Neuroscience Assesment Paper

5986 • The Journal of Neuroscience, July 24, 2019 • 39(30):5986 – 6000
Neurobiology of Disease
Neuromodulatory Action of Picomolar Extracellular A␤42

Oligomers on Presynaptic and Postsynaptic Mechanisms
Underlying Synaptic Function and Memory
X Walter Gulisano,1 Marcello Melone,2,3 Cristian Ripoli,4,5 X Maria Rosaria Tropea,1 Domenica D. Li Puma,4,5
Salvatore Giunta,1 Sara Cocco,4 Daniele Marcotulli,2 Nicola Origlia,6 Agostino Palmeri,1 XOttavio Arancio,7
Fiorenzo Conti,2,3,8 X Claudio Grassi,4,5 and X Daniela Puzzo1,9
1Department Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, Italy, 2Section of Neuroscience and Cell Biology, Department
Experimental and Clinical Medicine, Università Politecnica delle Marche, Ancona 60020, Italy, 3Center for Neurobiology of Aging, IRCCS Istituto Nazionale
Ricovero e Cura Anziani (INRCA), Ancona 60020, Italy, 4Institute of Human Physiology, Università Cattolica del Sacro Cuore, Rome 00168, Italy,
5Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome 00168, Italy, 6Neuroscience Institute, Italian National Research Council, Pisa 56100, Italy,
7Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, New York 10032, 8Foundation for Molecular
Medicine, Università Politecnica delle Marche, Ancona 60020, Italy, and 9Oasi Research Institute-IRCCS, Troina, 94018, Italy
Failure of anti-amyloid-␤ peptide (A␤) therapies against Alzheimer’s disease (AD), a neurodegenerative disorder characterized by high
amounts of the peptide in the brain, raised the question of the physiological role of A␤ released at low concentrations in the healthy brain.
To address this question, we studied the presynaptic and postsynaptic mechanisms underlying the neuromodulatory action of picomolar
amounts of oligomeric A␤42 (oA␤42 ) on synaptic glutamatergic function in male and female mice. We found that 200 pM oA␤42 induces
an increase of frequency of miniature EPSCs and a decrease of paired pulse facilitation, associated with an increase in docked vesicle
number, indicating that it augments neurotransmitter release at presynaptic level. oA␤42 also produced postsynaptic changes as shown
by an increased length of postsynaptic density, accompanied by an increased expression of plasticity-related proteins such as cAMP-
responsive element binding protein phosphorylated at Ser133, calcium-calmodulin-dependent kinase II phosphorylated at Thr286, and
brain-derived neurotrophic factor, suggesting a role for A␤ in synaptic tagging. These changes resulted in the conversion of early into late
long-term potentiation through the nitric oxide/cGMP/protein kinase G intracellular cascade consistent with a cGMP-dependent switch
from short- to long-term memory observed in vivo after intrahippocampal administration of picomolar amounts of oA␤42. These effects
were present upon extracellular but not intracellular application of the peptide and involved ␣7 nicotinic acetylcholine receptors. These
observations clarified the physiological role of oA␤42 in synaptic function and memory formation providing solid fundamentals for
investigating the pathological effects of high A␤ levels in the AD brains.
Key words: amyloid precursor protein; amyloid-beta oligomers; neurotransmitter release; nicotinic receptors; synaptic plasticity; syn-
aptic transmission
Significance Statement
High levels of oligomeric amyloid-␤42 (oA␤42 ) induce synaptic dysfunction leading to memory impairment in Alzheimer’s disease
(AD). However, at picomolar concentrations, the peptide is needed to ensure long-term potentiation (LTP) and memory. Here, we
show that extracellular 200 pM oA␤42 concentrations increase neurotransmitter release, number of docked vesicles, postsynaptic
density length, and expression of plasticity-related proteins leading to the conversion of early LTP into late LTP and of short-term
memory into long-term memory. These effects require ␣7 nicotinic acetylcholine receptors and are mediated through the nitric
oxide/cGMP/protein kinase G pathway. The knowledge of A␤ function in the healthy brain might be useful to understand the
causes leading to its increase and detrimental effect in AD.
Gulisano et al. • Physiological Role of A␤ Oligomers at the Synapse J. Neurosci., July 24, 2019 • 39(30):5986 – 6000 • 5987
Introduction Materials and Methods

Abnormal elevation of amyloid-␤ peptide (A␤) in the brain is Ethical approval
considered one of the main pathogenetic events in Alzheimer’s All experiments involving animals were approved by the University of
disease (AD) (Hardy and Selkoe, 2002). Most of the studies in the Catania (#327/2013-B, #119-2017-PR) and the Università Cattolica del
Sacro Cuore in Rome (#626-2016-PR) in accordance with the respective
field have focused on A␤ production, aggregation, and degrada- regulations of local Institutional Animal care and Use Committee and
tion, resulting in therapeutic strategies aimed at decreasing A␤ with European Union Directive 2010/63/EU. The experiments complied
levels in the brain. However, lack of correlation between insolu- with the ARRIVE guidelines and were conducted to minimize the ani-
ble A␤ deposits and cognitive impairment, presence of A␤ depos- mals’ pain and suffering.
its in plaques or soluble oligomers in nondemented individuals
(Aizenstein et al., 2008; Maarouf et al., 2011; Lesné et al., 2013), Animals
and failure of A␤-reducing therapies (Gulisano et al., 2018a) have WT (C57BL/6J; RRID:IMSR_JAX:000664), App-KO (B6.129S7-App tm1Dbo/J;
RRID:IMSR_JAX:004133) and ␣7-KO (B6.129S7-Chrna7 tm1Bay/J; RRID:
prompted the neuroscience community to reconsider the patho- IMSR_JAX:003232) mice were purchased from The Jackson Laboratory and
genetic role of the peptide (Herrup, 2015; Puzzo et al., 2015; bred in the animal facilities at University of Catania and Università Cattolica
Gulisano et al., 2018a). To this end, the concept that A␤ is not del Sacro Cuore. Mice were maintained in stable hygrometric and thermic
solely a toxic product derived from amyloid precursor protein conditions (50%; 21°C ⫾ 1°C) on 12 h light/dark cycle with ad libitum access
(APP) processing, already proposed in the early 1990s (Koo et al., to food and water. For electrophysiological recordings, male animals were
1993), has been strengthened by studies investigating the physi- used at 3– 4 months of age (field recordings) or 21 d (whole-cell recordings).
ological role of the peptide in the CNS. Nonetheless, information Dual patch-clamp recordings experiments were performed on organotypic
slices from P4 –P7 Wistar rats. For behavioral experiments, sex-balanced
regarding A␤-dependent regulation of synaptic activity in the groups of mice were used at 3– 4 months of age.
healthy brain is still fragmentary, precluding a clear insight into
the mechanisms underlying the switch from function to dysfunc- A␤ preparation, concentration, and characterization
tion (Koppensteiner et al., 2016). Oligomeric A␤ was prepared as described previously (Stine et al., 2003;
In addition to its neuroprotective activity (Pearson and Peers, Puzzo et al., 2008; Ripoli et al., 2013). Briefly, the lyophilized peptide
2006), A␤ is likely to act as a neuromodulator at the synapse, (American Peptide) was suspended in 1,1,1,3,3,3-hexafluoro-2-
where it is released during neuronal activity (Kamenetz et al., propanol (HFIP; Sigma-Aldrich) to 1 mM. After the complete evapora-
tion of HFIP to allow complete monomerization, the A␤ film was
2003; Brody et al., 2008) and is dynamically regulated by presyn- dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich), sonicated for
aptic mechanisms involving vesicle cycling (Cirrito et al., 2005, 15 min, aliquoted, and stored at ⫺20°C. DMSO-A␤ solution was incu-
2008). Our previous studies have shown that A␤ production in- bated in PBS at 4°C for 12 h to allow oligomerization. The oligomerized
creases during the induction of long-term potentiation (LTP) A␤ solution was then diluted in ACSF or saline solution (0.9% NaCl) to
(Palmeri et al., 2017) and contextual fear learning (Puzzo et al., the final concentration, calculated based on the MW of the monomeric
2011) and that blocking its function in the healthy brain impairs peptide.
LTP and memory (Garcia-Osta and Alberini, 2009; Morley et al., Western blot of 200 pM and 200 nM A␤ solutions was routinely per-
formed to assess oligomers presence (Gulisano et al., 2018b). A␤ solu-
2010; Puzzo et al., 2011). Moreover, administration of picomolar
tions were incubated for 20 min at 29°C to reproduce the experimental
concentrations of the peptide, mimicking its physiological con- conditions of electrophysiological experiments. After this step, NuPAGE
tent in the brain (Schmidt et al., 2005; Puzzo et al., 2008), en- LDS sample buffer 4⫻ was added and A␤ preparations (at the final
hances LTP and memory (Puzzo et al., 2008; Morley et al., 2010), concentration of 200 nM and 200 pM) were separated on 10 –20% Novex
suggesting that A␤ acts in a hormetic fashion exerting a biphasic Tricine precast gels (Invitrogen). Proteins were then transferred onto 0.2
effect depending upon its concentration (Puzzo et al., 2012). ␮m nitrocellulose membranes (GE Healthcare) that were incubated
Recently, we have demonstrated that both the positive and overnight at 4°C with the mouse monoclonal antibody 6E10 (1:1000;
Covance). The next day, membranes were revealed with HRP-
negative effects exerted by A␤42 at the synapse need the presence
conjugated secondary antibodies (Cell Signaling Technology) using the
of oligomers (Gulisano et al., 2018b), suggesting that oligomeric SuperSignal West Femto Maximum Sensitivity Substrate (Thermo
A␤42 (oA␤42) plays a key role in physiological synaptic func- Fisher Scientific) and documented using an automated imaging system
tion. These findings prompted us to investigate in depth how (UVItec, Cambridge Alliance). Low-range rainbow molecular weight
picomolar concentrations of oA␤42 affect the presynaptic and markers (GE Healthcare Life Sciences) were used to assess the protein
postsynaptic mechanisms underlying synaptic transmission size.
and plasticity, as well as memory, highlighting similarities and Dose–response (DR) curves were performed to choose the dose of A␤
mimicking the physiological effects of oligomers on synaptic plasticity.
differences between the physiological and pathological roles of
Based on our previous work (Puzzo et al., 2012), hippocampal slices were
the peptide. treated with A␤ doses of 2, 20, and 200 pM and 2, 20, and 200 nM for 20
min before to induce LTP. Based on these results, we used 200 pM A␤ for
in vitro experiments, corresponding to 0.903 pg for in vivo injections into
each hippocampus. DR curves were also performed with the same exper-
Received Jan. 19, 2019; revised April 9, 2019; accepted April 28, 2019. imental design to assess the dose of A␤ capable of modifying paired-pulse
Author contributions: W.G., M.M., C.R., O.A., F.C., C.G., and D.P. designed research; W.G., M.M., C.R., M.R.T., facilitation (PPF).
