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Research Articles: Neurobiology of Disease
Amyloid-beta modulates low-thresholdactivated voltage-gated L-type calciumchannels of arcuate neuropeptide Y neuronsleading to calcium dysregulation andhypothalamic dysfunction
https://doi.org/10.1523/JNEUROSCI.0617-19.2019
Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0617-19.2019
Received: 13 March 2019Revised: 17 July 2019Accepted: 11 September 2019
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Title: Amyloid-beta modulates low-threshold activated voltage-gated L-type calcium channels of 1
arcuate neuropeptide Y neurons leading to calcium dysregulation and hypothalamic dysfunction 2
3
Abbreviated Title: Ca2+ dysregulation in NPY neurons by amyloid-beta 4
5
Makoto Ishii1,2*, Abigail J. Hiller1, Laurie Pham1, Matthew J. McGuire1, Costantino Iadecola1,2, 6
and Gang Wang1 7
8
1 Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, 10065 9
2 Department of Neurology, Weil Cornell Medicine, New York, NY, 10065 10
* Corresponding author 11
12
Corresponding Author: 13
Makoto Ishii, M.D., Ph.D. 14
Feil Family Brain and Mind Research Institute 15
Weill Cornell Medicine 16
407 E. 61st Street 17
3rd Floor 18
New York, NY 10065, USA 19
Tel: (646) 962-8256 20
Fax: (646) 962-0535 21
Email: [email protected] 22
23
Number of figures: 8 24
Number of tables: 0 25
26
1
Number of words 27
Abstract: 245 28
Significance Statement: 120 29
Introduction: 639 30
Discussion: 1499 31
32
Conflict of Interest: The authors declare no competing financial interests. 33
34
Acknowledgements: This work was supported by the National Institutes of Health Grant 35
NS37853 to C.I. and AG051179 to M.I. We thank Dr. Anjali Rajadhyaksha for helpful 36
discussions, Mr. Daniel Lee for technical support, and Ms. Alicia Savage-Nieves, Sophy Aguilar, 37
Heather Blades, and Karen Carter for administrative support. M.J.M. is currently at Department 38
of Neurosurgery, Jacobs School of Medicine and Biomedical Science, Buffalo, NY, USA. 39
1
Abstract 40
Weight loss is an early manifestation of Alzheimer’s disease that can precede the cognitive 41
decline, raising the possibility that amyloid-beta (Aβ) disrupts hypothalamic neurons critical for 42
the regulation of body weight. We previously reported that in young transgenic mice 43
overexpressing mutated amyloid precursor protein (Tg2576) Aβ causes dysfunction in 44
neuropeptide Y (NPY)-expressing hypothalamic arcuate neurons prior to plaque formation. In 45
this study, we examined whether Aβ causes arcuate NPY neuronal dysfunction by disrupting 46
intracellular Ca2+ homeostasis. Here, we found that the L-type Ca2+ channel blocker nimodipine 47
could hyperpolarize the membrane potential, decrease the spontaneous activity, and reduce the 48
intracellular Ca2+ levels in arcuate NPY neurons from Tg2576 brain slices. In these neurons, 49
there was a shift from high to low voltage-threshold activated L-type Ca2+ currents resulting in 50
increased Ca2+ influx closer to the resting membrane potential, an effect recapitulated by Aβ1-42 51
and reversed by nimodipine. These low voltage-threshold activated L-type Ca2+ currents were 52
dependent in part on calcium/calmodulin-dependent protein kinase II and IP3 pathways. 53
Furthermore, the effects on intracellular Ca2+ signaling by both a positive (ghrelin) and negative 54
(leptin) modulator were blunted in these neurons. Nimodipine pre-treatment restored the 55
response to ghrelin-mediated feeding in young (3-5 months) but not older (10 months) female 56
Tg2576 mice, suggesting that intracellular Ca2+ dysregulation is only reversible early in Aβ 57
pathology. Collectively, these findings provide evidence for a key role of low-threshold activated 58
voltage gated L-type Ca2+ channels in Aβ-mediated neuronal dysfunction and in the regulation 59
of body weight. 60
61
62
63
64
65
2
66
Significance Statement 67
Weight loss is one of the earliest manifestations of Alzheimer’s disease (AD), but the underlying 68
cellular mechanisms remain unknown. Disruption of intracellular Ca2+ homeostasis by amyloid-69
beta is hypothesized to be critical for the early neuronal dysfunction driving AD pathogenesis. 70
Here, we demonstrate that amyloid-beta causes a shift from high to low voltage-threshold 71
activated L-type Ca2+ currents in arcuate neuropeptide Y neurons. This leads to increased Ca2+ 72
influx closer to the resting membrane potential resulting in intracellular Ca2+ dyshomeostasis 73
and neuronal dysfunction, an effect reversible by the L-type Ca2+ channel blocker nimodipine 74
early in amyloid-beta pathology. These findings highlight a novel mechanism of amyloid-beta-75
mediated neuronal dysfunction through L-type Ca2+channels and the importance of these 76
channels in the regulation of body weight. 77
78
79
80
3
Introduction 81
Weight loss is a common manifestation of Alzheimer’s disease (AD) and since the 1984 82
consensus statement has been a criterion consistent with the diagnosis of AD (McKhann et al., 83
1984). In epidemiological studies, early weight loss in AD was associated with worsening 84
disease progression and increased mortality, while weight gain was protective (White et al., 85
1998), suggesting that the factors involved in maintaining body weight are likely to be intrinsic to 86
AD pathogenesis. Importantly, weight loss can precede the cognitive decline in AD, suggesting 87
that systemic metabolic dysfunction occurs during the preclinical stage of AD, where 88
neuropathological abnormalities like hyperphosphorylated tau and amyloid-beta (Aβ) have 89
started to accumulate but have not caused significant cognitive impairment (Johnson et al., 90
2006; Gao et al., 2011; Emmerzaal et al., 2015). Despite the evidence supporting the 91
importance of early systemic metabolic dysfunction in AD, the underlying mechanisms have not 92
been elucidated but could conceivably be due to disruption of brain circuits regulating body 93
weight (Ishii and Iadecola, 2015). 94
Neurons that co-express neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the 95
arcuate nucleus of the hypothalamus are major orexigenic or positive regulators of body weight 96
(Loh et al., 2015). Leptin is an adipocyte-derived hormone that maintains body weight 97
homeostasis by acting as the negative afferent signal to the brain, where it can inhibit arcuate 98
NPY neurons (McGuire and Ishii, 2016). Conversely, ghrelin is a stomach-derived peptide that 99
can increase feeding and alter systemic metabolism by stimulating arcuate NPY neurons 100
(Yanagi et al., 2018). Therefore, leptin and ghrelin regulate body weight in large part by 101
modulating arcuate NPY neuronal function. We previously found that a transgenic mouse model 102
of Aβ accumulation (Tg2576 mice), at a young age (3 months) prior to developing amyloid 103
plaques, exhibited early body weight deficits and low plasma leptin levels that were due to 104
increased energy expenditure and not from changes in feeding behavior (Ishii et al., 2014). 105
Furthermore, the systemic metabolic deficits in this mouse model were associated with 106
4
depolarized membrane potentials in arcuate NPY neurons and a blunted response to both leptin 107
and ghrelin (Ishii et al., 2014). Collectively, these results are consistent with Aβ causing arcuate 108
