filled vesicles. The total amount of
216
releasable fluorescence at each synaptic bouton was calculated from the difference
217
between fluorescence intensity after staining (and before destaining) and after
218
depolarization induced destaining (ΔF = Fstaining-Fdestaining). The fraction of fluorescence
219
intensity decrease after depolarization was calculated as (Fdecrease%=ΔF/Fstaining). The
9
220
number and size of FM1-43 positive puncta before and after high K+ depolarization were
221
measured and compared along vHipp axons from Nrg1tm1.1Lwr wild type (+/+) vs.
222
heterozygous (+/-) mice using Metamorph software. The lengths of axonal projections
223
were also measured by tracing vHipp projections in MetaMorph.
224
For analyzing the time course of high K+ depolarization induced FM1-43 destaining, all
225
frames of the raw FM1-43 fluorescence images were saved as Slidebook files and then
226
exported as a series of TIF format images that were then imported to MetaMorph software
227
and transferred as Z-stack images for further analyses. After setting the threshold of the
228
FM1-43 fluorescence, the integrated intensity of the FM1-43 signals along vHipp axons
229
before and after high K+ depolarization was calculated. FM1-43 fluorescence data are
230
displayed as a normalized integrated intensity: [ΔF/F0 = (F - F0)/F0], where F0 is the
231
background-corrected pre- high K+ depolarization FM1-43 fluorescence. Data were
232
analyzed further using Excel software.
233
Electron Microscopy
234
After 5-7 days in vitro, vHipp microslices grown on Thermanox© plastic coverslips
235
(NUNC/Electron Microscopy Sciences) were fixed for 30 minutes in a mixture of cold 2%
236
paraformaldehyde and 2% glutaraldehyde in 0.1M phosphate buffer (PB), pH 7.4. After
237
several washes in PB, explants were post-fixed with 2% osmium tetroxide for 30 minutes,
238
en bloc stained with aqueous 1% uranyl acetate for 30 minutes, dehydrated through an
239
ascending series of ethanols, followed by acetonitrile, and embedded in Embed 812 resin
240
(Electron Microscopy Sciences) for 48 hours at 60°C. Ultrathin sections (60-90nm) were
241
cut and then stained with 1% methanolic uranyl acetate and 0.3% aqueous lead citrate,
242
and observed with a JEOL 1200EX transmission electron microscope.
10
243
Photoconversion of FM1-43
244
The photoconversion procedure was modified from a prior study (Harata et al., 2001).
245
After FM1-43 staining, cultures on glass coverslips containing vHipp axons were fixed with
246
2% glutaraldehyde in 100 mM sodium phosphate buffer (PBS) for 20 min, and washed
247
with glycine (100 mM in PBS) for 1 h, and then washed in ammonium chloride (100 mM in
248
distilled water) for 5 min. After brief rinsing in PBS, axons were incubated in DAB (1 mg/ml
249
in PBS) for 20 min. Fluorescence excitation light was then continuously applied for 10–20
250
min in DAB solution. Cultures were then washed in ice-cold PBS, and processed for EM.
251
After DAB photoconversion, microslices cultures were briefly rinsed with PBS and then
252
post-fixed with 1% osmium tetroxide containing 0.8% potassium ferricyanide for 30
253
minutes. The coverslips were en bloc stained with aqueous 2% uranyl acetate for 20
254
minutes, dehydrated through an ascending series of ethanols, and embedded in Durcupan
255
resin for 48 hours at 60oC.
256
Glass coverslips were removed by submerging in liquid nitrogen. Ultrathin sections (60-
257
90 nm) were cut and then stained with 1% methanolic uranyl acetate and 0.3% aqueous
258
lead citrate, and observed with a JEOL 1200EX transmission electron microscope.
259
Statistical analysis
260
All data were expressed as mean ± s.e.m unless otherwise indicated and analyzed with
261
StatView (SAS Institute Inc.) or Microsoft Excel (Microsoft Corp.) software. Superscript
262
letters listed with p-values correspond to the statistical tests shown in Table 1.
263
11
264
Results
265
We employed a gene chimeric co-culture system to examine whether and how
266
alteration of presynaptic Type III Nrg1 signaling modulates glutamatergic transmission.
267
The ventral hippocampus (vHipp) microslices extend glutamatergic axons that synapse on
268
dispersed medium spiny neurons (MSNs) from nucleus accumbens (nAcc) (Fig. 1A). Post-
269
synaptic currents were recorded from nAcc dispersed neurons innervated by either WT
270
(+/+) or Type III Nrg1 heterozygous (+/-) vHipp. First, we tested the spontaneous
271
glutamate-receptor mediated synaptic activity, i.e. miniature excitatory postsynaptic
272
currents (Glu mEPSCs: bicuculline and TTX resistant; CNQX-APV sensitive), in nAcc
273
MSNs from either WT vHipp (+/+)  WT nAcc (+/+) (Fig. 1B, Top) or Het vHipp (+/-)
274
WT nAcc (+/+) (Fig. 1B, Bottom) co-cultures. Both the Glu mEPSC frequency (~4 Hz vs
275
1.5 Hz; Fig. 1C; n= 8 WT  WT vs. 8 Het  WT; P=0.005a) and amplitude (~35 pA vs.
276
18 pA; Fig. 1D P=0.008b) were significantly reduced when measured in WT MSNs
277
receiving Type III Nrg1 heterozygote vHipp input compared to WT input. These data, along
278
with other published results, demonstrate that the level of presynaptic Type III Nrg1 affects
279
glutamatergic synaptic transmission (Zhong et al., 2008; Jiang et al., 2013).