D.D.L.P., S.G., S.C., N.O., and D.P. performed research; W.G., M.M., C.R., M.R.T., D.D.L.P., S.G., S.C., D.M., and D.P.
analyzed data; W.G., M.M., C.R., M.R.T., D.D.L.P., S.C., D.M., N.O., A.P., O.A., F.C., C.G., and D.P. edited the paper; O.A., Drugs
F.C., C.G., and D.P. wrote the paper; D.P. wrote the first draft of the paper. N␻-nitro-L-arginine methyl ester hydrochloride (L-NAME, Sigma-
This work was supported by Alzheimer’s Association (IIRG-09-134220 to D.P. and NIRG-14-321307 to C.R.),
Aldrich, 100 ␮M) (Johnstone and Raymond, 2011), 8- Bromoguanosine-
University of Catania (Bando Chance to A.P. and D.P.), Italian Ministry of University and Research (PRIN 2010-JFYFY2
to F.C.), INRCA IRCCS (intramural funds to F.C.), NIH (NS-049442 and AG034248 to O.A.).
3⬘, 5⬘- cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS,
The authors declare no competing financial interests. Biolog, 10 ␮M for electrophysiology and 1 ␮g/1 ␮l for behavioral studies)
Correspondence should be addressed to Daniela Puzzo at [email protected]. (Bollen et al., 2014), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
https://doi.org/10.1523/JNEUROSCI.0163-19.2019 (ODQ, Sigma-Aldrich, 100 ␮M) (Puzzo et al., 2005), anisomycin (ANI,
Copyright © 2019 the authors Sigma-Aldrich, 20 ␮M) (Johnstone and Raymond, 2011) (2R)-amino-5-
5988 • J. Neurosci., July 24, 2019 • 39(30):5986 – 6000 Gulisano et al. • Physiological Role of A␤ Oligomers at the Synapse
phosphonovaleric acid (APV, Tocris Bioscience, 50 ␮M) (Puzzo et al., for 1 h before equilibration at room temperature. During recordings,
2008), and methyllycaconitine (MLA, Sigma-Aldrich, 10 ␮M based on slices were placed in a recording chamber perfused with heated ACSF
DR curves obtained in our laboratory) were dissolved in DMSO, ali- (32°C) and bubbled with 95% O2/5% CO2. All recordings were made
quoted, and stored at ⫺20°C. All drugs were diluted in ACSF or saline with the GABAA receptor antagonist picrotoxin (50 ␮M) added to the
solution (0.9% NaCl) to the desired final concentration right before ACSF. Whole-cell recording pipettes (3–5 M⍀) were filled with the same
electrophysiological or behavioral experiments. 6E10 (Covance, catalog internal solution used for dual-patch recordings in organotypic hip-
#SIG-39320, RRID:AB_662798, 1:300) and M3.2 (Covance, catalog pocampal slices. Whole-cell recordings were performed with a Multi-
#SIG-39155, RRID:AB_2028758, 2 ␮g/ml) antibodies used for electro- clamp 700B amplifier (Molecular Devices). A Digidata 1440A series
physiological experiments were directly diluted in ACSF according to interface and pClamp 10 software were used for data acquisition and
previous studies (Ripoli et al., 2014; Palmeri et al., 2017). stimulation protocols. Data were filtered at 1 kHz, digitized at 10 kHz,
and analyzed online and offline.
Dual whole-cell recordings Experimental design. To study LTP in CA1 pyramidal cells, the stimu-
Hippocampal organotypic slice cultures were prepared from P4 –P7 rats lation intensity that elicited one-third of the maximal response ampli-
through a McIllwain tissue chopper and placed on semiporous mem- tude of AMPAR EPSC was used for delivering test pulses every 20 s. CA1
branes (Millipore) for 5–7 d before recordings, as described previously pyramidal cells were held at ⫺60 mV. LTP was induced by two trains of
(Spinelli et al., 2017). Hippocampal subfields and electrode positions HFS (100 Hz, 1 s) separated by 20 s, with the patched cells depolarized to
were identified with the aid of 4⫻ and 40⫻ water-immersion objectives 0 mV. This induction protocol was always applied within 5–7 min of
on an upright microscope equipped with differential interference con- achieving whole-cell configuration, to avoid “washout” of LTP. Re-
trast optics under infrared illumination (BX51WI; Olympus) and video sponses to test pulse were recorded for 30 min to assess LTP. The mag-
observation (BTE-B050-U CMOS camera; Mightex). Neighboring pairs nitude of LTP was calculated on basis of the averaged EPSC values during
of pyramidal cells were recorded simultaneously in CA1 by single stim- the last 5 min of post-HFS recordings (from minute 25 to minute 30).
ulating bipolar tungsten electrode (FHC) placed on the Schaffer collat- LTP magnitude was expressed as the percentage change in the mean
eral fibers, as described previously (Barone et al., 2019). Slices were EPSC peak amplitude normalized to baseline values, taken as 100% (i.e.,
incubated in artificial CSF (ACSF) containing the following (in mM): 119 mean values for the 5 min of recording before HFS).
NaCl, 2.5 KCl, 4 CaCl2, 4 MgCl2, 1 NaH2PO4, 26 NaHCO3, 11 D-glucose,
and 0.005 2-chloroadenosine, gassed with 95% O2/5% CO2. Whole-cell Electrophysiological field recordings
recording pipettes (3– 4 M⍀) were filled with a solution containing the Extracellular electrophysiological field recordings were performed on
following (in mM): 135 CsMeSO3, 8 NaCl, 10 HEPES, 0.25 EGTA, 2 400 ␮m transverse hippocampal slices as described previously (Puzzo et
Mg2ATP, 0.3 Na3GTP, 0.1 spermine, 7 phosphocreatine, and 5 QX-314, al., 2008). After a cutting procedure using a manual tissue chopper, slices
pH 7.25–7.30 (osmolarity 300 mOsm). Data were collected with a Mul- were transferred to a recording chamber and perfused (1–2 ml/min) with
tiClamp 700B amplifier (Molecular Devices), digitized at 10 kHz using ACSF containing the following (in mM): 124 NaCl, 4.4 KCl, 1 Na2HPO4,
the Digidata 1440A data acquisition system (Molecular Devices), and 25 NaHCO3, 2 CaCl2, 2 MgCl2, 10 glucose kept at 29°C and continuously
analyzed offline using pClamp 10 software (RRID:SCR_011323; Molec- bubbled with an O2/CO2 mixture at 95% and 5%. After 120 min recov-
ular Devices). ery, field EPSPs (fEPSPs) were recorded in CA1 stratum radiatum by a
Experimental design. The AMPA/NMDA ratio was calculated as the glass electrode filled with ACSF in response to Schaffer collateral stimu-
peak averaged AMPAR-mediated EPSC at holding potential of ⫺70 mV lation by a bipolar tungsten electrode.
divided by the averaged NMDAR-mediated EPSC at holding potential of Experimental design. Recordings were performed and analyzed in
⫹40 mV at a latency at which AMPAR-mediated EPSC responses were pClamp 10. We first measured basal synaptic transmission (BST) by
fully decayed (50 ms after stimulation). Miniature EPSC (mEPSC) am- stimulating with a series of increasing voltage pulses (from 5 to 35 V).
plitude and frequency were evaluated in 60 s recordings in the presence of This allowed us to preliminarily select healthy slices to be used for elec-
tetrodotoxin (0.5 ␮M) at ⫺70 mV. trophysiological recordings. For LTP, baseline was recorded every min-
We used two different experimental settings to evaluate whether ute by stimulating at a voltage able to evoke a response of 35% of the
oA␤42 exerted an extracellular and/or an intracellular effect. To study the maximum evoked response in BST. After 30 – 45 min, slices with a stable
extracellular effect, slices were perfused with extracellular 200 pM oA␤42 baseline (slope variation ⫾ 5%) were used. We recorded for 15 min, and
with one patch pipette filled with vehicle and the other with the 6E10 then perfused with vehicle or drugs for the appropriate time. LTP was
antibody that blocks the possible effect due to intracellular oA␤42. To induced by a theta-burst stimulation (TBS): trains of 10 ⫻ 100 Hz bursts
study the intracellular effect, slices were perfused with extracellular vehi- with five pulses per burst with a 200 ms interburst interval at the test pulse
cle with one patch pipette filled with vehicle and the other with 200 pM intensity. To elicit early LTP (E-LTP), we delivered a single TBS train
oA␤42. This design also allowed us to concurrently compare the effects of (weak tetanic stimulation), whereas for late LTP (L-LTP, we delivered
vehicle and 200 pM oA␤42 injected into adjacent neurons through patch three TBS trains with a 15 s intertrain interval (strong tetanic stimula-
pipettes. tion). Analysis of the fEPSP slope was performed offline and results were
To confirm the ability of 6E10 to block oA␤42 action, slices were per- expressed by normalizing on the first 15 min of baseline recordings. In
fused with extracellular 200 pM oA␤42 paired with 6E10, 6E10 alone, or another series of experiments, we evaluated PPF. After BST assessment,
200 pM oA␤42 alone; the patch pipette was filled with vehicle. slices were perfused with the NMDA receptor antagonist (2R)-amino-5-
phosphonovaleric acid (APV; 50 ␮M) for 45 min, and then treated with
Whole-cell LTP recordings in hippocampal brain slices vehicle or drugs in APV for the appropriate time. Two paired pulses
Experiments examining LTP were performed from single CA1 pyramidal within a time interval of 10, 20, 30, 40, 50, 100, 200, 500, and 1000 ms
cells after stimulating the Schaffer collateral fibers by means of a bipolar were delivered. PPF was measured as percentage of the synaptic response
tungsten electrode (FHC) in acute hippocampal brain slices (300 ␮m of the second against the first delivered stimulus.