NPY neuronal dysfunction leading to early systemic metabolic deficits; however, how Aβ causes 109
dysfunction in these neurons is not known. 110
Since intracellular Ca2+ signaling mechanisms play a key role in the regulation of arcuate 111
NPY neurons by leptin and ghrelin (Kohno et al., 2003; Wang et al., 2008), intracellular Ca2+ 112
dyshomeostasis may underlie the Aβ-mediated dysfunction of these neurons. Decades of 113
accumulating evidence have led to the hypothesis that early disruption of intracellular Ca2+ 114
signaling is critical for the neuronal dysfunction and driving AD pathogenesis (Alzheimer's 115
Association Calcium Hypothesis Workgroup, 2017). This disruption of intracellular Ca2+ 116
homeostasis by Aβ is thought to begin at the plasma membrane (Goodison et al., 2012). 117
Therefore, in this study, we tested the hypothesis that Aβ pathology can cause arcuate NPY 118
neuronal dysfunction by disrupting intracellular Ca2+ homeostasis through the modulation of 119
voltage-gated Ca2+ channels. Using brain slices or dissociated neurons from young Tg2576 120
mice, we found that arcuate NPY neurons have aberrant hyperactivity and increased 121
intracellular Ca2+ levels, an effect recapitulated in wild-type (WT) arcuate NPY neurons treated 122
with exogenous Aβ1-42 and reversed by the dihydropyridine L-type Ca2+ channel blocker 123
nimodipine. Whole-cell recordings demonstrated that these arcuate NPY neurons switched from 124
high to low voltage-threshold activated L-type Ca2+ currents, resulting in increased Ca2+ influx 125
and neuronal dysfunction. Furthermore, nimodipine rescued ghrelin-mediated feeding in young 126
(3-5 months) but not older (10 months) Tg2576 mice, suggesting that Aβ-mediated Ca2+ 127
neurotoxicity is reversible only during the early stages of Aβ pathology. These findings show 128
that Aβ disrupts intracellular Ca2+ homeostasis and neuronal function through low voltage-129
threshold activated L-type Ca2+ currents and that L-type Ca2+ channels in arcuate NPY neurons 130
play an important role in the regulation of body weight. 131
132
5
Materials & Methods 133
Animals. All procedures were approved by the Institutional Animal Care and Use Committee of 134
Weill Cornell Medical College. Experiments were performed in the Tg2576 transgenic line, 135
which overexpresses the Swedish mutant (K670N, M671L) human amyloid precursor protein 136
gene (APPswe) driven by the hamster prion protein promoter (RRID: MGI:3710766) (Hsiao et al., 137
1996). For NPY-GFP mice, we used the previously well-characterized BAC transgenic NPY-138
hrGFP mice line (B6.FVB-Tg(NPY-hrGFP)1Lowl/J, The Jackson Laboratory, catalog #006417, 139
RRID:IMSR_JAX:006417) (van den Pol et al., 2009; Ishii et al., 2014). To generate Tg2576 140
mice with GFP labeling in NPY neurons, we crossed male Tg2576 mice with female NPY-GFP 141
mice to produce hemizygous NPY-GFP mice with or without a copy of the APPswe transgene. 142
Wild-type (WT) littermates lacking the APPswe transgene were used as controls. All mice were 143
derived from an in-house colony and housed in climate controlled 12 h light-dark cycle rooms 144
with free access to water and standard rodent chow (LabDiet, catalog #5053). 145
146
Preparation of hypothalamic slices and whole-cell patch-clamp studies. Three to 4 month-old 147
young male and female NPY-GFP hemizygous mice with or without a copy of the APPswe 148
transgene were used in all electrophysiology experiments. The mice were anesthetized with 2% 149
isoflurane, and their brains were rapidly removed and immersed into ice-cold sucrose (s)-ACSF 150
composed of (in mM): 248, sucrose; 26, NaHCO3; 1, NaH2PO4; 5, KCl; 5, MgSO4; 0.5, CaCl2; 151
10, glucose; gassed with 95% O2 and 5% CO2, pH 7.3. Coronal slices (200 m thickness) 152
containing the hypothalamic arcuate nuclei were obtained using a Leica VT1000s Vibratome 153
(Leica Microsystems) and stored in a self-designed chamber containing lactic acid (l)-ACSF 154
composed of (in mM): 124, NaCl; 26, NaHCO3; 5, KCl; 1, NaH2PO4; 2, MgSO4; 2, CaCl2; 10, 155
glucose; 4.5, lactic acid, gassed with 95% O2 and 5% CO2 and pH 7.4, at 32 C for one hour, 156
and then the hypothalamic slice was transferred to a glass-bottom recording chamber (P-27; 157
6
Warner Instrument, Hamden, CT) mounted on an E600 epifluorescence microscope (Nikon) 158
stage. The slices were continuously perfused with the oxygenated l-ACSF at 2 mL/min. Under 159
the microscope equipped with 40X water-immersion lens and the FITC filter (Chroma 160
Technology), the arcuate nuclei were identified in the ventromedial portion near the base of the 161
third ventricle with GFP-labeled NPY neurons in slices consistently displaying intense green 162
fluorescence and the visualized whole-cell recordings were conducted only on GFP-labeled 163
neurons (Ishii et al., 2014). The patch glass electrode was pulled using borosilicate capillaries 164
(OD 1.5 mm/ID 0.86 mm; World Precision Instruments) and P-80 micropipette puller (Sutter 165
Instruments Company). 166
167
Current-clamp mode was used to record the membrane potential and spontaneous discharges 168
in arcuate NPY neurons in slices. The resistance of the pipette was 5–7 mΩ when filled with an 169
intracellular solution containing (in mM) the following: 130, K-gluconate; 10, NaCl; 1.6, MgCl2; 170
0.1, EGTA; 10, HEPES; and 2, Mg-ATP; adjusted to pH 7.3. The GFP-positive arcuate neurons 171
were current-clamped using an Axopatch 200A amplifier (filtered at 2 kHz, digitized at 10 kHz) 172
and Digidata 1320A (Molecular Devices). After a Giga Ω seal formation, brief negative pressure 173
was further applied to obtain the whole-cell configuration. The recording began with the 174
membrane test for monitoring the access resistance and the membrane, which was 175
continuously monitored through the recording. Only those cells in which access resistance was 176
stable (change <10%) were included in data analysis. The voltage and current signals were 177
filtered at 2 kHz, digitalized online at a sampling rate of 10 kHz and stored for offline analysis 178
using pClamp 10 software (Molecular Devices). Stable baseline recordings were achieved 179
before local application of vehicle (PBS), ghrelin (100 nM), -Cgtx-GVIA (1 M), -AgaIVA (1 180
M), or nimodipine (2 M) to the bath. The concentration of ghrelin was based on previously 181
7
published studies showing good physiological responses in arcuate NPY neurons (Cowley et 182
al., 2003). 183
. 184
Voltage-clamp mode was used to record L-type voltage-gated Ca2+ currents in arcuate NPY 185
neurons in slices. The resistance of the pipette was 3 5 M when filled with an intracellular 186
solution containing (in mM) 100, CsCl; 30, TEA-Cl; 1, MgCl2; 10, EGTA; 4, NaCl; 10, HEPES; 3, 187
Na2-ATP; adjusted to pH 7.3. Voltage-gated Ca2+ currents were recorded from GFP-labeled 188
arcuate NPY neurons using an Axopatch 200A amplifier (filtered at 2 kHz, digitized at 10 kHz), 189
Digidata 1320A (Molecular Devices) and pClamp 10 software (Molecular Devices). Following a 190
Giga seal formation with a further brief negative pressure applied to obtain the whole-cell 191
configuration. Using 5 mmol/L Ba2+ as the charge carrier, the voltage-gated Ca2+ channel 192
currents were elicited from the holding potential of -60 mV to stepping potentials ranging from -193
50 to +20 mV. The recording began with the membrane test for monitoring the access 194