280
The observed decrease in mEPSC frequency could reflect either a decrease in the total
281
number of synapses, or/and a decrease in the probability of spontaneous glutamatergic
282
synaptic vesicle release. To determine whether reduced presynaptic Type III Nrg1
283
expression altered the number of glutamatergic synapses formed on WT MSNs, vHipp-
284
nAcc co-cultures were fixed, permeabilized, and stained with antibodies recognizing the
285
presynaptic marker, vGluT1 and the post-synaptic marker, PSD95. Clusters of vGluT1
286
(Fig. 1E, red) were co-localized with the post-synaptic marker PSD95 (green) along
12
287
neurites of dispersed nAcc MSNs innervated by +/+ (Fig. 1E, Left top) or +/- (Fig. 1E,
288
Left bottom) vHipp axons. The number of PSD95+ / vGluT1+ clusters along nAcc MSN
289
neurites was significantly decreased (Fig. 1E, middle left) when the WT nAcc MSNs were
290
innervated by Type III Nrg1 Het (+/-) vHipp axons (17 ± 3 per 100 μm nAcc neurites)
291
compared to WT nAcc MSNs innervated by Nrg1 WT (+/+) vHipp axons (28 ± 4 per 100
292
μm nAcc neurites; P=0.001c). There was no significant difference (Fig. 1E, right P=0.45d)
293
in the number of vGluT1 cluster numbers along nAcc neurites when innervated by WT
294
vHipp axons (31 ± 4 per 100 μm nAcc neurites) compared to Nrg+/- vHipp axons (29 ± 8
295
per 100 μm nAcc neurites). The PSD95 cluster numbers along nAcc neurites innervated
296
by Nrg+/- vHipp axons (54 ± 10 per 100 μm nAcc neurites) compare to innervated by
297
Nrg+/+ vHipp axons (60 ± 10 per 100 μm nAcc neurites) showed a slight, but not
298
significant, decrease (Fig. 1E, middle right P=0.08e). This indicates that reduced
299
presynaptic Type III Nrg1 expression decreased the numbers of synaptic contacts formed
300
on Nrg1 WT post-synaptic MSNs in these vHipp-nAcc co-cultures. Neither the frequency
301
nor the amplitude of TTX-resistant inhibitory post-synaptic currents (mIPSCs) were altered
302
by the Nrg1 genotype of the vHipp explants (not shown), indicating that formation of
303
GABAergic synapses between nAcc MSNs was not affected by Type III Nrg1 on the vHipp
304
glutamatergic inputs.
305
The decreased amplitude of individual mEPSCs could result from a decrease in the
306
number of post-synaptic glutamate receptors (most likely AMPA type receptors under
307
these recording conditions). To determine whether alterations of pre-synaptic Type III Nrg1
308
signaling affected the numbers of post-synaptic AMPARs, we quantified the ratio of
309
surface/total GluA2-containing AMPARs on WT nAcc MSNs innervated by either WT
13
310
vHipp or Type III Nrg1 Het vHipp explants. Live co-cultures were stained with a GluA2
311
antibody that recognizes extracellular epitopes, after which the co-cultures were fixed,
312
permeabilized and stained with a different GluA2 antibody. Clusters of surface GluA2
313
(green) and total GluA2 (red) were present on both soma and neurites of WT nAcc MSNs
314
innervated by vHipp axons (Fig. 1F). The overall ratio of surface GluA2 to total GluA2
315
integrated fluorescence intensity along neurites of dispersed nAcc neurons was
316
significantly decreased (Fig. 1F, right; P=0.003f) when the neurons were innervated by
317
Type III Nrg1 Het (+/-) vHipp axons (0.18 ± 0.02) compared to WT nAcc MSNs innervated
318
by WT Nrg1 (+/+) vHipp axons (0.36 ± 0.07). The ratio of surface to total GluA2 quantified
319
over MSN soma was not as affected by presynaptic Type III Nrg1 genotype (WT ~4.5 vs
320
Het ~3.5; not shown).
321
To determine whether the surface GluA2 staining along nAcc neurites were at synapses,
322
we stained co-cultures of nAcc neurons innervated by WT vHipp axons for sGluA2 and
323
vGluT1 (Fig. 1G). The majority of sGluA2 staining along the neurites was co-localized with
324
vGluT1 clusters indicating that many of these clusters were likely to be sites of synaptic
325
contacts (Fig. 1G).
326
Taken together, these data demonstrate that reduction in the levels of pre-synaptic
327
Type III Nrg1 results in a 40-60% decrease in excitatory synapse formation on WT nAcc
328
MSNs, and that the level of post-synaptic receptors present on the synapses that do form
329
is decreased as well.
330
Formation of glutamatergic synapses onto striatal MSNs requires glutamatergic
331
transmission (Kozorovitskiy et al., 2012). Given that It has been demonstrated that
332
presynaptic Type III Nrg1 back-signaling is critical for targeting various receptors to
14
333
presynaptic sites (Hancock et al., 2008; Zhong et al., 2008; Hancock et al., 2011; Canetta
334
et al., 2011), we next asked whether alterations of presynaptic Type III Nrg1 signaling
335
could affect neurotransmitter release. To focus specifically on the effect of Type III Nrg1
336
signaling in vHipp axons, the next series of experiments utilized vHipp micro-slices that
337
were cultured in the absence of post-synaptic nAcc MSNs. vHipp microslices from either
338
WT or Type III Nrg1 Het mice were loaded with FM1-43 and then depolarization induced
339
destaining of the readily releasable pool of vesicles (RRP) was quantified by live cell
340
imaging. FM1-43 labeled vesicle puncta in both WT (+/+) (Fig. 2A, left, top) and Type III
341
Nrg1 Het (+/-) (Fig. 2A, right, top) vHipp axons showed no differences in the initial FM1-
342
43 fluorescence intensities after K+-dependent loading (Fig. 2B, P=0.35g). Also there are
343
no significant changes in FM1-43 fluorescence intensities and cluster numbers for at least
344
30 min in the absence of stimulation indicating that there was minimal photo-bleaching
345
under our imaging conditions (data not shown). After high K+ (56 mM) ACSF application,
346
the fluorescence intensity of all puncta along WT (+/+) vHipp axons (Fig. 2A, left, bottom;
347
Fig. 2B) decreased by ~80%, reflecting depolarization induced exocytosis of dye from
348
synaptic vesicles. In contrast, the decrease in depolarization induced fluorescence
349
intensity of FM1-43 puncta from Type III Nrg1 Het (+/-) axons was significantly reduced
350
compared to WT (Fig. 2A, right, bottom; Fig. 2C; ~50% reduction in fluorescence,
351
P=0.005h). Over 80% of FM1-43 loaded vesicle clusters along WT vHipp axons were
352
totally destained by depolarization (Fig 2D) and the size of the partially destained clusters
353
decreased by 70% (Fig 2E). In contrast only ~20% of FM1-43 clusters totally destained
354
along Type III Nrg1 Het (+/-) axons (Fig. 2D; WT vs. Het P=0.001i), and there was only
355
about a 25% decrease in overall cluster size (Fig 2E; WT vs. Het P=0.001j).