thick) obtained by 21-d-old male C57BL/6 mice, as described previously
(Ripoli et al., 2013, 2014). Animals were anesthetized with isoflurane, Intrahippocampal injections of oA␤42
decapitated, and the brains were rapidly placed in ice-cold cutting solu- Mice underwent stereotaxic surgery for cannulas implantation. After
tion containing the following (in mM): 124 NaCl, 3.2 KCl, 1 NaH2PO4, 2 anesthesia with tiletamine ⫹ zolazepam (60 mg/kg) and medetomidine
MgCl2, 1 CaCl2, 26 NaHCO3, 10 glucose, 2 Na-pyruvate, and 0.6 ascorbic (40 ␮g/kg), mice were implanted with a 26-gauge guide cannula into the
acid, pH 7.4, 95% O2/5% CO2. Slices were sectioned on a vibratome dorsal part of the hippocampi (coordinates from bregma: posterior ⫽
(VT1200S; Leica Microsystems) and rapidly transferred to an incubation 2.46 mm, lateral ⫽ 1.50 mm to a depth of 1.30 mm). The cannulas were
chamber filled with ACSF containing the following (in mM): 124 NaCl, fixed to the skull with acrylic dental cement (RelyX TM Unicem, 3M) and
3.2 KCl, 1 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 glucose, pH mice were allowed to recover for a minimum of 6 – 8 d. Twenty minutes
7.4, gassed with 95% O2/5% CO2. Slices were allowed to recover at 32°C before the training phase (T1), mice were bilaterally infused with oA␤42
solution or vehicle or oA␤42 ⫹ Rp-8-Br-cGMPS in a final volume of 1 ␮l vesicle pool, number of docked vesicles, area of spines, length of the
over 1 min with a microsyringe connected to the cannulas via polyethyl- postsynaptic density (PSD), and of the proportion of perforated synapses
ene tubing. During infusion, animals were handled gently to minimize (Shepherd and Harris, 1998; Pozzo-Miller et al., 1999; Geinisman, 2000;
stress. After infusion, the needle was left in place for another minute to Bourne et al., 2013; Babits et al., 2016) was performed. Randomly se-
allow diffusion. In some animals, after behavioral studies, a solution of lected electron microscopical fields of the stratum radiatum (vehicle, n ⫽
4% methylene blue was infused for localization of infusion cannulas. 87; vehicle ⫹ 1 TBS, n ⫽ 116; oA␤42 200 pM, n ⫽ 92; oA␤42 200 pM ⫹ 1
TBS, n ⫽ 113) with at least one identifiable axospinous synapse (Shep-
Novel object recognition (NOR) test herd and Harris, 1998) were acquired at original magnification of
The NOR test was performed as described previously (Bollen et al., 2014; 36,000⫻.
Palmeri et al., 2017) in sex-balanced WT mice. Mice underwent 3 d of Axospinous synapses were identified by the presence of a presynaptic
habituation to the arena, objects, and intrahippocampal injections; 1 d of terminal with vesicles, including those nearby the presynaptic density
training (T1); and 1 d of testing (T2). The arena was a plastic white box (i.e., the active zone), a synaptic cleft displaying electrodense material,
(50 ⫻ 35 ⫻ 45 cm), and objects (e.g., pyramid, cube, truncated sphere, and a postsynaptic membrane associated with a prominent PSD within
etc.) were designed by a computer-aided design software (Solidworks) the postsynaptic spines (Peters et al., 1991; DeFelipe et al., 1999). The
and printed in polylactic acid with a Prusa-inspired 3D printer of our distinction between vesicle pools (which comprise the reserve vesicle
design. After each trial, the box and the objects were cleaned with 70% pool) and docked vesicles (which are thought to be part of the readily
ethanol and dried with absorbent paper. releasable pool), was made according to Shepherd and Harris 1998;
Experimental design. During the first day of habituation the mouse was Pozzo-Miller et al., 1999; Bourne et al., 2013. Briefly, vesicle pools were
put into the empty arena and allowed to explore it for 5 min. During the determined by counting the total number of small vesicles (⬃50 nm) per
second and the third day (familiarization with objects), the mouse was terminal; the docked vesicles pool by counting the vesicles touching the
put into the arena containing two different objects, randomly chosen membrane of the presynaptic active zone. Spines profile area and PSD
among our object collection and changed from day to day, for 5 min. length were measured by ImageJ software tools (Schneider et al., 2012;
During the fourth day, NOR training session (T1) was performed with Babits et al., 2016). Spine profiles and PSD were traced along the mem-
two different protocols: short T1 and long T1. The mouse was put into branes and measured; PSD length corresponded to the distance between
the arena and allowed to explore two identical objects placed in the the edges of PSD. Perforated synapses were identified based on the pres-
central part of the box, equally distant from the perimeter and the center, ence of a discontinuous PSD (Geinisman, 2000; Babits et al., 2016). Ul-
for 3 min (short T1) or 10 min (long T1). During the fifth day, the mouse trathin sections and microscopical features of axospinous synapses were
underwent the second trial (T2) to test memory retention for 10 min. The examined and analyzed in a blinded manner.
long delay interval of 24 h between T1 and T2 did not allow storage of
memory information (natural forgetting) in mice that were previously Western blot on hippocampal slices
exposed to a short T1 (no discrimination between the familiar and the Western blot (WB) analysis was performed as described previously
novel objects). Conversely, long-term memory (LTM) could be formed (Caraci et al., 2015). Tissues were homogenized in RIPA buffer (Thermo
in mice that were previously exposed to a long T1. In T2, mice were Fisher Scientific) in the presence of phosphatase and protease inhibitors
presented with two different objects, respectively a “familiar” (i.e., the (Thermo Fisher Scientific), and sonicated 3 times for 10 min. Protein
one used for T1) and a “novel” object. Animal exploration was defined as concentrations were determined by Pierce BCA protein assay kit
the mouse pointing its nose toward the object from a distance not ⬎2 cm (Thermo Fisher Scientific) and equal amounts of proteins (30 –50 ␮g)
and was measured in T2 to analyze: (1) the discrimination index, explo- were then loaded onto 10% or 12% Tris-glycine polyacrylamide gels for
ration of novel object minus exploration of familiar object/total explo- electrophoretic separation. Membranes were blocked for 1 h, at room
ration time, and (2) total exploration time, the time spent exploring the temperature, in a solution of 5% nonfat dry milk in Tris-buffered saline
objects was scored using a personal computer by an experimenter who containing 0.1% Tween 20 or SEAblock (Thermo Fisher Scientific) be-
was blinded to the conditions tested. We excluded from the analyses mice fore incubation overnight at 4°C with the following primary antibodies:
with a total exploration time ⬍5 s. mouse anti-neuronal nitric oxide synthase (anti-nNOS) (Thermo Fisher
Scientific, catalog #37-2800, RRID:AB_2533308; 1:1000); rabbit anti-p-
Electron microscopy of hippocampal slices CREB (ser133) (Millipore, catalog #06-519, RRID:AB_310153; 1:1000);
Hippocampal slices (n ⫽ 2 for each conditions from 8 animals) were mouse anti-CREB (Cell Signaling Technology, catalog #9104, RRID:
quickly immersed (within ⬃30 s after 120 min of electrophysiological AB_490881; 1:500); rabbit anti-p-CaMKII (thr286) (Cell Signaling
recording) in a solution containing 4% paraformaldehyde and 0.5% glu- Technology, catalog #12716, RRID:AB_2713889; 1:1000); mouse anti-
taraldehyde in phosphate buffer (PB) and then stored for 6 weeks in the CaMKII␣ (Cell Signaling Technology, catalog #50049, RRID:
same fixative solution at 4°C. Subsequently, slices were exposed to an AB_2721906; 1:1000); rabbit anti-BDNF (Millipore, catalog #AB1534,
embedding procedure as described previously (Melone et al., 2011, RRID:AB_90746; 1:500). Mouse anti tubulin (Sigma Aldrich, catalog
2015). Briefly, they were postfixed in 1% osmium tetroxide in PB for 45 #T5293, RRID:AB_477580; 1:1000) was used as loading control. Molec-
min and contrasted with 1% uranyl acetate in maleate buffer, pH 6.0, for ular weights for immunoblot analysis were determined using precision
1 h. Dehydrated sections were immersed in propylene oxide, infiltrated Plus Protein Dual color Standards (Bio-Rad), PAGE-MASTER Protein
with a mixture of Epon/Spurr resins (Electron Microscopy Sciences) Standard (Genscript). Protein detection was performed by using a sec-
sandwiched between Aclar films and polymerized at 60°C for 48 h. A ondary infrared fluorescent dye conjugated antibody absorbing at 800 or
small block of tissue containing CA1 stratum radiatum was selected by 680 nm. The secondary antibody goat anti-rabbit IRDye 680 (Li-Cor
light microscopic inspection, glued to blank epoxy, and sectioned with an Biosciences, catalog #926-68021, RRID:AB_10706309) and goat anti-
ultramicrotome (MTX; Research and Manufacturing Company). Ultra- mouse IRDye 800CW (Li-Cor Biosciences, catalog #926-32210, RRID:
thin sections (⬃60 nm; for a total of 20 –25 ultrathin sections for each AB_621842) were used at 1:20,000 and 1:30,000, respectively. Blots were
small selected block) were mounted on 200 mesh copper grids, stained visualized using an Odyssey Infrared Imaging Scanner (Li-Cor). In other
with Sato’s lead, and examined with Philips EM 208 and/or CM10 elec- experiments, after incubation with appropriate secondary horseradish
tron microscopes coupled to a MegaView-II high resolution CCD cam- peroxidase-conjugated antibodies (1:2500; Cell Signaling Technology),
era (Soft Imaging System). visualization was performed with ECL plus (GE Healthcare) using
Experimental design. Electron microscopy was performed on hip- UVItec (Cambrige Alliance). Densitometric analysis was performed with
pocampal slices treated with vehicle, vehicle ⫹ 1 TBS, oA␤42 200 pM, or either Odyssey Infrared Imaging Scanner or UVItec or ImageJ software
A␤ 200 pM ⫹ 1 TBS. These slices were randomly collected during elec- after normalization with loading controls.
trophysiological field recording experiments 120 min after tetanic stim- Experimental design. We collected slices from electrophysiological field
ulation and analysis of recordings was performed for each sample recordings experiments 120 min after tetanic stimulation. For each ex-
following the sample protocol described above. Quantitative analysis of perimental condition (vehicle, 1 TBS, A␤, A␤ ⫹ 1 TBS; or vehicle, 3 TBS
for nNOS), 4 slices from different animals were collected to obtain a confirmed that blocking oA␤42 with extracellular 6E10 prevented
sample; 2–3 samples for a total of 8 –12 slices were used to evaluate the oA␤42 to increase mEPSC frequency (Fig. 1H ), whereas 6E10 did
expression of each protein of interest. not affect mEPSC per se.