resistance and the membrane, which was continuously monitored through the recording. Stable 195
baseline recordings were achieved before local application of vehicle (PBS), leptin (100 nM, 196
Sigma-Aldrich) or oligomeric Aβ1-42 (100 nM) to the bath. The concentrations of leptin and 197
oligomeric Aβ1-42 were chosen based on previously published studies showing good 198
physiological responses in arcuate NPY neurons (Berman et al., 2008; Baver et al., 2014; Ishii 199
et al., 2014). Only those cells in which access resistance was stable (change < 5%) were 200
included for data analysis. The Ca2+ channel current amplitudes were expressed as mean 201
SEM with N as the number of neurons tested from a minimum of 3 mice per group. The L-type 202
Ca2+ current was measured as the amplitude of Ca2+ currents at the end of the 500 msec 203
depolarizing pulse, whereas the amplitude of the transient fast inactivating Ca2+ current was 204
obtained by subtracting the amplitude of the L-type Ca2+ current from the peak transient Ca2+ 205
currents measured at 30 msec after initiation of depolarization pulse. The current-voltage 206
8
relationship (I/V) curves were plotted for each recording. The cell area was determined using 207
the cell membrane capacitance (pF) and the density of the Ca2+ channel currents (pA) was 208
expressed as pA/pF. 209
210
Assessment of cytoplasmic Ca2+ levels in arcuate NPY-GFP neurons. Three to 4 month-old 211
young male or female NPY-GFP hemizygous mice with or without a copy of the APPswe 212
transgene were used. Cytoplasmic free Ca2+ was measured using Fura-2 AM as the indicator 213
(Molecular Probes), as previously described (Koizumi et al., 2018). The isolated arcuate NPY-214
GFP neurons were obtained using enzymatic digestion with 0.05% thermolysin plus 0.05% 215
pronase (Sigma-Alderich Chemicals). Fura-2 AM (25 M) was loaded to the cells for 45 min at 216
37°C, and then transferred to polyornithine-coated glass-bottom Petri dish (Warner Instruments, 217
CT). Images were acquired on a Nikon 300 inverted microscope by using a fluorite oil-218
immersion lens (Nikon CF UV-F X40; N.A., 1.3). Fura-2 AM was alternately excited through 219
narrow bandpass filters (340 and 380 nm). An intensified CCD camera (Retiga ExI) recorded 220
the fluorescence emitted by the indicator (510±40 nm). Fluorescent images were digitized and 221
backgrounds were subtracted. GFP labeled cells were simultaneously recorded in a randomly 222
selected field. Fluorescence measurements were obtained from cell body and ratios were 223
calculated for each pixel by using a standard formula. Results were expressed as 340/380 nm 224
ratio. Leptin (100 nM), ghrelin (100 nM), Aβ1-42 (100 nM) and nimodipine (2 M) were perfused 225
for 20 min, respectively. 226
227
Preparation of oligomeric Aβ1-42. Soluble oligomeric Aβ1-42 was freshly prepared from lyophilized 228
solid human synthetic Aβ1-42 (rPeptide, catalog #A-1002) as previously described (Ishii et al., 229
2014). Briefly, lyophilized Aβ1-42 was suspended in 1,1,1, 3,3,3 hexafluoro-2-propanol (Sigma-230
Aldrich) to 1 mM using a gas-tight syringe. The peptide solution was aliquoted and lyophilized 231
9
using a Speed-Vac. Dried peptide was stored at -80°C until additional processing. Immediately 232
prior to the electrophysiology or calcium imaging experiments, dried peptide was resuspended 233
in anhydrous DMSO (Sigma-Aldrich), bath-sonicated for 10 min, and diluted to 100 mM in PBS. 234
The Aβ1-42 was then allowed to oligomerize by incubation at 4°C for 24 h. After incubation, the 235
peptide solution was centrifuged to remove insoluble aggregates. The supernatant containing 236
the soluble oligomeric Aβ1-42 was removed and used for the electrophysiology and calcium 237
imaging experiments as described. 238
239
Ghrelin-induced feeding experiments. A randomized controlled crossover design was used for 240
the feeding experiments. 3 to 5 month-old female Tg2576 or WT littermates were randomly 241
assigned to receive either vehicle (PBS) or ghrelin (0.5 mg per kg body weight in PBS, Phoenix 242
Pharmaceuticals). Ghrelin dosage was determined based on previously published experiments 243
in mice (Wang et al., 2002; Ueno et al., 2004). Each mouse was lightly restrained and given a 244
single intraperitoneal injection of PBS or ghrelin 3 hours into the light-cycle, when mice are in a 245
satiated state. Immediately after the injection, each mouse was individually housed in a 246
standard cage with free access to water and a pre-measured standard rodent chow (LabDiet, 247
catalog #5053). One hour after the injection, food intake was measured with the cage carefully 248
inspected to ensure there were no missing or spilled pellets. After the experiment, mice were 249
group housed with the same littermates as before. One week later, mice that had been given 250
previously PBS received ghrelin and those that were given ghrelin received PBS injections. 251
Food intake was measured as before. For the nimodipine pre-treatment experiment, 3 to 5 252
month-old mice were first administered a single intraperitoneal injection of nimodipine (10 mg 253
per kg body weight in 2% DMSO and PBS, Sigma-Aldrich). 30 minutes after the nimodipine 254
injection, mice were randomly given an intraperitoneal bolus injection of either ghrelin (0.5 mg 255
per kg body weight in PBS) or PBS alone. Food intake was measured as before. The following 256
week, all mice were pre-treated as before with nimodipine and those that received PBS the prior 257
10
week received ghrelin and those that received ghrelin received PBS. Experiments were 258
repeated in 10-month old Tg2576 and WT littermate mice. Since adult male Tg2576 mice have 259
to be single-housed at all times due to aggressive behavior, female mice were used in all 260
feeding experiments to minimize any confounding effects caused by stress due to prolonged 261
social isolation. The food intake measured was normalized to the body weight (per 100 g) of 262
each mouse to minimize any variations due to differences in body weight as Tg2576 mice as a 263
group have lower body weight compared to WT littermates throughout adulthood (Ishii et al., 264
2014). 265
266
Experimental Design and Statistical Analyses. Experiments were designed to determine 267
changes associated with experimental manipulations (e.g., vehicle and drug) and/or genotype 268
(i.e., Tg2576 mice compared to WT littermate mice). Unless otherwise indicated, all data are 269
presented as mean SEM and analyzed using Prism version 8 (GraphPad software). Two-270
group comparisons were analyzed by the two-tailed unpaired Student’s t test. Two-tailed paired 271
Student’s t test was used for comparisons between two different treatments (i.e., vehicle and 272
drug) used in the same cell or mouse. Multiple three or more group comparisons were 273
evaluated by one-way or two-way ANOVA followed by post hoc Tukey or Sidak’s test for 274
multiple comparisons. Differences were considered statistically significant for p values < 0.05. * 275
p < 0.05, ** p < 0.01, and *** p < 0.001. The data that support the findings of this study are 276
available from the authors upon reasonable request. 277
278
Results 279
Arcuate NPY neurons from Tg2576 mice exhibit nimodipine-sensitive changes in 280
membrane potential and spike frequency 281