15
356
We also quantified the time course of depolarization induced FM1-43 release from
357
individual synaptic vesicular clusters. Images of FM1-43 along vHipp axons were recorded
358
every 1.5 seconds for 5 min using live spinning disk confocal microscopy, and changes in
359
FM1-43 fluorescence intensities were used to calculate decay time constants (Fig 3B).
360
The fraction of normalized integrated intensity decrease at single clusters (3 pixels ≈ 1 µm)
361
along vHipp axons were plotted vs. time (Fig. 3A). Following depolarization, WT axons
362
destained with a time constant of ~4.8 sec, whereas the Type III Nrg1 Het (+/-) axons
363
destained at a significantly slower rate and as noted above, failed to fully destain (Fig. 3B;
364
time constant ~8.5 sec; WT vs. Het P=0.001k). Based on these measures, it appears that
365
presynaptic Type III Nrg1 contributes to the normal pattern and rate of synaptic vesicle
366
cycling and neurotransmitter release along vHipp axons; the majority of the loaded
367
vesicles in the Type III Nrg1 Het axons either failed to destain, or partially destained at a
368
slower rate, upon subsequent depolarizations.
369
In addition to overall alterations in vesicle cycling in Type III Nrg1 Het vHipp axons, the
370
total number of FM1-43 labeled clusters was reduced compared to Nrg1 WT axons (Fig.
371
4A&B). In contrast, the number (P=0.56n) and size (P=0.580) of vGluT1 containing clusters
372
recognized by antibody staining did not differ by genotype (Fig. 4C-E). VGluT1 cluster
373
number (Fig. 4D) along vHipp axons from Nrg1 WT (+/+) (28.1 ± 4.2 per 100 μm) were
374
comparable to axons from Type III Nrg1 Hets (+/-) (28.5 ± 5.5 per 100 μm). After loading
375
with FM1-43, we quantified the numbers of vesicle clusters (puncta) in WT (Fig. 4A, top)
376
and Type III Nrg1 Het (+/-) (Fig. 4A, middle) vHipp axons. Analyses of pooled data (Fig.
377
4B) shows a significant decrease in the number of FM1-43 stained clusters along axons
378
from Type III Nrg1 Het (+/-) (22 ± 2 per 100 μm vHipp axon) compared to WT (+/+) (29 ± 1
16
379
per 100 μm vHipp axon, P=0.008l); there was no statistically significant difference in the
380
size of FM1-43 positive clusters between the genotypes (data not shown, but see Fig 5
381
below). To confirm that presynaptic Type III Nrg1 back-signaling contributes to the
382
regulation of synaptic vesicle clustering, we stimulated Type III Nrg1 back-signaling in
383
Type III Nrg1 Het cultures by adding soluble ErbB4 extracellular domain to the media.
384
After treatment with soluble ErbB4-ECD for 24 hours, microslices were loaded with FM1-
385
43 (Fig. 4A, bottom). Stimulation of Type III Nrg1 back-signaling in vHipp axons from
386
Type III Nrg1 Het animals restored synaptic vesicle cluster numbers to wild type levels (28
387
± 1 per 100 μm vHipp axon, Fig. 4B, P=0.005m).
388
To investigate the effect of reducing presynaptic Type III Nrg1 signaling on the
389
ultrastructural organization of synaptic vesicles, we used transmission electron
390
microscopic examination of ultrathin sections of vHipp micro-slices cultures with extensive
391
axonal arbors. Varicosities containing clusters of apparent synaptic vesicles were clearly
392
seen along WT axons (Fig. 5A and 5B). Following FM1-43 loading and photoconversion,
393
(Harata et al., 2001), we found electron-dense materials resulting from photoconversion of
394
FM1-43 dye were essentially confined to the lumen of photoconverted (PC+) synaptic
395
vesicles (Fig. 5A bottom), whereas non-photoconverted (PC-) synaptic vesicles have a
396
typical translucent appearance (Fig. 5A top), indicating that FM1-43 labeled puncta
397
correspond to vesicle clusters visualized by electron microscopy.
398
Although clusters of 20 to 60, 30nm vesicles were seen along axons from both WT (+/+)
399
and Type III Nrg1 Het (+/-) vHipp micro-slices (Fig. 5B top, middle), there were fewer
400
clusters per 100 µm of Type III Nrg1 Het axon compared to the Nrg1 WTs (Fig 5C): 13 ± 3
401
per 100 μm of Type III Nrg1 Het vHipp axon compared to 31 ± 4 per 100 μm of WT vHipp
17
402
axon (P=0.001p). The organization of vesicles into clusters along the axon was under the
403
control of Type III Nrg1 back-signaling – a 24 hour treatment of Type III Nrg1 Het (+/-)
404
microslices cultures with soluble ErbB4-ECD, increased the number of vesicle clusters
405
(Fig. 5B, bottom, Fig. 5C) to WT levels (34 ± 4 per 100 μm vHipp axon, P=0.001q). Taken
406
together, these findings suggest that reduced presynaptic Type III Nrg1 signaling impairs
407
the organization of synaptic vesicles into distinct clusters, and this deficit can be rescued
408
by stimulating Type III Nrg1 back-signaling.