Statistical analyses Further experiments in which LTP was studied through
For each experiment, sample size relied on power analyses (␣ ⫽ 0.05, patch-clamp technique showed that 200 pM oA␤42 increased
power 1-␤ ⫽ 0.80) calculated by G-Power 3.1 software suggesting for EPSC amplitude recorded for 30 min after a high-frequency stim-
each condition a minimum of 6 slices (electrophysiology) and 8 mice ulation (2 trains at 100 Hz for 1 s separated by 20 s) only when
(behavioral studies) to obtain an effect size ⫽ 0.62. Experimenters were extracellularly applied, not when injected into CA1 pyramidal
blinded to treatment. All data are expressed as mean ⫾ SEM. neurons through the patch pipette (Fig. 1I ). These findings dem-
After data collection, statistical analysis was performed by SigmaPlot onstrate that picomolar concentrations of oA␤42 extracellularly
12.0 (RRID:SCR_003210), Systat 9 (RRID:SCR_010455) and GraphPad
modulate glutamatergic transmission and plasticity and it they
Prism 7 (RRID:SCR_002798) software. A preliminary analysis of normal
distribution was performed by Shapiro–Wilk normality test. We also
do act through a direct intracellular mechanism.
used the following tests: (1) ANOVA for repeated measures to analyze
PPF and LTP (120 min after tetanus) curves; (2) one-way ANOVA with Picomolar concentrations of oA␤42 affect PPF and convert
Bonferroni’s or Tukey’s post hoc correction for LTP graphs displaying
E-LTP into L-LTP and short-term memory (STM) into LTM
residual potentiation (average of the last 5 min of LTP recording) for
analyses of discrimination index and total exploration time among con- The increase of mEPSC frequency prompted us to investigate
ditions in NOR and for WB; (3) two-samples t test to compare conditions whether oA␤42 affects PPF, a presynaptic form of short-term
in dual-patch recording; and (4) one-sample t test to compare discrimi- plasticity linked with changes in release probability (Zucker and
nation index with zero. Given the non-normal distribution of electron Regehr, 2002). We found that 20 min perfusion with 200 pM
microscopical data, as assessed by D’Agostino and Pearson normality oA␤42 decreased PPF (Fig. 2A), thus suggesting an increase of the
test, comparison of the number of pool vesicles, docked vesicles, area of release probability (Dobrunz and Stevens, 1997). A DR curve
spines, and PSD length between all groups was made by nonparametric confirmed that 200 pM was the dose capable of affecting PPF (Fig.
Kruskal–Wallis test with Dunn’s multiple-comparisons test. The per- 2B). To further investigate the role of A␤ in PPF, we suppressed
centages of nonperforated and perforated synapses were compared using
endogenous A␤ through the anti-A␤ antibody M3.2, specifically
nonparametric contingency analysis with Fisher’s test. The level of sig-
nificance was set at p ⬍ 0.05.
targeting murine A␤. This induced the opposite effect, an en-
hancement of PPF, which was rescued by concomitant perfusion
Results with human 200 pM oA␤42 (Fig. 2C), not recognized by M3.2.
Picomolar concentrations of extracellular oA␤42 affect These findings are consistent with a positive modulatory role of
spontaneous neurotransmitter release and synaptic plasticity A␤ onto neurotransmitter release probability.
Intraneuronal uptake and accumulation of high doses of oA␤42 Next, we investigated whether 200 pM oA␤42 influenced the
are key events leading to impairment of synaptic transmission, E-LTP, a form of protein-synthesis-independent plasticity that
LTP, and memory (Ripoli et al., 2014; Puzzo et al., 2017). How- involves a change in presynaptic neurotransmitter release and
ever, it is not known whether the capability of oA␤42 to enhance short-term kinase activity (Huang, 1998). We found that pre-
synaptic function when at low concentrations is triggered by an treatment with 200 pM oA␤42 converted E-LTP obtained through
extracellular mechanism or if it requires protein internalization. a weak tetanic stimulation (1 TBS) into L-LTP (Fig. 2D). The
To address this issue, we first characterized our oA␤42 prepa- potentiation induced by pairing 200 pM oA␤42 with 1 TBS was
rations and confirmed that they contained both monomers and comparable to that induced by a strong tetanic stimulation con-
oligomers (Fig. 1A), as recently shown (Gulisano et al., 2018b). sisting of 3 TBSs elicited with a 15 s intertrain interval. To confirm
DR curves obtained by treating hippocampal slices with different that 200 pM oA␤42 converted E-LTP into the traditional protein-
doses of oA␤42 (from 2 pM to 200 nM) for 20 min before tetanus synthesis-dependent L-LTP (Johnstone and Raymond, 2011),
confirmed that 200 pM oA␤42 was the concentration inducing the slices were continuously perfused with the translation inhibitor
maximum enhancement of LTP (Fig. 1B), consistent with our ANI (20 ␮M; 30 min before and 25 after tetanus), which pre-
previous findings (Puzzo et al., 2008, 2012). vented the oA␤42-induced L-LTP (Fig. 2E). Additionally, we con-
Next, we studied the effect of extracellular and intracellular firmed that ANI perfusion inhibits 3-TBS-induced L-LTP
200 pM oA␤42 on glutamatergic basal synaptic transmission without affecting 1-TBS-induced E-LTP (Fig. 2E), as the latter is
through dual patch-clamp whole-cell recordings of adjacent CA1 known to not involve new protein synthesis (Johnstone and Ray-
pyramidal neurons of organotypic slice cultures in the following mond, 2011). These observations support a role for oA␤42 in
experimental conditions: (1) extracellular 200 pM oA␤42, with triggering new protein synthesis and gene transcription.
one patch pipette filled with vehicle and the other with an anti- Because LTP represents the cellular surrogate of memory, we
body raised against human A␤42 (6E10) and (2) extracellular assessed whether bilateral intrahippocampal injections with 200
vehicle with one patch pipette filled with vehicle and the other pM oA␤42 20 min before training were able to convert STM into
with 200 pM oA␤42 (Fig. 1C). We found no effect of oA␤42 onto LTM. To this end, we used an NOR protocol in which the short
AMPA and NMDA glutamatergic receptor evoked currents when time exposure (3 min) during the training phase (T1) does not
the peptide was administered either extracellularly or intracellu- allow animals to discriminate between the old and the new object
larly (Fig. 1 D, E). Conversely, extracellular administration of after a 24 h long-term interval due to natural forgetting (Bollen et al.,
oA␤42 increased the spontaneous release of neurotransmitter 2014; Palmeri et al., 2017). The analyses of the discrimination
from the presynaptic terminal measured through the mEPSC index indicated that mice receiving a weak stimulus (i.e., short
frequency without altering their amplitude 20 min after its appli- exposure in T1) paired with 200 pM oA␤42 were able to discrim-
cation (Fig. 1 F, G). Remarkably, intracellular application of 6E10 inate between the old and the familiar object after 24 h compared
did not block the effect of extracellular oA␤42, confirming that with vehicle-treated animals showing natural forgetting. This
the oA␤42-induced modification of glutamatergic transmission oA␤42-induced LTM was comparable to that induced by a stron-
does not require peptide internalization. Control experiments ger training stimulus (10 min of exposure in T1) (Fig. 2F ). Total
Figure 1. Extracellular, but not intracellular, picomolar concentrations of oA␤42 affect spontaneous release of neurotransmitter and synaptic plasticity. A, Characterization of synthetic human
oA␤42 solutions by WB analysis showing the presence of monomers, dimers, trimers, and tetramers for 200 nM oA␤42 solution and the presence of monomers and tetramers for 200 pM oA␤42. B,
DR curve for the effect of oA␤42 (from 2 pM to 200 nM, n ⫽ 5 for each concentration) on CA1-LTP indicates that the peptide has a maximum stimulatory effect at 200 pM (321.04 ⫾ 17.92% of baseline
vs 228.63 ⫾ 20.29% of baseline; F(6,28) ⫽ 9.882, p ⬍ 0.0001; Bonferroni’s p ⫽ 0.031) and an inhibitory effect at 200 nM (133.25 ⫾ 11.59% of baseline; Bonferroni’s p ⫽ 0.023). The dotted
horizontal line corresponds to treatment with vehicle. The residual potentiation was calculated by averaging the last 5 min of LTP. C, Schematic representation of dual whole-cell recordings from
adjacent (#1 and #2) CA1 hippocampal pyramidal neurons (see Materials and Methods for details). D, Representative AMPAR- and NMDAR-EPSCs at time 0 (T0) and 20 min after extracellular