Using whole-cell current-clamp recordings, we investigated the electrophysiological properties 282
of arcuate NPY neurons in brain slices of Tg2576 mice expressing GFP in NPY neurons (Ishii et 283
11
al., 2014). Since voltage-gated Ca2+ channels are involved in Aβ-mediated intracellular Ca2+ 284
dysregulation in other systems (Mattson, 2007; Alzheimer's Association Calcium Hypothesis 285
Workgroup, 2017; Frere and Slutsky, 2018), we tested whether these channels could be 286
responsible for the aberrant electrophysiological properties of arcuate NPY neurons in Tg2576 287
brain slices. The L-type Ca2+ channel antagonist nimodipine (2 M) significantly hyperpolarized 288
the membrane potential and lowered the spike frequency in arcuate NPY neurons from Tg2576 289
slices (Fig. 1A-C); however, antagonists against other types of voltage-gated Ca2+ channels 290
such as -Cgtx-GVIA (1 M) and -AgaIVA (1 M), which respectively block N-type and P/Q-291
type Ca2+ channels, had no effect (Fig. 1D-E). 292
293
Arcuate NPY neurons from Tg2576 mice exhibit nimodipine-sensitive increases in 294
cytoplasmic Ca2+ levels 295
Since the electrical activities in Tg2576 arcuate NPY neurons were sensitive to the L-type Ca2+ 296
channel blocker nimodipine, we next used Fura-2 AM to image and quantitate cytoplasmic Ca2+ 297
levels in dissociated arcuate NPY neurons identified by GFP. Arcuate NPY neurons from 298
Tg2576 mice had a higher cytoplasmic Ca2+ levels at baseline compared to those from WT mice 299
(Fig. 2A). Incubation with nimodipine (2 M) decreased cytoplasmic Ca2+ levels in arcuate NPY 300
neurons from Tg2576 mice but not in WT mice (Fig. 2A). Next, we incubated arcuate NPY 301
neurons dissociated from WT mice with oligomeric Aβ1-42 (100 nM) and found a significant dose-302
related increase in cytoplasmic Ca2+ levels that was reversed by nimodipine (Fig. 2B). 303
304
The peak L-type Ca2+ current-voltage (I-V) curve of arcuate NPY neurons from Tg2576 305
mice is shifted to the left 306
Due to the increased cytoplasmic Ca2+ levels and altered activity in arcuate NPY neurons from 307
Tg2576 and WT slices treated with Aβ1-42, we next sought to examine the role of Ca2+ currents 308
12
in these neurons. Using a whole-cell voltage-clamp configuration, voltage-gated calcium 309
currents were elicited in the presence of the Na+ channel blocker TTX and the N-type Ca2+ 310
channel blocker -Cgtx-GVIA (1 M) (Fig. 3A). Interestingly, there was a left-shift in the peak L-311
type Ca2+ current I-V curve of arcuate NPY neurons from Tg2576 brain slices compared to WT 312
slices (Fig. 3B), an effect also observed in arcuate NPY neurons of WT slices incubated with 313
oligomeric Aβ1-42 (100 nM) (Fig. 3C). The left-shift in the I-V curves suggests a propensity for L-314
type Ca2+ channels to open at a lower voltage-threshold, closer to the resting membrane 315
potentials, resulting in increased Ca2+ influx into the neurons. Nimodipine (2 M) blocked L-type 316
Ca2+ currents at -10 mV in NPY neurons from brain slices of Tg2576 mice as well as WT brain 317
slices incubated with oligomeric Aβ1-42. (Fig. 3D). 318
319
CaMKII and IP3 inhibitors can partially reverse the left-shift in the L-type Ca2+ current I-V 320
curve of arcuate NPY neurons from Tg2576 mice 321
To gain initial insight into the signaling pathways contributing to the Aβ-mediated changes in L-322
type Ca2+ currents, we investigated whether common modulators of intracellular Ca2+ signaling 323
were involved (Gao et al., 2006; Zamponi et al., 2015; Zamponi, 2016). KN93 (10 M), an 324
inhibitor of calcium/calmodulin-dependent kinase II (CaMKII), and 2APB (50 M), inhibitor of IP3 325
and transient receptor potential (TRP) channels, partially reversed the left-shift in the peak L-326
type Ca2+ current I-V curve of Tg2576 slices, but the phospholipase C inhibitor U733122 (10 327
M) had no effect (Fig. 4A, B, C). This observation suggests that the intracellular Ca2+ 328
dyshomeostasis in arcuate NPY neurons caused by Aβ is mediated in part through CaMKII and 329
IP3-dependent mechanisms. 330
331
Ghrelin and leptin fail to modulate cytoplasmic Ca2+ levels in arcuate NPY neurons of 332
Tg2576 mice 333
13
Next, we tested whether a known activator (ghrelin) and inhibitor (leptin) of arcuate NPY 334
neurons could modulate cytoplasmic Ca2+ levels in these neurons from WT and Tg2576 mice 335
(McGuire and Ishii, 2016; Yanagi et al., 2018). Using Ca2+ imaging in arcuate NPY neurons 336
dissociated from WT mice, we found that ghrelin (100 nM) increased the cytoplasmic free Ca2+ 337
levels (Fig. 5A). However, ghrelin had no effect on cytoplasmic Ca2+ levels of arcuate NPY 338
neurons from Tg2576 mice (Fig. 5A). Similarly, leptin (100 nM) decreased the cytoplasmic free 339
Ca2+ levels in WT, but not in Tg2576 arcuate NPY neurons (Fig. 5B). We then sought to 340
determine if leptin regulates cytoplasmic free Ca2+ levels in these neurons through L-type Ca2+ 341
channels. Using whole-cell voltage-clamp recordings, we found that leptin decreased the peak 342
L-type Ca2+ current amplitudes in WT, but not in Tg2576 arcuate NPY neurons (Fig. 5C, D), 343
attesting to the leptin insensitivity of these neurons. 344
345
Aβ1-42 attenuates the neurophysiological responses to ghrelin in arcuate NPY neurons 346
from WT mice, an effect mediated by L-type Ca2+ channels 347
Since the neurophysiological responses to ghrelin were attenuated in Tg2576 arcuate NPY 348
neurons, we examined whether Aβ1-42 mediated this effect by L-type Ca2+ channels. Using a 349
whole-cell current-clamp configuration, ghrelin could significantly depolarize the membrane 350
potential and increase the spike frequency in WT arcuate NPY neurons, an effect that was 351
attenuated when pre-incubated with oligomeric Aβ1-42 (100 nM) (Fig. 6A, B). Incubation with 352
nimodipine (2 M) restored the effects of ghrelin on the membrane potentials (Fig. 6A, B). 353
Similarly, using Ca2+ imaging in arcuate NPY neurons dissociated from WT mice, the increased 354
cytoplasmic free Ca2+ levels mediated by ghrelin was attenuated when pre-incubated with 355
oligomeric Aβ1-42 (100 nM) and subsequently restored with nimodipine (2 M) (Fig. 6C). 356
357
14
Ghrelin induces feeding behavior in WT but not in Tg2576 mice, an effect mediated by L-358
type Ca2+ channels 359
We next tested whether the acute orexigenic effect of ghrelin, which is mediated by arcuate 360
NPY neurons, was also altered in Tg2576 mice in vivo. Using a randomized controlled 361
crossover design, young (3 to 5-month old) female Tg2576 and WT littermate mice were 362
injected with a single bolus of ghrelin (0.5 mg per kg body weight, i.p.) or vehicle (PBS) in the 363
first week and then switched to vehicle or ghrelin as appropriate in the second week. As 364
expected, WT mice had an acute increase in food intake after ghrelin injection; however, 365
Tg2576 mice had no significant increase in food intake after ghrelin injection (Fig. 7A). We then 366
examined whether nimodipine, which readily crosses the blood-brain barrier (Ingwersen et al., 367
2018), could restore the acute orexigenic effects of ghrelin in the young Tg2576 mice. Pre-368
treatment with nimodipine (10 mg per kg body weight, i.p.) led to a robust increase in food 369
intake after ghrelin injection in Tg2576 mice that were similar to those seen in WT littermates, 370