409
410
411
412
413
414
415
416
417
418
419
420
421
18
422
Discussion
423
We have utilized a gene chimeric, synaptic co-culture between vHipp microslices (pre-
424
synaptic input) and dispersed nAcc MSNs (as the post-synaptic target) to examine the
425
effect of selective reduction of pre-synaptic Type III Nrg1 expression on glutamatergic
426
transmission. Reduction of presynaptic Type III Nrg1 expression had multiple effects on
427
glutamatergic synaptic transmission, including decreases in both amplitude and frequency
428
of MSN mEPSCs. These changes were associated with decreased morphological synapse
429
numbers (vGluT1 x PSD95 staining), decreased trafficking of GluA2 containing AMPA
430
receptors to dendritic surfaces and disrupted organization of functional synaptic vesicles in
431
the vHipp axons.
432
One major mechanism to modify glutamatergic synaptic transmission is to alter the
433
number of synapses at the glutamatergic terminals (Chao et al., 2007). We used co-
434
localized puncta of the presynaptic and postsynaptic markers, vGluT1 and PSD95 (Melone
435
et al., 2005; Chao et al., 2007; Stevens et al., 2007) to quantify vHipp-nAcc glutamatergic
436
synapse number and found decreases at nAcc MSNs innervated by Type III Nrg1
437
heterozygous vHipp axons. These results are consistent with prior findings that have
438
examined the role of Nrg1/ErbB4 signaling in the establishment of excitatory input to
439
cortical GABAergic interneurons (Chen et al., 2010). Nrg1 activation of ErbB4 on axons of
440
cortical GABAergic interneurons has been shown to enhance activity dependent GABA
441
release (Woo et al., 2007). This provides an additional mechanism by which Nrg1 / ErbB4
442
signaling can modulate the function of GABAergic neurons. Although we did not see
443
changes in mIPSC frequency or amplitude in our gene chimera co-cultures, we cannot rule
444
out a similar interaction between axonal Type III Nrg1 and MSN axonal ErbB4. Thus,
19
445
perturbation of Nrg1 / ErbB4 signaling impairs maturation of glutamatergic synaptic input
446
to both GABAergic interneurons and striatal GABAergic projection neurons, possibly
447
contributing to widespread glutamatergic hypofunction (Li et al., 2007).
448
The decreased mEPSC amplitude recorded at nAcc neurons innervated by Type III
449
Nrg1
heterozygous
vHipp
axons
indicates
decreased
postsynaptic
receptor
450
responsiveness. When co-cultures were stained with an antibody recognizing cell surface
451
GluA2 containing AMPA receptors, it was clear that reduction of pre-synaptic Type III Nrg1
452
levels impaired post-synaptic trafficking of surface GluA2 receptors to neurites or reduced
453
accumulation of surface GluA2 at post-synaptic sites. The decrease in surface to total
454
GluA2 levels was only seen on neurites and not on MSN soma, indicating that the effect
455
on trafficking is not to the cell surface per se. Previous studies (Gu et al., 2005; Hahn et
456
al., 2006; Li et al., 2007; Pitcher et al., 2011; Abe et al., 2011; Fenster et al., 2012), largely
457
in hippocampus or cortex, have shown that alterations of Nrg1 / ErbB4 signaling affect
458
glutamatergic synaptic transmission and plasticity by modulating the expression,
459
internalization and insertion of NMDA and AMPA receptors. Here we found that genetic
460
defects in vHipp Type III Nrg1 signaling impaired AMPA receptor trafficking from
461
intracellular sites to the MSN surface.
462
It is likely that the decrease in mEPSC frequency recorded from nAcc MSNs reflects
463
both a decrease in total synapse number as noted above, and a reduction of spontaneous
464
glutamate release at presynaptic terminals. Using both live imaging and high resolution
465
EM, we have demonstrated that pre-synaptic Type III Nrg1 signaling contributes to the
466
formation of functional clusters of synaptic vesicles and contributes to the recycling of
467
vesicle pools.
20
468
Neurotransmitter release involves a cycle of calcium dependent exocytotic fusion of
469
synaptic vesicles (SVs) followed by local endocytic recycling (Alabi and Tsien, 2012;
470
Südhof, 2013; Gauthier-Kemper et al., 2015). Physiologically, SVs are distinguished based
471
on their release probability: there is a so-called readily releasable pool (RRP) consisting of
472
vesicles that are released first following depolarization and there is a reserve pool (RP)
473
which replenishes the RRP upon its depletion. Activity-dependent FM1-43 loading and
474
destaining of the RRP has been widely used to examine the property of neurotransmitter
475
release (Tokuoka and Goda, 2008; Blundon et al., 2011; Upreti et al., 2012). Using FM1-
476
43, we found both an absolute decrease in exocytosis of synaptic vesicles and a
477
decreased rate of release at Type III Nrg1 heterozygous presynaptic specializations after
478
depolarization. Given that FM1-43 loading initially required depolarization-induced vesicle
479
fusion followed by endocytosis, the subsequent defects in FM1-43 destaining are likely to
480
reflect problems in the recycling of vesicles, either within the RRP or between the reserve
481
pool and the RRP. Whether this represents an ongoing role of Type III Nrg1 back-signaling
482
during transmitter release per se or is secondary to other deficits in the formation of active
483
release sites, cannot be distinguished at this time.
484
Quantification of the number of SV clusters, either by FM1-43 loading or at the
485
ultrastructural level revealed significant decreases in Type III Nrg1 heterozygous axons
486
compared to wild type. Similar decreases were not seen when we quantified vesicle
487
clusters by vGlutT antibody staining. It is possible that antibody staining is identifying
488
secretory vesicles that are trafficking along axons that have not yet contributed to
489
formation of mature, functional synaptic vesicles (Ahmari et al., 2000).
21
490
It has been shown before that presynaptic Type III Nrg1 also is required for targeting of
491
nicotinic acetylcholine receptors to presynaptic sites at vHipp-nAcc, and at cortical-BLA
492
synapses where they are important modulators of glutamate release (Zhong et al., 2008;
493
Jiang et al., 2013). Here, we demonstrate an additional level at which Nrg1 / ErbB4
494
signaling modulates neurotransmitter release; by regulating the vesicle recycling process.