application of vehicle or 200 pM oA␤42 (T20). The amplitude of NMDA currents recorded at ⫹40 mV (holding potential) was measured at 50 ms poststimulus (red crosses). E, Neither intraneuronal
(in) nor extracellular (ex) 200 pM oA␤42 affected AMPAR/NMDAR ratios. Slices perfused with exVehicle: inVehicle T0 ⫽ 0.74 ⫾ 0.16, T20 ⫽ 0.97 ⫾ 0.28, n ⫽ 10; in 200 pM oA␤42 T0 ⫽ 0.92 ⫾
0.14, T20 ⫽ 0.90 ⫾ 0.16, n ⫽ 10. Slices perfused with ex 200 pM oA␤42: inVehicle T0 ⫽ 0.80 ⫾ 0.19, T20 ⫽ 0.86 ⫾ 0.16, n ⫽ 15; in6E10 T0 ⫽ 0.92 ⫾ 0.20, T20 ⫽ 1.04 ⫾ 0.21, n ⫽ 15. F,
Representative mEPSC traces recorded in neurons treated with vehicle and 200 pM inA␤42 at T0 and T20. Intraneuronal 200 pM oA␤42 had no significant effect on mEPSC frequency (from 0.82 ⫾ 0.18
Hz at T0 to 0.63 ⫾ 0.10 Hz at T20, n ⫽ 14; p ⫽ 0.10) or amplitude (from 11.33 ⫾ 2.58 pA at T0 to 8.64 ⫾ 2.06 pA at T20, n ⫽ 14; p ⫽ 0.07) compared with slices in which cells were injected with
vehicle and extracellularly perfused with vehicle (mEPSC frequencies from 0.87 ⫾ 0.13 Hz at T0 to 0.78 ⫾ 0.10 Hz at T20, n ⫽ 14; p ⫽ 0.07; mEPSC amplitudes from 13.58 ⫾ 2.77 pA at T0 to
10.73 ⫾ 2.97 pA at T20, n ⫽ 14; p ⫽ 0.19). G, Extracellular application of 200 pM oA␤42 significantly increased mEPSC frequency both in vehicle-injected neurons (from 0.79 ⫾ 0.08 Hz at T0 to
1.10 ⫾ 0.10 Hz at T20, n ⫽ 14; p ⫽ 0.002) and adjacent 6E10-injected neurons (from 0.85 ⫾ 0.11 Hz at T0 to 1.18 ⫾ 0.15 Hz at T20, n ⫽ 14; p ⫽ 0.0008) without altering the mEPSC amplitude
(mean amplitude) of: (1) vehicle: T0 ⫽ 10.6 ⫾ 1.9 pA, T20 ⫽ 12.2 ⫾ 1.6 pA, n ⫽ 14; p ⫽ 0.20 or (2i) 6E10: T0 ⫽ 9.7 ⫾ 1.4 pA, T20 ⫽ 11.2 ⫾ 2.7 pA, n ⫽ 14; p ⫽ 0.51. H, Extracellular application
of 200 pM oA␤42 paired with 6E10 or 6E10 alone did not modify mEPSC frequency in vehicle-injected neurons (exA␤⫹6E10: from 0.96 ⫾ 0.08 Hz at T0 to 1.00 ⫾ 0.15 Hz at T20, n ⫽ 18; p ⫽ 0.746;
6E10: from 0.83 ⫾ 0.07 Hz at T0 to 0.77 ⫾ 0.10 Hz at T20, n ⫽ 10; p ⫽ 0.543). In interleaved experiments, extracellular 200 pM oA␤42 was still capable of incresing the mEPSC frequency (from
1.00 ⫾ 0.10 Hz at T0 to 1.31 ⫾ 0.15 Hz at T20, n ⫽ 8; p ⫽ 0.029). I, Extracellular oA␤42 (n ⫽ 11) enhanced LTP elicited through a high-frequency stimulation (two trains at 100 Hz for 1 s separated
by 20 s) compared with vehicle (n ⫽ 10) (Bonferroni’s p ⫽ 0.003). By contrast, intracellular oA␤42 did not modify potentiation (Bonferroni’s p ⬎ 0.05). ANOVA among all: F(2,28) ⫽ 10.846; p ⫽
0.001. **p ⬍ 0.005; ***p ⬍ 0.001. Data are expressed as mean ⫾ SEM.
exploration time was similar in all groups (Fig. 2F ). Thus, 200 pM cific ultrastructural changes (i.e., vesicle pool, number of docked
oA␤42 converts STM into LTM. vesicles, area of spines, PSD length, and percentage of perforated
synapses; Table 1) at axospinous synapses of the CA1 stratum
Picomolar concentrations of oA␤42 induce ultrastructural radiatum in slices used for electrophysiological experiments col-
changes of hippocampal CA1 synapses at both presynaptic lected and stored at 120 min after A␤ treatment. Quantitative
and postsynaptic levels electron microscopy showed an increase of docked vesicles in
Synaptic plasticity is accompanied by structural changes occur- axon terminals from slices treated with 200 pM oA␤42 and of PSD
ring at both the presynaptic and postsynaptic levels (Bourne et al., length in spines from slices treated with oA␤42 paired with a weak
2013). We therefore investigated whether oA␤42 determines spe- stimulation (Fig. 3). Together, these ultrastructural changes
Figure 2. Picomolar concentrations of oA␤42 decrease PPF and convert E-LTP into L-LTP, as well as STM into LTM. A, PPF was decreased in slices perfused with 200 pM oA␤42 for 20 min
compared with control slices (facilitation at 100 ms interval here and in following panels: vehicle ⫽ 179.58 ⫾ 7.47%, n ⫽ 9; 200 pM oA␤42 ⫽ 145.67 ⫾ 8.74%, n ⫽ 10; ANOVA for
repeated measures for the entire curve F(1,17) ⫽ 6.262, p ⫽ 0.023; Bonferroni’s p ⫽ 0.041 at 100 ms. B, DR curve for the effect of oA␤42 (from 2 pM to 200 nM, n ⫽ 7 for each
concentration) on PPF indicates that the peptide has a maximum stimulatory effect at 200 pM. Curve showing the percentage of facilitation at 100 ms stimulus interval (Bonferroni’s p ⫽
0.022 between vehicle and 200 pM oA␤42). The dotted horizontal line corresponds to treatment with vehicle. C, Increase of PPF caused by the anti-murine A␤ antibody M3.2 mAb
(193.75 ⫾ 8.43%, n ⫽ 11; F(1,18) ⫽ 4.749, p ⫽ 0.043 vs vehicle; Bonferroni’s p ⫽ 0.007, 0.011 and 0.006 at 20, 30 and 40 ms) was rescued by 200 pM human oA␤42 (175.39 ⫾ 17.22%,
n ⫽ 10; F(1,19) ⫽ 5.111, p ⫽ 0.036 vs M3.2 mAb; F(1,17) ⫽ 0.006, p ⫽ 0.939 vs vehicle). D, Twenty-minute perfusion with 200 pM oA␤42 converted E-LTP elicited through a weak tetanic
stimulation (1 TBS) to L-LTP (134.24 ⫾ 5.44% of baseline vs 220.10 ⫾ 17.74% of baseline, n ⫽ 7/7; F(1,12) ⫽ 9.883, p ⫽ 0.008), inducing a potentiation similar to that obtained with
a strong tetanic stimulation (3 TBS) (219.91 ⫾ 7.75% of baseline, n ⫽ 7; F(1,12) ⫽ 0.321, p ⫽ 0.582 comparing oA␤42 ⫹ 1 TBS vs vehicle ⫹ 3 TBS). E, Perfusion (30 min before and 25
min after tetanus) with the translation inhibitor ANI blocked the oA␤42-induced L-LTP (127.48 ⫾ 8.71% of baseline, n ⫽ 7; F(1,12) ⫽ 11.946, p ⫽ 0.005 vs oA␤42 ⫹ 1 TBS). ANI did not
modify E-LTP induced by 1 TBS (137.92 ⫾ 8.05% of baseline, n ⫽ 7; F(1,11) ⫽ 3.091; p ⫽ 0.106 vs vehicle ⫹ 1 TBS) but blocked L-LTP induced by 3 TBS (146.41 ⫾ 14.77% of baseline,
n ⫽ 6; F(1,12) ⫽ 25.976, p ⬍ 0.0001 vs vehicle ⫹ 3 TBS). Shaded area with dashed line corresponds to mean ⫹ SEM of the 5 last recorded point in slices treated with vehicle ⫹ 3 TBS
as in D. F, Evaluation of recognition memory indicated a difference in discrimination index (D; the exploration of novel object minus exploration of familiar object/total exploration time)
between vehicle-treated mice that underwent a 3 min exposition in T1 (short T1) or a 10 min exposition in T1 (long T1) (0.04 ⫾ 0.02 vs 0.31 ⫾ 0.03 comparing short T1 vs long T1, n ⫽
10/10 sex-balanced mice; Bonferroni’s p ⬍ 0.0001). Comparison of D with zero confirmed that a long T1 was able to induce LTM in a 24-h-delay novel object recognition task (t(9) ⫽
9.604, p ⬍ 0.0001), whereas a short T1 did not (t(9) ⫽ 1.62, p ⫽ 0.140). Intrahippocampal injections with 200 pM oA␤42 20 min before a short T1 converted STM into LTM (D ⫽ 0.38 ⫾
0.01, n ⫽ 10 sex-balanced mice; t(9) ⫽ 19.79, p ⬍ 0.0001 comparing D with zero in oA␤42 ⫹ Short T1). One-way ANOVA with Bonferroni’s post hoc corrections confirmed the significant
difference between D in vehicle- and oA␤42-treated mice previously exposed to a short T1 ( p ⬍ 0.0001). The increase of D induced by treatment with oA␤42 was similar to that obtained
in vehicle-treated mice that spent a longer period in T1 ( p ⫽ 0.314 comparing D in oA␤42 ⫹ short T1 vs vehicle ⫹ long T1). Right, Total exploration time was comparable in the 3 groups
of mice (F(2,27) ⫽ 0.086, p ⫽ 0.918). *p ⬍ 0.05, **p ⬍ 0.01, ****p ⬍ 0.0001; # difference from 0. Data are expressed as mean ⫾ SEM.
Table 1. Detailed results of TEM performed on hippocampal slices stored at 120 min after electrophysiological recordings in the following experimental conditions: vehicle
(V), vehicle ⴙ 1 TBS tetanus (V ⴙ T), oA␤42 200 pM (A␤), oA␤42 200 pM ⴙ 1 TBS tetanus (A␤ ⴙ T)
Experimental
condition V V⫹T A␤ A␤⫹T Statistical analysesa ( p value)
Asymmetric
synapses (n) n ⫽ 172 n ⫽ 151 n ⫽ 124 n ⫽ 113 V vs V⫹T V vs A␤ V vs A␤⫹T V⫹T vs A␤ A␤ vs A␤⫹T V⫹T vs A␤⫹T
Vesicle pool (n) 74.8 ⫾ 2.4 70.2 ⫾ 2.9 72.9 ⫾ 3.1 77.3 ⫾ 3.0 0.48 ⬎0.99 ⬎0.99 ⬎0.99 ⬎0.99 0.73
Docked vesicles (n) 1.7 ⫾ 0.08 3.7 ⫾ 0.12 2.6 ⫾ 0.10 4.2 ⫾ 0.14 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.13
Area of spines (␮m 2) 0.09 ⫾ 0.004 0.11 ⫾ 0.006 0.10 ⫾ 0.005 0.10 ⫾ 0.006 0.08 0.055 ⬎0.99 ⬎0.99 0.32 0.51
PSD length (␮m) 0.212 ⫾ 0.005 0.214 ⫾ 0.005 0.207 ⫾ 0.006 0.249 ⫾ 0.008 ⬎0.99 ⬎0.99 0.0037 ⬎0.99 0.0041 0.0398
Perforated PSD 4.07% 11.26% 5.65% 14.20% 0.018b 0.58b 0.0013b 0.132b 0.021b 0.508b
a
Statistical analyses performed by nonparametric Kruskal–Wallis test with Dunn’s test for multiple comparison. Number of microscopical fields: V (n ⫽ 87); V⫹T (n ⫽ 116); A␤ (n ⫽ 92); A␤ ⫹ T (n ⫽ 113). For vesicle pool, docked vesicles,
area of spines, and PSD length values are shown as mean ⫾ SEM. For perforated PSD, % indicates the proportion of asymmetric synapses with perforated PSD.
b
Statistical analysis performed by contingency analysis with Fisher’s test.