suggesting that blocking L-type Ca2+ channels could restore the orexigenic pathways in vivo in 371
the Tg2576 mice (Fig. 7B). However, pre-treatment of nimodipine did not restore the effects of 372
ghrelin on feeding behavior in 10-month old Tg2576 mice (Fig. 7C). 373
374
Hypothalamic neurons in the paraventricular nucleus from Tg2576 mice do not have 375
increased cellular activity and have no change in L-type Ca2+ currents 376
Next, we sought to determine if Aβ-mediated intracellular Ca2+ dysregulation is a general 377
pathological process affecting all hypothalamic neurons. To this end, we selected random 378
hypothalamic neurons in the paraventricular nucleus from WT and Tg2576 mice to determine if 379
these neurons were similarly affected as arcuate NPY neurons. Using whole-cell voltage-clamp, 380
we found that Tg2576 paraventricular hypothalamic neurons display baseline 381
electrophysiological properties similar to those of WT mice and no changes in the amplitudes or 382
the peak L-type Ca2+ current I-V curve (Fig. 8). 383
15
384
Discussion 385
We investigated the cellular mechanisms underlying how Aβ mediates dysfunction of 386
hypothalamic neurons that are essential for the regulation of body weight. Our prior studies had 387
found that overexpression of APP or application of oligomeric Aβ1-42 in arcuate NPY neurons 388
depolarized the membrane potential resulting in a lack of neurophysiological response to 389
positive (ghrelin) and negative (leptin) modulators (Ishii et al., 2014). In this study, we found that 390
the L-type Ca2+ channel antagonist nimodipine hyperpolarized the membrane potential, 391
decreased the spike frequency, and reduced the intracellular Ca2+ levels due to overexpression 392
of APP in arcuate NPY neurons. Furthermore, compared to arcuate NPY neurons from WT 393
slices, there was a left-shift in the peak L-type Ca2+ current I-V curve in arcuate NPY neurons 394
from Tg2576 slices, an effect recapitulated in WT slices treated with oligomeric Aβ1-42. This left-395
shift in the peak L-type Ca2+ current I-V curve suggests that Aβ can alter the L-type Ca2+ influx 396
in arcuate NPY neurons to one that is low-voltage threshold activated with an increased 397
propensity of channel opening around the resting membrane potential, leading to more frequent 398
firing and disruption of intracellular Ca2+ homeostasis. These effects were due in part to CaMKII 399
and IP3-dependent mechanisms. We further show evidence that leptin can decrease, whereas 400
ghrelin can increase cytoplasmic free Ca2+ levels in arcuate NPY neurons from WT mice but not 401
in Tg2576 mice. In vivo, pre-treatment with nimodipine restored the acute hyperphagic effects of 402
ghrelin in young (3-5 months) but not older (10 months) Tg2576 mice. Finally, not all 403
hypothalamic neurons are affected, as paraventricular neurons from Tg2576 mice have L-type 404
Ca2+ currents similar to those of WT mice. Altogether, these observations provide, for the first 405
time, evidence that Aβ disrupts intracellular Ca2+ homeostasis and neurophysiological 406
characteristics of select hypothalamic neurons by shifting from a high-threshold to a low-407
threshold activated L-type Ca2+ current. 408
409
16
Aβ mediated intracellular Ca2+ dysregulation and the role of L-type Ca2+ channels 410
Accumulating evidence support the calcium hypothesis of AD, which postulates that the 411
disruption of mechanisms that normally regulate intracellular Ca2+ signaling plays a critical role 412
in triggering the neuronal dysfunction and driving AD pathogenesis (Alzheimer's Association 413
Calcium Hypothesis Workgroup, 2017). Multiple mouse models of Aβ pathology exhibit 414
intracellular Ca2+ dyshomeostasis including the Tg2576 mice (Lopez et al., 2008; Kastanenka et 415
al., 2016). Furthermore, immunotherapy with aducanumab, an Aβ antibody developed for the 416
treatment of AD, restored intracellular Ca2+ in Tg2576 mice, strengthening the hypothesis that 417
Aβ directly disrupts intracellular Ca2+ homeostasis (Kastanenka et al., 2016). However, the 418
mechanisms of these effects have remained elusive. Early studies have implicated L-type Ca2+ 419
channels as having a significant role in the Aβ mediated Ca2+ neurotoxicity particularly in cortical 420
and hippocampal neurons (Weiss et al., 1994; Ueda et al., 1997; Fu et al., 2006). Additionally, 421
whole cell recordings from HEK293 cells recombinantly expressing L-type Ca2+ channels 422
demonstrated that Aβ peptides can increase L-type Ca2+ currents (Scragg et al., 2005; Kim and 423
Rhim, 2011). Here, we provided a mechanistic insight into this process by reporting that such 424
Aβ-induced Ca2+ dysregulation could be attributed to a shift from high to low voltage-threshold 425
activated L-type Ca2+currents. 426
Despite the significant evidence linking L-type Ca2+ channels to Aβ mediated Ca2+ 427
neurotoxicity in cortical and hippocampal neurons, to the best of our knowledge, nothing was 428
known about its effects on hypothalamic neurons. We present the first evidence that Aβ can 429
cause intracellular Ca2+ dyshomeostasis in select hypothalamic neurons, which could be an 430
underlying mechanism for the early metabolic and non-cognitive manifestations of AD mediated 431
by hypothalamic dysfunction (Ishii and Iadecola, 2015). Additionally, we show that 432
overexpression of APP or oligomeric Aβ1-42 in arcuate NPY neurons can trigger a shift from high 433
to low voltage-threshold-activated L-type Ca2+ currents leading to an increase in intracellular 434
Ca2+ levels. This results in neuronal hyperactivity that likely destabilizes the firing synchrony of 435
17
these neurons and disrupts the network similar to the effects of Aβ on hippocampal neurons 436
(Frere and Slutsky, 2018; Zott et al., 2019). This effect appears to be selective since 437
paraventricular neurons in the hypothalamus of Tg2576 mice were unaffected. However, it is 438
likely that other neuronal populations are affected either independent of the arcuate NPY 439
neuronal dysfunction or directly such as the postsynaptic neurons in the parabrachial nucleus 440
(Wu et al., 2009). Furthermore, the exact source of Aβ causing arcuate NPY neuronal 441
dysfunction has not been elucidated. We have previously shown that APP overexpression can 442
be detected in the hypothalamus of Tg2576 mice (Ishii et al., 2014); however, whether Aβ is 443
expressed directly in arcuate NPY neurons is not known. Ex-vivo experiments with application 444
of exogenous Aβ1-42 to WT slices suggest that Aβ does not need to be expressed in arcuate 445
NPY neurons to cause dysfunction of these neurons. Finally, ghrelin-mediated feeding was 446
restored in young Tg2576 mice after pre-treatment with nimodipine, but importantly, nimodipine 447
could not restore ghrelin function in older Tg2576 mice. Therefore, neuronal dysfunction caused 448
by Ca2+ overload through L-type Ca2+ channels may be reversible only early in Aβ pathology; 449
however, it is unclear how early in the pathological process the dysfunction occurs. 450
How Aβ elicits a low voltage-threshold activated L-type Ca2+ current in arcuate NPY 451
neurons remains elusive. Our studies did not specifically investigate the molecular mechanisms 452
leading to this shift. L-type Ca2+ channels consist of five subunits with α1 subunit forming the 453