495
496
497
Conclusion
498
We conclude that at vHipp-nAccs synapses, pre-synaptic Type III Nrg1 bi-directional
499
signaling controls the establishment of vHipp-nAcc glutamatergic synapses. Type III Nrg1
500
back-signaling contributes to functional synaptic vesicle clustering and neurotransmitter
501
vesicle release. Presynaptic Type III Nrg1, either acting through post-synaptic ErbB4
502
receptors, or secondary to presynaptic glutamate release (or both) controls the extent of
503
post-synaptic glutamatergic synapse formation and the synaptic recruitment of GluA2
504
AMPA receptors. Genetic perturbation of Nrg/ErbB signaling contributes to dysfunction of
505
glutamatergic
506
mechanisms.
synaptic
transmission
through
507
508
509
510
22
both
presynaptic
and
postsynaptic
511
References
512
1. Abe Y, Namba H, Kato T, Iwakura Y, Nawa H (2011) Neuregulin-1 signals from the
513
periphery regulate AMPA receptor sensitivity and expression in GABAergic
514
interneurons in developing neocortex. J Neurosci 31: 5699-709.
515
516
517
518
2. Ahmari SE, Buchanan J, Smith SJ (2000) Assembly of presynaptic active zones
from cytoplasmic transport packets. Nat Neurosci 3: 445-51.
3. Alabi AA, Tsien RW (2012) Synaptic vesicle pools and dynamics. Cold Spring Harb
Perspect Biol 4: a013680.
519
4. Allam SL, Bouteiller JM, Hu EY, Ambert N, Greget R, Bischoff S, Baudry M, Berger
520
TW (2015) Synaptic Efficacy as a Function of Ionotropic Receptor Distribution: A
521
Computational Study. PLoS One 10: e0140333.
522
5. Banerjee A, Macdonald ML, Borgmann-Winter KE, Hahn CG (2010) Neuregulin 1-
523
erbB4 pathway in schizophrenia: From genes to an interactome. Brain Res Bull 83:
524
132-9.
525
6. Blundon JA, Bayazitov IT, Zakharenko SS (2011) Presynaptic gating of
526
postsynaptically expressed plasticity at mature thalamocortical synapses. J
527
Neurosci 31: 16012-25.
528
7. Canetta SE, Luca E, Pertot E, Role LW, Talmage DA (2011) Type III Nrg1 back
529
signaling enhances functional TRPV1 along sensory axons contributing to basal
530
and inflammatory thermal pain sensation. PLoS One 6: e25108.
531
532
8. Chao HT, Zoghbi HY, Rosenmund C (2007) MeCP2 controls excitatory synaptic
strength by regulating glutamatergic synapse number. Neuron 56: 58-65.
23
533
9. Chen YJ, Johnson MA, Lieberman MD, Goodchild RE, Schobel S, Lewandowski N,
534
Rosoklija G, Liu RC, Gingrich JA, Small S, Moore H, Dwork AJ, Talmage DA, Role
535
LW (2008) Type III neuregulin-1 is required for normal sensorimotor gating,
536
memory-related behaviors, and corticostriatal circuit components. J Neurosci 28:
537
6872-83.
538
10. Chen YJ, Zhang M, Yin DM, Wen L, Ting A, Wang P, Lu YS, Zhu XH, Li SJ, Wu CY,
539
Wang XM, Lai C, Xiong WC, Mei L, Gao TM (2010) ErbB4 in parvalbumin-positive
540
interneurons is critical for neuregulin 1 regulation of long-term potentiation. Proc
541
Natl Acad Sci U S A 107: 21818-23.
542
11. Coyle JT, Basu A, Benneyworth M, Balu D, Konopaske G (2012) Glutamatergic
543
synaptic dysregulation in schizophrenia: therapeutic implications. Handb Exp
544
Pharmacol 213: 267-95.
545
12. Fazzari P, Paternain AV, Valiente M, Pla R, Luján R, Lloyd K, Lerma J, Marín O,
546
Rico B (2010) Control of cortical GABA circuitry development by Nrg1 and ErbB4
547
signalling. Nature 464: 1376-80.
548
13. Fenster C, Vullhorst D, Buonanno A (2012) Acute neuregulin-1 signaling influences
549
AMPA receptor mediated responses in cultured cerebellar granule neurons. Brain
550
Res Bull 87: 21-9.
551
14. Gaspar PA, Bustamante ML, Silva H, Aboitiz F (2009) Molecular mechanisms
552
underlying glutamatergic dysfunction in schizophrenia: therapeutic implications. J
553
Neurochem 111: 891-900.
554
555
15. Gauthier-Kemper A, Kahms M, Klingauf J (2015) Restoring synaptic vesicles during
compensatory endocytosis. Essays Biochem 57: 121-34.
24
556
16. González-Forero D, Montero F, García-Morales V, Domínguez G, Gómez-Pérez L,
557
García-Verdugo JM, Moreno-López B (2012) Endogenous Rho-kinase signaling
558
maintains synaptic strength by stabilizing the size of the readily releasable pool of
559
synaptic vesicles. J Neurosci 32: 68-84.
560
17. Grossmann KS, Wende H, Paul FE, Cheret C, Garratt AN, Zurborg S, Feinberg K,
561
Besser D, Schulz H, Peles E, Selbach M, Birchmeier W, Birchmeier C (2009) The
562
tyrosine phosphatase Shp2
563
throughout Schwann cell development. Proc Natl Acad Sci U S A 106: 16704-9.
564
18. Gu Z, Jiang Q, Fu AK, Ip NY, Yan Z (2005) Regulation of NMDA receptors by
565
566
(PTPN11)
directs Neuregulin-1/ErbB signaling
neuregulin signaling in prefrontal cortex. J Neurosci 25: 4974-84.
19. Hahn CG, Wang HY, Cho DS, Talbot K, Gur RE, Berrettini WH, Bakshi K, Kamins J,
567
Borgmann-Winter KE, Siegel SJ, Gallop RJ, Arnold SE (2006) Altered neuregulin 1-
568
erbB4 signaling contributes to NMDA receptor hypofunction in schizophrenia. Nat
569
Med 12: 824-8.