Figure 3. Picomolar concentrations of oA␤42 increase the number of docked vesicles and PSD length. A, Representative asymmetric axospinous synapses of CA1 stratum radiatum from
hippocampal slices previously treated for electrophysiological recordings. Framed regions enlarged (bottom) show the synaptic contact for each synapse from different experimental conditions.
Arrows indicate docked vesicles (dv) at the active zone of axon terminals; arrowheads are the edges of PSD. AxT, Axon terminal; sp, spine. Scale bars: 500 nm for top, 100 nm for bottom. B, oA␤42
alone increased the number (n) of docked vesicles compared with vehicle ( p ⬍ 0.0001). The number of docked vesicles increased in tetanized slices treated with oA␤42 compared with nontetanized
slices treated with the peptide, as well as in tetanized slices treated with vehicle versus vehicle-treated nontetanized ones ( p ⬍ 0.0001). C, oA␤42 paired with a weak tetanic stimulation (1 TBS)
increased PSD length compared with other conditions. See Table 1 for detailed results and statistical analyses. V, Vehicle, A␤ ⫽ 200 pM oA␤42, 1 TBS ⫽ weak tetanic stimulation. *p ⬍ 0.05; ***p ⬍
0.005; ****p ⬍ 0.0001. Data are expressed as mean ⫾ SEM.
robustly support the electrophysiological data. Most impor- molar concentrations of A␤, pM concentrations of the peptide
tantly, this body of evidence indicates that synapses undergo a did not require APP to produce their synaptic effects.
series of coordinated changes occurring both at the presynap- We then turned on ␣7nAChRs because of their interplay with
tic and postsynaptic site following application of oA␤42 at pM A␤ in physiological conditions (Puzzo et al., 2008, 2011; Zappet-
concentrations. tini et al., 2012; Lawrence et al., 2014). When slices from
␣7nAChRs KO (␣7-KO) mice were treated with 200 pM oA␤42,
Effect of picomolar concentrations of oA␤42 on short-term the decrease of PPF and the conversion of E-LTP to L-LTP were
and long-term synaptic plasticity and memory requires ␣7 not elicited (Fig. 4C,D). To confirm that the effects of 200 pM
nicotinic acetylcholine receptors (␣7nAChRs) oA␤42 were mediated by ␣7nAChRs, we acutely blocked these
Because recent studies indicated that high nanomolar concentra- receptors by the selective antagonist MLA. This prevented 200 pM
tions of extracellular oA␤42 require APP to impair LTP and mem- oA␤42 from affecting PPF and LTP (Fig. 4 E, F ), suggesting that
ory (Puzzo et al., 2017; Wang et al., 2017), we tested whether the oA␤42 needs ␣7nAChRs to exert its enhancing effects.
plasticity-enhancing effect of extracellular picomolar concentra- To corroborate our findings indicating that ␣7nAChRs, but
tions of oA␤42 was APP dependent. We used APP-KO mice to not APP, were needed for 200 pM oA␤42 to enhance plasticity, we
determine whether the absence of endogenous APP prevented repeated experiments after the acute block of ␣7nAChR function
200 pM oA␤42 to exert its effects on PPF and LTP. We found that with MLA, which prevented the 200 pM oA␤42-mediated decrease
200 pM oA␤42 was still able to decrease PPF (Fig. 4A) and convert of PPF and conversion of E-LTP into L-LTP in slices from
E-LTP into L-LTP (Fig. 4B) in APP-KO mice. Thus, unlike nano- APP-KO mice (Fig. 4G,H ).
Next, we validated our in vitro and ex

vivo results in vivo by investigating
whether APP and/or ␣7nAChRs were
needed for 200 pM oA␤42 to convert STM
into LTM. APP-KO and ␣7-KO mice were
injected intrahippocampally with vehicle
or 200 pM oA␤42 before training (short
exposure in T1) and memory was evalu-
ated after 24 h. Analyses of the discrimi-
nation index revealed that 200 pM oA␤42
was capable to induce LTM in APP-KO
but not in ␣7-KO mice (Fig. 4I ), without
modifying total exploration time (Fig.
4J ). Thus, ␣7nAChRs, but not APP, were
necessary for pM oA␤42 to convert STM
into LTM.
oA␤42-induced conversion of early-LTP

into late-LTP and STM into LTM
depends upon the nitric oxide (NO)/
cGMP/protein kinase G (PKG) pathway
and involves plasticity-related proteins
(PRPs)
To gain insight into the intracellular
mechanisms involved in the oA␤42-
induced enhancement of LTP, we focused
on the NO/cGMP/PKG pathway, which
plays a key role in presynaptic and post-
synaptic mechanisms of plasticity and in
4
mean ⫹ SEM of the last recorded point of 1 TBS-induced LTP in
WT slices treated with vehicle or 200 pM oA␤42 as shown in
Figure 2; the arrow indicates 1 TBS in this and the following
panels. C, 200 pM oA␤42 did not affect PPF in slices from ␣7-KO
mice (177.42 ⫾ 13.67% vs 178.03 ⫾ 8.52%, n ⫽ 8/8; F(1,16)
⫽ 0.388, p ⫽ 0.542). D, 200 pM oA␤42 did not convert E-LTP
into L-LTP in slices from ␣7-KO mice (131.12 ⫾ 3.46% of
baseline vs 124.43 ⫾ 11.09% of baseline, n ⫽ 7/7; F(1,12) ⫽
0.002, p ⫽ 0.964). E, 200 pM oA␤42 did not affect PPF in slices
from WT mice treated with the ␣7nAChR antagonist MLA (10
nM, 10 min before tetanus) (192.99 ⫾ 13.84% vs 188.02 ⫾
10.54%, n ⫽ 11/11; F(1,20) ⫽ 0.154, p ⫽ 0.699). F, 200 pM
oA␤42 did not convert E-LTP into L-LTP in slices from WT mice
treated with MLA (137.45 ⫾ 12.25% of baseline vs 128.57 ⫾
3.81% of baseline, n ⫽ 6/7; F(1,11) ⫽ 0.009, p ⫽ 0.927). G,
200 pM oA␤42 did not modify PPF in APP-KO mice treated with
MLA (189.90 ⫾ 6.86% vs 197.38 ⫾ 12.64%, n ⫽ 6/10; F(1,14)
⫽ 0.001, p ⫽ 0.973). H, 200 pM oA␤42 did not convert E-LTP
into L-LTP in slices from APP-KO mice treated with MLA
(130.70 ⫾ 9.68% of baseline vs 121.18 ⫾ 7.35% of baseline,
n ⫽ 6/7; F(1,11) ⫽ 0.589, p ⫽ 0.459). I, Intrahippocampal
injections with 200 pM oA␤42 20 min before a short T1 con-
verted STM into LTM in APP-KO mice (D ⫽ 0.06 ⫾ 0.04 vs
0.30 ⫾ 0.03; n ⫽ 10/10 sex-balanced mice; Bonferroni’s p ⫽
0.022 comparing APP-KO mice treated with vehicle or 200 pM
oA␤42; t(9) ⫽ 7.645, p ⬍ 0.0001 comparing D vs zero in APP
KO ⫹ 200 pM oA␤42). Conversely, 200 pM oA␤42 administra-
Figure 4. The ␣7-nAChR, but not APP, is needed for picomolar concentrations of oA␤42 to decrease PPF and convert both E-LTP tion did not induce LTM in ␣7-KO mice (D ⫽ ⫺0.07 ⫾ 0.07 vs
into L-LTP and STM into LTM. A, Twenty-minute perfusion with 200 pM oA␤42 was capable of decreasing PPF in slices from APP-KO 0.02 ⫾ 0.02; n ⫽ 10/10 sex-balanced mice; Bonferroni’s p ⫽
mice (% facilitation at 100 ms interval here and in following panels: APP KO ⫹ vehicle ⫽ 189.34 ⫾ 7.51%, n ⫽ 8; 200 pM oA␤42 1 comparing ␣7-KO mice treated with vehicle or 200 pM
⫽ 159.57 ⫾ 8.32%, n ⫽ 8; ANOVA for repeated measures F(1,14) ⫽ 5.234, p ⫽ 0.038; Bonferroni’s p ⫽ 0.013 at 50 ms and 0.044 oA␤42; t(9) ⫽ 0.999, p ⫽ 0.344 comparing D vs zero in ␣7 KO
at 100 ms). Gray shaded curves represent mean ⫹ SEM of PPF curves in WT slices treated with vehicle or 200 pM oA␤42 as shown ⫹ 200 pM oA␤42). J, Total exploration time was comparable in
in Figure 2. B, 200 pM oA␤42 converted E-LTP into L-LTP in slices from APP-KO mice (124.69 ⫾ 13.13% of baseline vs 210.28 ⫾ the 4 groups of mice (F(3,36) ⫽ 0.515, p ⫽ 0.674). *p ⬍ 0.05;
12.60% of baseline, n ⫽ 7/7; ANOVA for repeated measures F(1,12) ⫽ 14.77, p ⫽ 0.002). Gray shaded areas with lines represent # difference from 0. Data are expressed as mean ⫾ SEM.