main ion-conducting pore of the channel (Zamponi et al., 2015). There are two distinct L-type 454
Ca2+ channel α1 subunits identified in the brain resulting in the two major isoforms Cav1.2 and 455
Cav1.3 (Zamponi et al., 2015). A major electrophysiological distinction between Cav1.2 and 456
Cav1.3 is that Cav1.3 is activated at lower threshold potentials than Cav1.2, triggering sub-457
threshold depolarization, high intracellular Ca2+ levels, spontaneous firings, and synaptic 458
transmission (Koschak et al., 2001; Xu and Lipscombe, 2001; Zamponi et al., 2015; Pinggera 459
and Striessnig, 2016). Therefore, one possibility is that Aβ upregulates Cav1.3 relative to 460
Cav1.2. Alternatively, there could be a shift in cellular localization of Cav1.3 leading to a 461
18
redistribution of the channel to the cell surface. This has been demonstrated in one study where 462
the neurotoxic Aβ25-35 fragment increased L-type Ca2+ currents by upregulating Cav1.3 surface 463
protein levels while Cav1.3 mRNA and total protein levels remained unchanged (Kim and Rhim, 464
2011). It has also been demonstrated that the short Cav1.3 isoform, but not the long isoform, 465
can associate to form cooperative clusters to help facilitate Ca2+ entry into neurons (Moreno et 466
al., 2016). Therefore, Aβ could cause an increase in the short form of Cav1.3 leading to 467
increased Ca2+ influx and subsequent neurotoxicity. Alternatively, Aβ pathology could modify the 468
Cav1.2 protein leading to changes in the electrophysiological properties of Cav1.2 that make it 469
appear more Cav1.3-like. Future studies utilizing genetic models will help to elucidate the 470
contribution of Cav1.2 and Cav1.3 to Aβ-mediated Ca2+ neurotoxicity. 471
Finally, the upstream mechanisms contributing to the Aβ-mediated shift to low-threshold 472
activated L-type Ca2+ currents in arcuate NPY neurons are not known, but our data suggests 473
that they involve CaMKII and IP3-dependent pathways. Therefore, one hypothesis would be that 474
Aβ first causes an increase in intracellular Ca2+ levels independent of L-type Ca2+ channels, 475
such as through NMDA receptors, leading to activation of CaMKII and IP3 pathways. The 476
activation of CaMKII and IP3 pathways could then in turn promote the switch to a low-threshold 477
activated L-type Ca2+ current and further disruption of intracellular Ca2+ homeostasis by Ca2+-478
dependent facilitation in a positive-feedback mechanism. 479
480
L-type Ca2+ channels and the regulation of body weight 481
While the major focus of this study has been on understanding how Aβ pathology in the brain 482
can lead to early systemic metabolic deficits, the results also highlight the role for L-type Ca2+ 483
channels as modulators of arcuate NPY neuronal function and potential therapeutic targets in 484
body weight disorders. Interestingly, L-type Ca2+ channels have been implicated in 485
neuropsychiatric disorders such as mood disorders that often have comorbid body weight 486
disorders, suggesting a common molecular pathway linking these disorders (Treasure et al., 487
19
2015; Kabir et al., 2016). Therefore, modulation of L-type Ca2+ channels in hypothalamic 488
neurons may serve as an important but under-appreciated pathway for regulating body weight. 489
490
Conclusions 491
We have demonstrated that Aβ disrupts intracellular Ca2+ homeostasis in arcuate NPY neurons 492
by shifting from normally high to low voltage-threshold activated L-type Ca2+ currents. While 493
additional studies are needed to elucidate the exact molecular mechanisms and whether Aβ can 494
similarly affect non-hypothalamic neurons, our findings suggest that Aβ disruption of arcuate 495
NPY neurons may contribute to the weight loss seen in the earliest stages of AD. Furthermore, 496
our finding that the L-type Ca2+ channel blocker nimodipine can restore ghrelin-mediated 497
feeding in Tg2576 mice, suggests a therapeutic strategy against the early hypothalamic 498
dysfunction caused by Aβ. Finally, since L-type Ca2+ channels may play a significant role in 499
neurological and psychiatric disorders ranging from autism to Parkinson’s disease (Zamponi, 500
2016), elucidating the mechanisms causing alterations in the function of L-type Ca2+ channels in 501
the brain could lead to a better understanding in the role of these channels in a variety of 502
pathophysiological conditions. 503
504
505
20
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24
Figure Legends: 629
630
Figure 1: The membrane potential and spike frequency in arcuate NPY neurons from Tg2576 631
mice are sensitive to the L-type Ca2+ channel blocker nimodipine. A, Brain slices containing the 632
hypothalamic arcuate nucleus from young (3-4 month old) NPY-GFP mice with APPswe 633
transgene (Tg2576) were used for whole-cell patch-clamp recordings. Representative traces are 634
shown before and after nimodipine (2 M). B, Application of the L-type voltage-gated Ca2+ 635
channel antagonist nimodipine (2 M) hyperpolarized the membrane potential of arcuate NPY 636
neurons in Tg2576 brain slices. n = 8 cells from 6 mice per group. Statistical analysis: two-tailed 637
paired Student’s t-test (t = 4.508, df = 7, p = 0.0028). C, Application of the L-type voltage-gated 638
Ca2+ channel antagonist nimodipine (2 M) reduced the spike frequency of arcuate NPY 639
neurons in Tg2576 brain slices. n = 8 cells from 6 mice per group. Statistical analysis: two-tailed 640
paired Student’s t-test (t = 4.548, df = 7, p = 0.0026). D, Application of the N-type ( -Cgtx-641
GVIA ,1 M) or the P/Q-type Ca2+ channel ( -AgaIVA, 1 M) antagonists had no effect on 642
membrane potential of arcuate NPY neurons in Tg2576 brain slices. n = 4 cells from 3 mice per 643
group. Statistical analysis: Repeated measures one-way ANOVA F(1.030, 3.089) = 0.968, p = 644
0.3993. E, Application of the N-type ( -Cgtx-GVIA ,1 M) or the P/Q-type Ca2+ channel ( -645
AgaIVA, 1 M) antagonists had no effect on the spike frequency of arcuate NPY neurons in 646
Tg2576 brain slices. n = 4 cells from 3 mice per group. Statistical analysis: Repeated measures 647
one-way ANOVA F(1.947, 5.841) = 1.168, p = 0.3727. Data are expressed as means SEM. 648
649
Figure 2: Arcuate NPY neurons from Tg2576 brain slices or exogenous Aβ1-42 treated WT brain 650
slices have increased cytoplasmic free Ca2+ levels that can be reversed by the L-type Ca2+ 651
channel blocker nimodipine (NMD). A, Arcuate NPY neurons from Tg2576 mice have increased 652
cytoplasmic free Ca2+ levels compared to WT mice that are decreased by nimodipine. 653
25
Cytoplasmic free Ca2+ was measured using Fura-2 AM in arcuate NPY neurons isolated by 654
enzymatic digestion from WT or Tg2576 mice. After obtaining baseline levels, cells were 655
perfused with nimodipine (2 M). n = 18 to 21 cells from ≥ 3 mice per group. Statistical analysis: 656
one-way ANOVA F(3, 74) = 9.738, p < 0.0001, followed by post-hoc Tukey’s multiple comparisons 657
** p < 0.01. B, Exogenous Aβ1-42 treated arcuate NPY neurons from WT mice have increased 658
cytoplasmic free Ca2+ levels that are decreased by nimodipine. Cytoplasmic free Ca2+ was 659
measured using Fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from WT 660
mice. After obtaining baseline levels, cells were first perfused with oligomeric Aβ1-42 (100 or 300 661
nM) followed by nimodipine (2 M). n = 4 to 5 cells from 2 mice per group. Statistical analysis: 662