570
20. Hancock ML, Canetta SE, Role LW, Talmage DA (2008) Presynaptic type III
571
neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to
572
axons. J Cell Biol 181: 511-21.
573
21. Hancock ML, Nowakowski DW, Role LW, Talmage DA, Flanagan JG (2011) Type
574
III neuregulin 1 regulates pathfinding of sensory axons in the developing spinal cord
575
and periphery. Development 138: 4887-98.
576
22. Harata N, Ryan TA, Smith SJ, Buchanan J, Tsien RW (2001) Visualizing recycling
577
synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion. Proc Natl
578
Acad Sci U S A 98 (22): 12748-53.
25
579
23. Hobbs SS, Coffing SL, Le AT, Cameron EM, Williams EE, Andrew M, Blommel EN,
2nd
580
Hammer RP, Chang H, Riese DJ
581
patterns of ErbB family receptor activation. Oncogene 21: 8442-52.
582
583
(2002) Neuregulin isoforms exhibit distinct
24. Ippolito DM, Eroglu C (2010) Quantifying synapses: an immunocytochemistrybased assay to quantify synapse number. J Vis Exp 45: pii2270.
584
25. Jiang L, Emmetsberger J, Talmage DA, Role LW (2013) Type III neuregulin 1 is
585
required for multiple forms of excitatory synaptic plasticity of mouse cortico-
586
amygdala circuits. J Neurosci 33: 9655-66.
587
26. Kim HG, Lee CK, Cho SM, Whang K, Cha BH, Shin JH, Song KH, Jeong SW (2013)
588
Neuregulin 1 up-regulates the expression of nicotinic acetylcholine receptors
589
through the ErbB2/ErbB3-PI3K-MAPK signaling cascade in adult autonomic
590
ganglion neurons. J Neurochem 124: 502-13.
591
592
27. Kozorovitskiy Y, Saunders A, Johnson CA, Lowell BB, Sabatini BL (2012)
Recurrent network activity drives striatal synaptogenesis. Nature 485: 646-50.
593
28. Kwon OB, Longart M, Vullhorst D, Hoffman DA, Buonanno A (2005) Neuregulin-1
594
reverses long-term potentiation at CA1 hippocampal synapses. J Neurosci 25:
595
9378-83.
596
597
598
599
29. Li B, Woo RS, Mei L, Malinow R (2007) The neuregulin-1 receptor erbB4 controls
glutamatergic synapse maturation and plasticity. Neuron 54: 583-97.
30. Lin CH, Lane HY, Tsai GE (2012) Glutamate signaling in the pathophysiology and
therapy of schizophrenia. Pharmacol Biochem Behav 100: 665-77.
26
600
31. Liu Y, Ford B, Mann MA, Fischbach GD (2001) Neuregulins increase alpha7
601
nicotinic acetylcholine receptors and enhance excitatory synaptic transmission in
602
GABAergic interneurons of the hippocampus. J Neurosci 21: 5660-9.
603
32. Melone M, Burette A, Weinberg RJ (2005) Light microscopic identification and
604
immunocytochemical characterization of glutamatergic synapses in brain sections.
605
J Comp Neurol 492: 495-509.
606
607
33. Okada M, Corfas G (2004) Neuregulin1 downregulates postsynaptic GABAA
receptors at the hippocampal inhibitory synapse. Hippocampus 14: 337-44.
608
34. Paz RD, Tardito S, Atzori M, Tseng KY (2008) Glutamatergic dysfunction in
609
schizophrenia: from basic neuroscience to clinical psychopharmacology. Eur
610
Neuropsychopharmacol 18: 773-86.
611
35. Pitcher GM, Kalia LV, Ng D, Goodfellow NM, Yee KT, Lambe EK, Salter MW (2011)
612
Schizophrenia
613
upregulation of NMDA receptors. Nat Med 17: 470-8.
614
615
616
susceptibility
pathway
neuregulin
1-ErbB4
suppresses
Src
36. Stefansson H et al. (2002) Neuregulin 1 and susceptibility to schizophrenia. Am J
Hum Genet 71: 877-92.
37. Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N,
617
Micheva KD, Mehalow AK, Huberman AD, Stafford B, Sher A, Litke AM, Lambris JD,
618
Smith SJ, John SW, Barres BA (2007) The classical complement cascade mediates
619
CNS synapse elimination. Cell 131: 1164-78.
620
621
38. Südhof
TC
(2013)
A
molecular
machine
synaptotagmin and beyond. Nat Med 19: 1227-31.
27
for
neurotransmitter
release:
622
39. Ting AK, Chen Y, Wen L, Yin DM, Shen C, Tao Y, Liu X, Xiong WC, Mei L (2011)
623
Neuregulin 1 promotes excitatory synapse development and function in GABAergic
624
interneurons. J Neurosci 31: 15-25.
625
40. Tokuoka H, Goda Y (2008) Activity-dependent coordination of presynaptic release
626
probability and postsynaptic GluR2 abundance at single synapses. Proc Natl Acad
627
Sci U S A 105: 14656-61.
628
41. Upreti C, Otero R, Partida C, Skinner F, Thakker R, Pacheco LF, Zhou ZY,
629
Maglakelidze G, Velíšková J, Velíšek L, Romanovicz D, Jones T, Stanton PK,
630
Garrido-Sanabria ER (2012) Altered neurotransmitter release, vesicle recycling and
631
presynaptic structure in the pilocarpine model of temporal lobe epilepsy. Brain 135:
632
869-85.
633
42. Wolpowitz D, Mason TB, Dietrich P, Mendelsohn M, Talmage DA, Role LW (2000)
634
Cysteine-rich domain isoforms of the neuregulin-1 gene are required for
635
maintenance of peripheral synapses. Neuron 25: 79-91.
636
43. Woo RS, Li XM, Tao Y, Carpenter-Hyland E, Huang YZ, Weber J, Neiswender H,
637
Dong XP, Wu J, Gassmann M, Lai C, Xiong WC, Gao TM, Mei L (2007) Neuregulin-
638
1 enhances depolarization-induced GABA release. Neuron 54: 599-610.