Figure 5. Conversion of E-LTP into L-LTP and STM into LTM by pM oA␤42 is mediated through the NO/cGMP/PKG cascade. A, 200 pM oA␤42 did not induce L-LTP in slices treated with the nNOS
inhibitor L-NAME (100 ␮M for 40 min) (118.96 ⫾ 12.57% of baseline, n ⫽ 7; F(1,12) ⫽ 18.645, p ⫽ 0.001 vs 200 pM oA␤42 ⫹ 1 TBS), the sGC inhibitor ODQ (100 ␮M for 40 min) (125.84 ⫾ 13.39%
of baseline, n ⫽ 6; F(1,11) ⫽ 8.903, p ⫽ 0.012), and the PKG inhibitor Rp-8-Br-cGMPS (10 ␮M for 10 min) (140.48 ⫾ 19.61% of baseline, n ⫽ 7; F(1,12) ⫽ 9.161, p ⫽ 0.011). Shaded area with line
corresponds to mean ⫾ SEM of the last recorded point in WT slices treated with 200 pM oA␤42 ⫹ 1 TBS as in Figure 2. B, Treatment with L-NAME, ODQ, or Rp-8Br-cGMPS alone did not modify residual
potentiation induced by a weak tetanic stimulation compared with vehicle (n ⫽ 4 for each condition, F(1,12) ⫽ 0.315, p ⫽ 0.814). C, Blocking the NO/cGMP/PKG pathway inhibits L-LTP induced by
a strong tetanic stimulation compared with vehicle (n ⫽ 4 for each condition, F(3,14) ⫽ 13.391, p ⬍ 0.0001 among all; vehicle: 228.15 ⫾ 18.80% of baseline; L-NAME: 148.44 ⫾ 7.45% of baseline,
Bonferroni’s p ⫽ 0.006; ODQ: 131.24 ⫾ 9.22% of baseline, Bonferroni’s p ⫽ 0.001; Rp-8Br-cGMPS: 126.15 ⫾ 2.54% of baseline, Bonferroni’s p ⫽ 0.001). D, Top, Representative images of WB assay
(cropped images based on MW) performed on hippocampal slices (n ⫽ 4 for each lane) treated for electrophysiological experiments, collected, and stored at 120 min after treatment (V, vehicle; T,
1 TBS; A␤ ⫽ oA␤42 200 pM). Bottom, Bar graph obtained by the average of two or three different membranes here in E and in G. ␤-Tubulin expression is shown as one example of loading control.
An increase of nNOS expression (F(3,8) ⫽ 4.570; p ⫽ 0.038; Tukey’s p ⫽ 0.042) was detected in slices treated with A␤ and a 1 TBS stimulation. E, nNOS expression increased in slices treated with
vehicle and 3TBS (F(1,2) ⫽ 52.412; p ⫽ 0.019). F, WB assay of PRPs. G, p-CREB expression was increased after treatment with A␤ alone and further enhanced in A␤ ⫹T (F(3,12) ⫽ 61.713; p ⬍
0.0001; Tukey’s p ⬍ 0.0001). H, No changes were detected in total CREB expression among different conditions (F(3,4) ⫽ 0.930; p ⫽ 0.504). I, J, A␤ paired with 1 TBS increased p-CaMKII levels (F(3,8)
⫽ 11.746; p ⫽ 0.003) compared with V ( p ⫽ 0.017) or V⫹T ( p ⫽ 0.004) (I), whereas CaMKII expression was not modified (J; F(3,4) ⫽ 2.758; p ⫽ 0.176). K, A␤ paired with 1 TBS increased BDNF
levels (F(3,8) ⫽ 5.686; p ⫽ 0.022) compared with V ( p ⫽ 0.034) and V⫹T ( p ⫽ 0.038). L, Evaluation of recognition memory indicated that 200 pM oA␤42 did not induce LTM in mice concurrently
treated with the PKG inhibitor Rp-8-Br-cGMPS [discrimination index (D) ⫽ 0.03 ⫾ 0.044, n ⫽ 10 sex-balanced mice for each condition; t(9) ⫽ 0.735, p ⫽ 0.481 (Figure legend continues.)
the conversion from E-LTP to L-LTP

(Bon and Garthwaite, 2003; Johnstone
and Raymond, 2011). To this end, hip-
pocampal slices were treated with 200 pM
oA␤42 paired with drugs able to inhibit
NO or cGMP production or PKG activa-
tion. When preventing NO production by
using the inhibitor of the nNOS L-NAME,
the inhibitor of soluble guanylyl cyclase
ODQ, or the cGMP analog Rp-8-Br-
cGMPS, oA␤42 failed to induce a long-
lasting potentiation (Fig. 5A). Control
experiments showed that L-NAME,
ODQ, and Rp-8-Br-cGMPS did not dis-
rupt LTP evoked by a weak tetanic stimu-
lation per se (Fig. 5B), whereas they
impaired L-LTP induced by a 3 TBS stim-
ulation (Fig. 5C), further suggesting that
pM oA␤42 acts via a pathway that is phys-
iologically involved in L-LTP formation.
Because electrophysiological data showed
an involvement of the NO/cGMP/PKG path-
way, we performed Western blots on slices
from electrophysiology experiments to
evaluate whether oA␤42 triggers NO pro-
duction by affecting nNOS expression.
We found a significant increase of nNOS
in slices treated with 200 pM oA␤42 paired
with a weak tetanus (Fig. 5D), similar to
that obtained in slices treated with vehicle
paired with a strong tetanus (Fig. 5E).
Furthermore, we explored the molecular
mechanisms underlying oA␤42-induced
L-LTP by analyzing the expression of PRPs,
known to be involved in synaptic plasticity
and memory (Puzzo et al., 2016), including
cAMP-responsive element binding protein
(CREB), calcium-calmodulin-dependent
protein kinase II ␣ (CaMKII), and brain-
derived neurotrophic factor (BDNF), in Figure 6. A␤-mediated events occurring at the synapse in physiological conditions: a working hypothesis. In physiological
the same hippocampal slices used for elec- conditions, A␤ activates ␣7nAChRs, leading to Ca 2⫹ entry and thus increasing neurotransmitter release. This, in turn, would
trophysiological experiments. We found trigger a cascade of intracellular events involving the NO/cGMP/PKG pathway and the plasticity-related molecules p-CREB,
that cAMP-responsive element binding p-CaMKII, and BDNF, leading to the enhancement of synaptic plasticity and memory.
protein (CREB) phosphorylated at Ser
133 (p-CREB) was increased in slices treated with oA␤42 alone or we found that treatment with the cGMP analog Rp-8-Br-cGMPS
oA␤42 paired with a weak tetanic stimulation without affecting prevented oA␤42 to induce LTM in mice that underwent a short
total CREB levels (Fig. 5F–H ). oA␤42 also increased the expres- exposure during the training phase (Fig. 5L), whereas Rp-8-Br-cGMPS
sion of the CaMKII phosphorylated at Thr 286 (p-CaMKII) (Fig. did not affect STM per se (Fig. 5L). Thus, PKG activation mediates
5 F, I,J ) and BDNF (Fig. 5 F, K ). oA␤42-induced conversion of STM into LTM.
Finally, because the NO/cGMP/PKG pathway has been widely Overall, our findings indicate that oA␤42 mainly acts via an
demonstrated to intervene in memory processes and previous intracellular signaling pathway involving the NO/cGMP/PKG
studies have suggested that PKG activity maintains CREB phos- pathway leading to PRP activation, synaptic potentiation, and
phorylation at Ser133 during memory consolidation (Puzzo et memory formation, as summarized in Figure 6.
al., 2016), we investigated whether the oA␤42-induced conver-
sion of STM into LTM depends upon PKG activity. As for LTP, Discussion
We focused on the role of picomolar concentrations of oA␤42 at
4 hippocampal synapses in the healthy rodent brain. We found that
200 pM oA␤42 increases neurotransmitter release, induces pre-
(Figure legend continued.) comparing D vs zero; Bonferroni’s p ⬍ 0.0001 comparing D between
200 p M oA␤ vs 200 pM oA ␤ ⫹ Rp-8-Br-cGMPS]. Rp-8-Br-cGMPS alone did not affect STM synaptic and postsynaptic ultrastructural changes, and increases
42 42
(Bonferroni’s p ⫽ 1 vs vehicle). Total exploration time (right) was not modified by treatments PRP expression, leading to the conversion of E-LTP into L-LTP
(F(2,27) ⫽ 0.086, p ⫽ 0.667). Shaded area with lines corresponds to mean ⫹ SEM of D in mice and of STM into LTM.
treated with vehicle or 200 pM oA␤42 20 min before a short T1 exposure. *p ⬍ 0.05; **p ⬍ This study was inspired by previous observations indicating
0.01; ****p ⬍ 0.0001. Data are expressed as mean ⫾ SEM. that oA␤42 exerts opposite effects onto synaptic plasticity and
memory depending upon its concentration (Puzzo et al., 2012; prevented both endogenous or exogenous A␤ to exert its effects
Gulisano et al., 2018b). Here, we first performed a DR curve to at the synapse.
confirm that 200 pM oA␤42 was the dose capable of enhancing Because the probability of transmitter release contributes to
LTP, whereas 200 nM impaired it, consistent with previous works LTP induction and maintenance (Kleschevnikov et al., 1997), we
(Puzzo et al., 2008, 2011, 2012; Garcia-Osta and Alberini, 2009; investigated whether oA␤42 converted E-LTP into L-LTP, two
Morley et al., 2010; Lazarevic et al., 2017). different forms of plasticity characterized by different temporal
Next, because high concentrations of extracellular oA␤42 en- and mechanistic features (Huang, 1998). E-LTP can be induced
ter neurons (Ripoli et al., 2014; Puzzo et al., 2017), leading to by a weak tetanic stimulation, decays in 1 or 2 h, does not depend
synaptic dysfunction through direct intracellular targets (Ripoli on new protein synthesis, and is mostly due to activation of ki-
et al., 2014), we investigated whether pM oA␤42 acts at extracel- nases. Conversely, L-LTP needs a stronger stimulation to be in-
lular and/or intracellular levels when enhancing synaptic trans- duced, lasts several hours, and requires new protein synthesis and
mission and plasticity. We demonstrated that the effect of pM gene transcription (Bliss and Collingridge, 1993; Nguyen et al.,
oA␤42 is exerted only when the peptide is applied extracellularly, 1994; Huang, 1998). Several studies have demonstrated that ad-
because its administration inside neurons did not affect LTP. equate physiological or pharmacological stimuli might convert
Dual-patch experiments, in which we applied oA␤42 from the E-LTP into L-LTP, a mechanism that parallels transformation of
extracellular space while one of the two adjacent neurons was STM into LTM (for review, see Puzzo et al., 2016). Here, we
injected with the 6E10 antibody to neutralize human oA␤42 pos- found that treatment with picomolar concentrations of oA␤42
sibly accumulated inside neurons, confirmed that only extracel- before a weak tetanic stimulation converted E-LTP into L-LTP
lular oA␤42 enhances mEPSC frequency. and STM into LTM, suggesting that, at this concentration, the
The increase in mEPSC frequency and the decrease in PPF peptide acts as a cognitive enhancer and further begging the ques-
suggest a presynaptic mechanism of neurotransmitter release, tion of how its removal from the brain might be beneficial.