one-way ANOVA F(3, 14) = 5.028, p = 0.0143, followed by post-hoc Tukey’s multiple comparisons 663
* p < 0.05. Data are expressed as means SEM. 664
665
Figure 3: Arcuate NPY neurons from Tg2576 brain slices or exogenous Aβ1-42 treated WT brain 666
slices have a left-shift in the peak L-type Ca2+ current I-V curve that can be reversed by the L-667
type Ca2+ channel blocker nimodipine. A, L-type Ca2+currents in arcuate NPY neurons from 668
Tg2576 brain slices show a propensity for channel opening close to the resting potential 669
compared to arcuate NPY neurons from WT brain slices. Representative traces are shown 670
using the holding potential -60 mV to different stepped potentials from -50 mV to +20 mV 671
(showing -30 mV to 10 mV). B, Current-voltage relationships for L-type Ca2+ currents in arcuate 672
NPY neurons from WT or Tg2576 brain slices. There was a left-shift in the peak L-type Ca2+ 673
current I-V curve in arcuate NPY neurons from Tg2576 brain slices compared to WT brain 674
slices. The peak L-type Ca2+ current in the I-V curve occurred at -20 mV for arcuate NPY 675
neurons from Tg2576 brain slices compared to 0 mV for arcuate NPY neurons from WT slices. 676
At -20 mV, arcuate NPY neurons from Tg2576 brain slices have significantly higher L-type Ca2+ 677
currents compared to arcuate NPY neurons from WT brain slices. Data are represented as L-678
26
type Ca2+ current density (pA/pF). n = 19 to 27 per group from ≥ 12 mice per group. Statistical 679
analysis: two-tailed unpaired Student’s t-test (t = 2.832, df = 45, p = 0.0069). C, The peak L-type 680
Ca2+ current I-V curve in arcuate NPY neurons from WT brain slices treated with oligomeric Aβ1-681
42 (100 nM) recapitulates the left-shift seen in arcuate NPY neurons from Tg2576 brain slices. 682
The peak L-type Ca2+ current in the I-V curve occurred between -10 mV and -20 mV for arcuate 683
NPY neurons after application of oligomeric Aβ1-42 (100 nM) slices compared to 0 mV at 684
baseline (vehicle treatment). At -20 mV, arcuate NPY neurons from oligomeric Aβ1-42 (100 nM) 685
treated WT brain slices have significantly higher L-type Ca2+ currents compared to arcuate NPY 686
neurons from vehicle treated WT brain slices. Data are represented as L-type Ca2+ current 687
density (pA/pF). n = 6 per group from ≥ 4 mice per group. Statistical analysis: two-tailed paired 688
Student’s t-test (t = 4.598, df = 5, p = 0.0059). D, L-type Ca2+ current induced at -10mV were 689
significantly decreased after application of nimodipine (2 M) in arcuate NPY neurons from WT 690
brain slices, Tg2576 brain slices, and WT brain slices treated with oligomeric Aβ1-42 (100 nM). n 691
= 5 to 8 per group from ≥ 4 mice per group. Statistical analysis: two-tailed paired Student’s t-test 692
(WT: t = 9.806, df =7, p < 0.0001; Tg2576: t = 4.694, df = 5, p = 0.0054; Aβ1-42: t = 2.912, df = 4, 693
p = 0.0436). Data are expressed as means SEM. 694
695
Figure 4: Pharmacologic inhibition of CaMKII or IP3 can partially reverse the left-shift in the 696
peak L-type Ca2+ current I-V curve in arcuate NPY neurons from Tg2576 brain slices. A, 697
Current-voltage relationships for L-type Ca2+ currents in arcuate NPY neurons from Tg2576 698
brain slices after application of the CaMKII antagonist KN93 (10 μM). The left-shifted I-V curve 699
was partially reversed after application of KN93. The peak L-type Ca2+ current in the I-V curve 700
occurred at -20 mV for arcuate NPY neurons from vehicle treated Tg2576 brain slices compared 701
to -10 mV after KN93 application. At -20 mV, arcuate NPY neurons from KN93 (10 μM) treated 702
Tg2576 brain slices have significantly lower L-type Ca2+ currents compared to vehicle treated 703
27
Tg2576 brain slices. Data are represented as L-type Ca2+ current density (pA/pF). n = 5 per 704
group from 4 mice per group. Statistical analysis: two-tailed paired Student’s t-test (t = 2.854, df 705
= 4, p = 0.0462). B, Current-voltage relationships for L-type Ca2+ currents in arcuate NPY 706
neurons from Tg2576 brain slices after application of the IP3 antagonist 2APB (50 μM). The left-707
shifted I-V curve was partially reversed after application of 2APB. The peak L-type Ca2+ current 708
in the I-V curve occurred at -20 mV for arcuate NPY neurons from Tg2576 brain slices 709
compared to -10 mV after 2APB application. At -20 mV, arcuate NPY neurons from 2APB (50 710
μM) treated Tg2576 brain slices have significantly lower L-type Ca2+ currents compared to 711
vehicle treated Tg2576 brain slices. Data are represented as L-type Ca2+ current density 712
(pA/pF). n = 4 per group from 3 mice per group. Statistical analysis: two-tailed paired Student’s 713
t-test (t = 3.656, df = 3, p = 0.0353). C, Current-voltage relationships for L-type Ca2+ currents in 714
arcuate NPY neurons from Tg2576 brain slices after application of the phospholipase C 715
antagonist U733122 (10 μM). The left-shifted I-V curve was unchanged after application of 716
U733122. The peak L-type Ca2+ current in the I-V curve occurred at -20 mV for arcuate NPY 717
neurons from Tg2576 brain slices before and after U733122 application. At -20 mV, arcuate 718
NPY neurons from U733122 (10 μM) treated Tg2576 brain slices have similar L-type Ca2+ 719
currents compared to vehicle treated Tg2576 brain slices. n = 5 cells per group from 3 mice per 720
group. Statistical analysis: two-tailed paired Student’s t-test (t = 0.3566, df = 4, p = 0.7394). 721
Data are expressed as means SEM. 722
723
Figure 5: Ghrelin and leptin fail to modulate cytoplasmic free Ca2+ levels in arcuate NPY 724
neurons from Tg2576 mice. A, Ghrelin increases cytoplasmic free Ca2+ levels in arcuate NPY 725
neurons from WT but not Tg2576 mice. Cytoplasmic free Ca2+ was measured using Fura-2 AM 726
in arcuate NPY neurons isolated by enzymatic digestion from WT or Tg2576 mice. After 727
obtaining baseline levels, cells were perfused with ghrelin (100 nM). n = 34 WT and 7 Tg2576 728
cells from ≥ 3 mice per group. Statistical analysis: two-tailed paired Student’s t-test (WT: t = 729
28
2.664, df = 34, p = 0.0117; Tg2576: t = 1.020, df = 7, p = 0.3471). B, Leptin decreases 730
cytoplasmic free Ca2+ levels in arcuate NPY neurons from WT but not Tg2576 mice. 731
Cytoplasmic free Ca2+ was measured using Fura-2 AM in arcuate NPY neurons isolated by 732
enzymatic digestion from WT or Tg2576 mice. After obtaining baseline levels, cells were 733
perfused with leptin (100 nM). n = 25 WT and 20 Tg2576 cells from ≥ 3 mice per group. 734
Statistical analysis: two-tailed paired Student’s t-test (WT: t = 3.032, df = 24, p = 0.0057; 735
Tg2576: t = 2.030, df = 19, p = 0.0566). C, Current-voltage relationships for L-type Ca2+ currents 736
in arcuate NPY neurons from WT brain slices after application of leptin (100 nM). Compared to 737
vehicle treatment, leptin significantly decreased the peak L-type Ca2+ current at 0 mV. Data are 738
represented as L-type Ca2+ current density (pA/pF). n = 7 cells per group. Statistical analysis at 739
0 mV: two-tailed paired Student’s t-test (t = 3.601, df = 6, p = 0.0114). D, Current-voltage 740
relationships for L-type Ca2+ currents in arcuate NPY neurons from Tg2576 brain slices after 741
application of leptin (100 nM). Compared to vehicle treatment, there is no significant difference 742
in L-type Ca2+ currents. Data are represented as L-type Ca2+ current density (pA/pF). n = 4 cells 743