639
44. Zhong C, Du C, Hancock M, Mertz M, Talmage DA, Role LW (2008) Presynaptic
640
type III neuregulin 1 is required for sustained enhancement of hippocampal
641
transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine
642
receptors. J Neurosci 28: 9111-6.
643
644
45. Zhong C, Talmage DA, Role LW (2013) Nicotine elicits prolonged calcium signaling
along ventral hippocampal axons. PLoS One 8 (12): e82719.
28
645
46. Zhong C, Talmage DA, Role LW (2015) Live Imaging of Nicotine Induced Calcium
646
Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons. J Vis
647
Exp 100: e52730.
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
29
665
666
Table 1. Statistical analyses used in this study.
Line
Data structure
Type of test
P
a
Normal distribution
two-sample Kolmagorov–Smirnov test
0.005
b
Normal distribution
two-sample Kolmagorov–Smirnov test
0.008
c
Normal distribution
Student’s t test
0.001
d
Normal distribution
Student’s t test
0.45
e
Normal distribution
Student’s t test
0.08
f
Normal distribution
Student’s t test
0.003
g
Normal distribution
Student’s t test
0.35
h
Normal distribution
Student’s t test
0.005
i
Normal distribution
Student’s t test
0.001
j
Normal distribution
Student’s t test
0.001
k
Normal distribution
Student’s t test
0.001
l
Normal distribution
One-way ANOVA tests
0.008
m
Normal distribution
One-way ANOVA tests
0.005
n
Normal distribution
Student’s t test
0.56
o
Normal distribution
Student’s t test
0.58
p
Normal distribution
One-way ANOVA tests
0.001
q
Normal distribution
One-way ANOVA tests
0.001
667
668
669
670
30
671
FIGURE LEGENDS
672
Figure 1: Reduced presynaptic Type III Nrg1 signaling impairs glutamatergic
673
synaptic transmission at vHipp-nAcc synapses.
674
(A) Schematic of genotype-specific in vitro circuits. Glutamatergic transmission at vHipp-
675
nAcc synapses was examined in gene chimeric co-cultures. In Figure 1 experiments
676
ventral hippocampus/subiculum slices from an individual Nrg+/+ or Nrg+/- mouse were
677
plated as micro thinned explants. Dispersed nucleus accumbens neurons from WT mice
678
were added 24 hrs. later. (B) Representative traces of spontaneous glutamate-receptor
679
mediated synaptic activity (Glu mEPSCs: Bicuculline and TTX resistant; CNQX-APV
680
sensitive) recorded from +/+ vHipp to +/+ nAcc synapses (Top) and from +/- vHipp to +/+
681
nAcc synapses (Bottom). (C) Box plots of mEPSC frequency data from +/+ vHipp to +/+
682
nAcc and from +/- vHipp to +/+ nAcc reveal more than a 3 fold difference (** P<0.01, n = 8
683
for each condition) at gene chimeric synapses compared with WT- WT synapses. (D) Box
684
plots of mEPSC amplitude data from +/+ vHipp to +/+ nAcc and from +/- vHipp to +/+ nAcc
685
reveal a 2 fold difference (** P<0.01, n = 8 for each condition) at gene chimeric synapses
686
compared with WT- WT synapses.
687
(E) Examination of pre and postsynaptic markers in gene chimeric vHipp-nAcc co-cultures.
688
After 5-7 days in vitro, co-cultures were fixed, permeabilized, and stained with antibodies
689
targeted to vesicular glutamate transporter 1 (vGluT1; red) and to PSD95 (green). Red
690
“clusters” of vGluT1 are co-localized with PSD95 (green) on neurites of dispersed nAcc
691
neurons innervated by +/+ vHipp (E, Top, left) or +/- vHipp (E, Bottom left; scale bar:
692
10μm). The arrows indicate co-localization of PSD95 with vGLuT1 (yellow puncta) along
31
693
neurites of nAcc MSN. The number of PSD95 positive /vGluT1 clusters along neurites of
694
dispersed nAcc neurons innervated by +/+ vHipp were significantly greater than those
695
innervated by +/- vHipp inputs (28 ± 4 per 100 μm, n= 9, 5 vs 17 ± 3 per 100 μm n= 6, 3;
696
where n= the number of samples/experiment and the number of separate experiments;
697
**P<0.01, E, middle, left). The number of PSD95 clusters along neurites of nAcc neurons
698
were counted and the bar graph (E, middle, right) showed no difference between +/+ and
699
+/- vHipp innervation (+/+vHipp to +/+nAcc: 60 ± 10, n=9, 5 vs +/-vHipp to +/+nAcc: 54 ±
700
10, n= 6, 3 P>0.05). The number of vGluT1 clusters along neurites of nAcc neurons were
701
also counted and the bar graph (E, right) showed no difference between +/+ and +/- vHipp
702
innervation (+/+ vHipp to +/+ nAcc: 31 ± 4, n=9, 5 vs +/- vHipp to +/+ nAcc: 29 ± 8, n= 6, 3
703
P>0.05).
704
(F) Examination of surface vs total glutamate A2/A3 receptor subtypes (GluA2) in gene
705
chimeric vHipp-nAcc co-cultures. After 5-7 days in vitro, co-cultures were stained with
706
antibodies targeted to GluA2. For labeling of surface GluA2 (green), the cultures were
707
incubated with anti-GluR2 antibody, extracellular, for 45 mins prior to fixation, and then,
708
cultures were fixed, permeabilized, and total GluA2 (Red) were recognized with anti-GluR2
709
+ GluR3 antibody, C-terminal. Clusters of surface (Green) vs total GluA2/3 (Red) can be
710
found on neurites of dispersed nAcc neurons innervated by vHipp axons (F, left panel
711
scale bar: 5 μm). The ratio of integrated intensities of surface GluA2 vs total GluA2
712
between gene chimeric conditions differed in a statistically significant manner (+/+ vHipp to
713
+/+ nAcc: 0.36 ± 0.07, n=10, 4 vs +/- vHipp to +/+nAcc: 0.18 ± 0.02, n= 8, 3; **P<0.01, F
714
right hand panel).
32
715
(G) Examination of synapse formation of surface GluA2 (red) and vGluT1 (green) along
716
nAcc neurites in vHipp-nAcc co-cultures. After 5-7 days in vitro, co-cultures were
717
incubated with anti-GluR2 antibody recognizing an extracellular epitope, for 45 mins to
718
label surface GluA2, and then, cultures were fixed, permeabilized, and stained with
719
antibody targeted to vGluT1. Representative micrographs of vGluT1 and sGluA2 co-
720
localization are shown, the arrows are examples of those co-localized sites (yellow puncta)
721
of sGluA2 (red) and vGLuT1 (green) along neurites of nAcc MSN (G, scale bar: 5 μm).
722
723
Figure 2: Reduced presynaptic Type III Nrg1 signaling decreases depolarization
724
induced FM1-43 destaining along vHipp axons. Cultures of vHipp microslices from WT
725
(Nrg+/+) or heterozygous (Nrg+/-) mice were loaded with FM1-43. Representative
726
micrographs of WT (Nrg+/+, A, left) and Nrg+/- (A, right) vHipp axons (loaded with FM1-
727
43, green) before (A, top) and after (A, bottom) depolarization with elevated extracellular
728
K+ are shown. Scale bar: 10μm. (B) Bar graph showed no difference in the initial FM1-43
729
fluorescence intensities after K+-dependent loading between +/+ and +/- vHipp axons. The
730
efficacy of depolarization in eliciting release was assayed from determinations of FM1-43
731
staining including measures of the % decrease with depolarization as well as any
732
differences in the number and/or size of the FM1-43 clusters (C, D, E). (C) The overall
733
FM1-43 fluorescence intensity decreased along vHipp axons after depolarization in both
734
Nrg+/+ and Nrg+/- vHipp axons, the magnitude of the effect is significantly lower for the
735
Nrg+/- vHipp axons compared with control whether assessed as the percent of total
736
fluorescence intensity (C) or the percent decrease in cluster number (D) or in the size of
737
the FM1-43 clusters (E). Bars graphs of mean ± s.e.m. of 8 independent experiments. The
33
738
effect of depolarization on transmitter release (assayed as FM1-43 destaining) significantly
739
depressed in the Nrg+/- vHipp compared with the Nrg+/+ controls (**P<0.01).
740
Figure 3: Reduced presynaptic Type III Nrg1 slows depolarization induced FM1-43
741
release. (A) Spinning disk confocal images of FM1-43 loaded axons from either +/+ or +/-
742
vHipp were collected every 1.5 seconds for 5 min and FM1-43 fluorescence intensities
743
were calculated and quantified as a normalized integrated intensity at each time point. The
744
percent decrease of normalized integrated intensity at an individual 1µm spot (3 pixels)
745
along vHipp axons were plotted vs. time. Representative plots of the release time course
746
from Nrg+/+ (red) and Nrg+/- (green) vHipp axon are shown before and after
747
depolarization. (B) Box plot of pooled data shows slower decay time constant (τ) of high
748
K+ depolarization induced FM1-43 destaining along axons from Nrg+/- (~8.5 sec, n=20, 8)
749
compared to Nrg+/+ (~4.8 sec, n=36, 10 experiments; **P<0.01).
750
751
Figure 4: ErbB4-induced back-signaling rescues presynaptic phenotype of Type III
752
Nrg+/- vHipp axons. (A) Representative micrographs of Nrg+/+ (top), Nrg+/- (middle)
753
and Nrg+/- with 24 hours treatment of soluble ErbB4 (bottom) vHipp axons loaded with
754
FM1-43 (green) to visualize sites of vesicle clusters (Scale bar: 10μm). (B) Box plot of
755
pooled data shows a significant decrease in the number of FM1-43 stained clusters along
756
axons from Nrg+/- compared to Nrg+/+ vhipp axons. Following 24 hours treatment of
757
Nrg+/- axons with 2 ng/ml soluble ErbB4-ECD, the number of FM1-43 stained clusters
758
along Nrg+/- axons was rescued to Nrg +/+ levels; **P<0.01.
34
759
(C) Representative micrographs of Nrg+/+ (top), Nrg+/- (bottom) vHipp axons stained
760
with antibodies recognizing vGluT1 (red) and a pan axonal marker (green) are shown.
761
Scale bar: 10μm. (D) Quantification of VGluT1 cluster numbers along vHipp axons from
762
Nrg+/+ (28 ± 4 per 100 μm) or Nrg+/- (28.5 ± 5.5 per 100 μm) shows that there is no
763
significant difference between conditions. (E) Box plot of pooled data shows no significant
764
difference in the size of vGluT1 clusters along axons from Nrg+/- compared to Nrg+/+.
765
766
Figure 5: ErbB4 induced back-signaling reverses ultrastructural defects in the
767
number of synaptic vesicle clusters. (A) Representative electron micrographs of control
768
(nonphotoconverted, PC-, top) and photoconverted (PC+, bottom) synaptic vesicles along
769
vHipp axons are shown (Scale bar: 500 nm). The arrows indicate electron-dense material
770
resulting from photoconversion of FM1-43 dye that is confined to the lumen of
771
photoconverted synaptic vesicles. (B) Representative electron micrographs of Nrg+/+
772
(top), Nrg+/- (middle) and Nrg+/- after 24 hours of treatment with soluble ErbB4 (bottom)
773
vHipp axons are shown. The arrows indicate the location of vesicle clusters (defined as ≥
774
15 vesicles within less than a vesicle diameter of one another, Scale bar: 500 nm). (C) Box
775
plot of pooled data (> 40 axons from 2 separate experiments) shows a significant
776
decrease in the number of vesicle clusters along axons from Nrg+/- compared with Nrg+/+;
777
**P<0.01. Following 24 hours treatment with 2 ng/ml soluble ErbB4-ECD, the total number
778
of vesicle clusters per 100 µm axon length along Nrg+/- vHipp axons was rescued to levels
779
comparable to Nrg+/+ control (**P<0.01).
780
35
781
782
783
36