per group. Statistical analysis at -20 mV: two-tailed paired Student’s t-test (t = 1.379, df = 3, p = 744
0.2617). Data are expressed as means SEM. 745
746
Figure 6: Nimodipine can restore the neurophysiological responses to ghrelin in arcuate NPY 747
neurons treated with oligomeric Aβ1-42. A, B, Brain slices containing hypothalamic arcuate 748
nucleus from young (3-4 month old) NPY-GFP mice were used for whole-cell patch-clamp 749
recordings. Application of ghrelin (100 nM) depolarized the membrane potential and increased 750
spike frequency of arcuate NPY neurons. Exogenous oliogmeric Aβ1-42 (100 nM) depolarized the 751
membrane potential of arcuate NPY neurons and inhibited its response to ghrelin (100 nM). 752
Nimodipine (NMD, 2 μM) restored the ghrelin-mediated depolarization of arcuate NPY neurons 753
treated with exogenous oligomeric Aβ1-42. Statistical analysis: Two-tailed paired Student test for 754
comparison of vehicle to ghrelin-treated arcuate NPY neurons (membrane potential: t = 6.444, 755
29
df = 15, p < 0.001, n = 16 cells from 13 mice per group; spike frequency: t = 3.742, df = 13, p = 756
0.0025, n = 14 cells from 12 mice per group). Repeated measures one-way ANOVA comparing 757
vehicle, Aβ1-42, and ghrelin treatments (membrane potential: F(1.579, 12.63) = 7.577, p = 0.0096, n = 758
9 cells from 5 mice per group; spike frequency: F(1.365, 9.557) = 3.941, p = 0.0679, n = 8 cells from 759
5 mice per group), followed by post-hoc Tukey’s multiple comparisons. Repeated measures 760
one-way ANOVA comparing vehicle, Aβ1-42, nimodipine, and ghrelin treatments (membrane 761
potential: F(1.883, 11.30) = 8.785, p = 0.0014, n = 7 cells from 4 mice per group; spike frequency: 762
F(2.125, 12.75) = 3.220, p = 0.0716, n = 7 cells from 4 mice per group) followed by post-hoc Tukey’s 763
multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are expressed as means 764
SEM. C, Nimodipine restored the ghrelin-mediated increase in cytosolic free Ca2 levels in 765
arcuate NPY neurons treated with exogenous oligomeric Aβ1-42. Cytoplasmic free Ca2+ was 766
measured using Fura-2 AM in arcuate NPY neurons isolated by enzymatic digestion from young 767
(3-4 month old) WT NPY-GFP mice. After obtaining baseline levels, cells were perfused 768
sequentially with oligomeric Aβ1-42 (100 nM), nimodipine (2 M), and then ghrelin (100 nM). 769
Statistical analysis: repeated measures one-way ANOVA comparing vehicle, Aβ1-42, nimodipine, 770
and ghrelin treatments (F(2.128, 44.69) = 13.90, p < 0.0001, n = 22 cells from 4 mice) followed by 771
post-hoc Tukey’s multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001. Data are 772
expressed as means SEM. 773
774
Figure 7: Pre-treatment with nimodipine can restore ghrelin-mediated feeding behavior in young 775
(3-5 months) but not older (10 months) Tg2576 mice. A, Ghrelin (0.5 mg per kg body weight, 776
i.p.) increased feeding in young (3-5 months) WT but not Tg2576 mice. n = 9 WT and 14 777
Tg2576 mice. Statistical analysis: two-tailed paired Student’s t-test (WT: t = 2.337, df = 8, p = 778
0.0476; Tg2576: t = 1.034, df = 13, p = 0.3201). B, Nimodipine (10 mg per kg body weight, i.p.) 779
administered one hour before ghrelin (0.5 mg per kg body weight, i.p.) restored the ghrelin-780
30
mediated feeding behavior in young (3-5 months) Tg2576 mice. n = 12 WT and 12 Tg2576 781
mice. Statistical analysis: two-tailed paired Student’s t-test (WT: t = 3.986, df = 11, p = 0.0021; 782
Tg2576: t = 4.284, df = 11, p = 0.0013). C, Nimodipine (10 mg per kg body weight, i.p.) 783
administered one hour before ghrelin (0.5 mg per kg body weight, i.p.) failed to restore the 784
ghrelin-mediated feeding behavior in older (10 months) Tg2576 mice. n = 12 WT and 14 785
Tg2576 mice. Statistical analysis: two-tailed paired Student’s t-test (WT: t = 4.128, df = 11, p = 786
0.0017; Tg2576: t = 1.754, df = 13, p = 0.103). Data are expressed as individual mice. * p < 787
0.05, ** p < 0.01, *** p < 0.001; paired t-test. 788
789
Figure 8: There are no significant differences in the L-type Ca2+ currents in hypothalamic 790
paraventricular neurons from Tg2576 or WT mice. I-V curves for L-type Ca2+ currents in 791
randomly selected hypothalamic paraventricular NPY neurons from WT or Tg2576 slices. There 792
are no significant differences between hypothalamic paraventricular NPY neurons from WT and 793
Tg2576 slices. The peak L-type Ca2+ current occurred at -10 mV for hypothalamic 794
paraventricular neurons in both WT and Tg2576 brain slices with no significant differences in 795
the amplitudes. Statistical analysis: two-tailed unpaired Student’s t-test (t = 0.2289, df = 6, p = 796
0.8266). Data are represented as L-type Ca2+ current density (pA/pF). n = 4 cells per group from 797
≥ 3 mice per group. Data are expressed as means SEM. 798
799
800
Vehicle Nimodipine
0 mV
-40 mV
10 sec
Tg2576
Vehicle NMD
-60
-40
-20
0
Mem
bran
e Po
tent
ial (
mV)
** Vehicle NMD0
2
4
6
Firin
g ra
te (H
z)
**
Vehicle -GVIA AgaIVA0
2
4
6
Firin
g ra
te (H
z)
Vehicle GVIA AgaIVA
-60
-40
-20
0
Mem
bran
e Po
tent
ial (
mV)
A.
B. C. D. E.
Vehicle 100 300 NMD0.0
0.1
0.2
0.3
340/
380
nm
A 1-42 (nM)
**
Vehicle NMD Vehicle NMD0.0
0.2
0.4
0.6
0.8
1.0
340/
380
nm
**
WT Tg2576
**A. B.
-60 -50 -40 -30 -20 -10 0 10 20 30
-100.0
-50.0
0.0
50.0
L-ty
pe I C
a den
sity
(pA/
pF)
WTTg2576
Stepped Potentials (mV)-60 -50 -40 -30 -20 -10 0 10 20 30
-200.0
-150.0
-100.0
-50.0
0.0
50.0
L-ty
pe I C
a den
sity
(pA/
pF)
VehicleA 42
Stepped Potentials (mV)
Vehi
cle
NM
D
Vehi
cle
NM
D
Vehi
cle
NM
D
0
50
100
150
% C
hang
e in
L-ty
pe I C
a de
nsity
WT Tg2576 A 42
*** ** *
Stepped potentials
-30 mV
-20 mV
-10 mV
0 mV
10 mV
WT Tg2576 A.
B. C.
D.
-60 -50 -40 -30 -20 -10 0 10 20
-60-50-40-30-20-10
010
L-ty
pe I C
a den
sity
(pA/
pF)
Stepped Potentials (mV)
VehicleKN93
-60 -50 -40 -30 -20 -10 0 10 20 30
-120
-100
-80
-60
-40
-20
0
L-ty
pe I C
a den
sity
(pA/
pF)
Vehicle2APB
Stepped Potentials (mV)-60 -50 -40 -30 -20 -10 0 10 20 30
-150
-100
-50
0
50
L-ty
pe I C
a den
sity
(pA/
pF)
VehicleU733122
Stepped Potentials (mV)A. B. C.
A. B.
C. D.
A. B.
C. D.
0.0
0.2
0.4
0.6
0.8
1.0
340/
380
nm
Vehicle Ghrelin Vehicle Ghrelin WT Tg2576
*
-60-50-40-30-20-10 0 10 20 30 40
-100
-50
0
50
L-ty
pe I C
a den
sity
(pA/
pF)
Stepped Potentials (mV)
LeptinVehicle
-60-50-40-30-20-10 0 10 20 30 40
-100
-50
0
50
L-ty
pe I C
a den
sity
(pA/
pF)
Stepped Potentials (mV)
LeptinVehicle
Vehicle Leptin Vehicle Leptin0.0
0.2
0.4
0.6
340/
380
nm**
WT Tg2576
-100
-80
-60
-40
-20
0
Mem
bran
e po
tent
ial (
mV)
*** *** ***
Vehicle + + + + + + + + + A 42 - - - + + - + + +
NMD - - - - - - - + + Ghrelin - + - - + - - - +
0
2
4
6
8
10
Firin
g ra
te (H
z)
**
Vehicle + + + + + + + + + A 42 - - - + + - + + +
NMD - - - - - - - + + Ghrelin - + - - + - - - +
0.0
0.1
0.2
0.3
0.4
0.5
340/
380
nm
****
**
**
*
Vehicle + + + + A 42 - + + +
NMD - - + + Ghrelin - - - +
A.
B.
C.
PBS Ghrelin PBS Ghrelin0
1
2
3
Food Inta
ke (
g)
/ B
odyw
eig
ht (1
00 g
)
*
WT Tg2576
3-5 Month Old
n.s.
PBS Ghrelin PBS Ghrelin0
1
2
3
4
Food Inta
ke (
g)
/ B
odyw
eig
ht (1
00 g
)
** **
WT Tg2576
3-5 Month Old - Nimodipine
PBS Ghrelin PBS Ghrelin0
1
2
3
Food Inta
ke (
g)
/ B
odyw
eig
ht (1
00 g
)
**
WT Tg2576
10 Month Old - Nimodipine
n.s.
A. B. C.
-60 -50 -40 -30 -20 -10 0 10 20 30
-200.0
-150.0
-100.0
-50.0
0.0
50.0
L-ty
pe I C
a den
sity
(pA/
pF)
WT Tg2576
Stepped Potentials (mV)A.