consistent with previous electrophysiological findings showing Ultrastructural analyses of hippocampal slices treated as for
that pM oA␤42 affect basal synaptic transmission, inducing an electrophysiology revealed that oA␤42 produces a series of con-
increase of fiber volley amplitude, an index of presynaptic re- comitant changes occurring both presynaptically and postsynap-
cruitment (Gulisano et al., 2018b), and posttetanic potentiation, tically, as one would expect for long-term normal synaptic
a form of short-term plasticity due to presynaptic calcium entry plasticity to occur (Antonova et al., 2001). Indeed, oA␤42 alone
(Puzzo et al., 2008). Furthermore, it has been recently demon- elevated the fraction of vesicles available for release (i.e., docked
vesicles) during the induction of plasticity (Bourne et al., 2013),
strated that A␤ exerts an opposite effect on synaptic vesicle recy-
whereas a longer PSD, which is suggestive of plastic changes oc-
cling depending upon the dose (Lazarevic et al., 2017), consistent
curring at the postsynaptic site (Babits et al., 2016), was observed
with other studies showing a sustained increase of mEPSC fre-
in slices treated with oA␤42 paired with a weak tetanus.
quency after prolonged exposure to 200 pM oA␤42 (Koppen-
In the present study, we also sought to explore the possible
steiner et al., 2016) or treatment with inhibitors of A␤
extracellular targets used by pM oA␤42 to affect PPF and convert
degradation (Abramov et al., 2009). In these circumstances, high
E-LTP into L-LTP. We first focused on APP because it binds
levels of A␤ may maintain neurotransmitter release for a longer
different A␤ species (Lorenzo et al., 2000; Shaked et al., 2006;
period, leading to vesicle depletion (Parodi et al., 2010), or enter
Fogel et al., 2014) and is involved in the enhancement of neu-
neurons directly, affecting presynaptic proteins such as synapto- rotransmitter release, leading to hippocampal hyperactivity in
physin, VAMP2, or synapsin I (Russell et al., 2012; Koppensteiner AD (Bakker et al., 2012; Busche et al., 2012).
et al., 2016). Furthermore, recent studies have shown that, at high concen-
Here, we show that both exogenously applied human A␤ at trations, extracellular A␤ needs APP to impair synaptic plasticity
low concentrations and endogenous A␤ are involved in neu- and memory (Puzzo et al., 2017; Wang et al., 2017). Here, we used
rotransmitter release. This is consistent with previous studies APP-KO mice at young age, when the impairment of LTP and
demonstrating that endogenous A␤ is needed for synaptic plas- memory is not yet manifested (Dawson et al., 1999; Tyan et al.,
ticity and memory to occur (Garcia-Osta and Alberini, 2009; 2012), to investigate whether A␤ needs APP to enhance synaptic
Morley et al., 2010; Puzzo et al., 2011) and that A␤ production plasticity and memory when at low concentration. Because pM
physiologically increases during neuronal activity and memory oA␤42 still enhanced neurotransmitter release, synaptic plastic-
induction (Kamenetz et al., 2003; Cirrito et al., 2005; Brody et al., ity, and memory in APP-KO mice, we concluded that APP inter-
2008; Puzzo et al., 2011; Palmeri et al., 2017). Interestingly, we venes in A␤ detrimental effects when the peptide is present at
have previously demonstrated that the impairment of LTP and high concentrations (Puzzo et al., 2017), but is not needed for
memory due to the inhibition of endogenous A␤ could be res- oA␤42 at low concentrations to enhance synaptic plasticity.
cued by concomitant application of exogenous 200 pM A␤, with Because A␤ induces a series of events mediated by an increase
300 pM A␤ producing a more pronounced enhancement similar of Ca 2⫹ entry in the presynaptic terminal (Puzzo et al., 2008;
to that obtained with the administration of 200 pM exogenous A␤ Lawrence et al., 2014), we focused on ␣7nAChRs, ionotropic
alone (Puzzo et al., 2011). In the same work, we demonstrated channels permeable to calcium that are highly expressed in the
that the threshold of A␤ needed for normal synaptic plasticity hippocampus and implicated in a variety of cognitive functions
and memory is ⬃330 –380 pM. Thus, considering that A␤42 levels such as learning and memory, attention and reward (for review,
in basal conditions are equal to 180 –200 pM (Puzzo et al., 2008), see Picciotto et al., 2000). The link between A␤ and ␣7nAChRs
adding 200 pM exogenous A␤ is likely to induce a further en- has been widely studied and it is known that A␤ binds ␣7nAChRs
hancement of synaptic plasticity and memory. Even if we cannot with high affinity, exerting an agonistic or antagonistic effect in a
exclude that exogenously applied and endogenous A␤ might use dose-dependent manner (Wang et al., 2000; Mura et al., 2012).
different mechanisms to act, previous works have suggested that Using genetic and pharmacological approaches, we showed that
a common target might be represented by ␣7-nAchRs (Puzzo et ␣7nAChRs are needed for pM oA␤42 to increase neurotransmitter
al., 2008, 2011), the genetic or pharmacological deletion of which release and to consolidate LTP. Experiments performed in hip-
pocampal slices from APP-KO mice treated with the ␣7nAChR whereas nM oA␤42 requires APP and intraneuronal internaliza-
antagonist MLA further confirmed that, at low concentrations, tion (Ripoli et al., 2014; Puzzo et al., 2017), even if it is not pos-
oA␤42 exerts its effect through ␣7nAChRs and not APP, in agree- sible to exclude an extracellular effect mediated by different
ment with previous studies (Puzzo et al., 2008, 2011; Mura et al., receptors. Conversely, low and high oA␤42 levels affect the same
2012; Lawrence et al., 2014). key intracellular pathways involved in synaptic plasticity and
The in vitro observation showing that ␣7nAChRs but not APP memory (i.e., the NO/cGMP/PKG/p-CREB cascade, CaMKII,
are necessary for pM oA␤42 to produce L-LTP was further con- BDNF), leading to their stimulation at picomolar concentrations
firmed by in vivo studies demonstrating that the oA␤42-induced or inhibition at nanomolar concentrations (Puzzo et al., 2005,
conversion of STM into LTM relies upon ␣7nAChRs and does 2009; Ghosh and Giese, 2015; Song et al., 2015; Opazo et al.,
not involve APP. 2018). The capability of oA␤42 to induce opposite effects depend-
Here, we also demonstrated that oA␤42 effects are mediated by ing upon its dose is consistent with previous studies (Calabrese,
the NO/cGMP/PKG cascade, already known to be involved in 2001; Puzzo et al., 2012), and this biphasic behavior characterized
LTP and memory induction and maintenance (Johnstone and by low-dose stimulation and high-dose inhibition might be
Raymond, 2011; Bollen et al., 2014). In fact, inhibition of this framed into the “hormesis” phenomenon (Calabrese, 2008;
pathway prevented oA␤42-induced L-LTP and LTM. Interest- Mattson, 2008). Indeed, even if the most used approach to model
ingly, we have recently demonstrated that in physiological con- a DR curve is the linear Hill model, several studies have pointed
ditions, cGMP stimulates A␤ production to induce L-LTP and out that it might not be sufficient to explain the multiphasic
memory (Palmeri et al., 2017). This, together with the present response exerted by a variety of endogenous or exogenous com-
results, indicates that the NO/cGMP pathway acts both upstream pounds (for review, see Prickaerts et al., 2017). Although differ-
and downstream of A␤, suggesting the existence of a cGMP-A␤- ent mechanisms may underlie the hormetic effect (Mattson,
cGMP loop as a preferential intracellular mechanism involved in 2008), it is widely accepted that a compound might interact with
A␤-mediated plastic effects. a certain target depending upon its concentration. Consistently,
Additionally, pM oA␤42 increased the expression of nNOS, A␤ binds different targets in a concentration-dependent manner
which is decreased by high concentrations of the peptide (Ven- (Mura et al., 2012; Zappettini et al., 2012). and, recently, it has
turini et al., 2002). This effect might be due to direct or indirect been shown that A␤ directly affects ␣7-nAchR conformation and
mechanisms. Indeed, previous studies have demonstrated that function by acting as an agonist when at picomolar concentra-
A␤ fragments can bind nNOS (Padayachee and Whiteley, 2011), tions or as an antagonist when at nanomolar concentrations (La-
suggesting the possibility of a direct interaction between the two sala et al., 2019). It should be also considered that at high A␤
proteins. Conversely, the oA␤42-induced increase of neurotrans- concentrations, the amount of oligomerization proportionally
mitter release might enhance Ca 2⫹ influx at the postsynaptic increases overrunning the physiological level (Gulisano et al.,
level, triggering increased nNOS expression (Sasaki et al., 2000). 2018a,b). This, in turn, might determine the interaction of A␤
The oA␤42-induced conversion of E-LTP into L-LTP suggests oligomers with different targets, including APP (Puzzo et al.,
a possible role for A␤ in the synaptic tagging mechanism. Syn- 2017).
apses that have received a weak stimulation enter a receptive state These observations might be useful to understand the mech-
(tagging) that, if associated with the synthesis of PRPs, will lead to anisms switching the positive into the negative effects exerted by
LTP maintenance. The initial changes in the synaptic weight are oA␤, and might be a basis for further studies to ensure novel, safe,
given by the efficacy of the presynaptic and postsynaptic elements and rational personalized therapeutic approaches to cure patients
and require a third factor to persist (Redondo and Morris, 2011). affected by AD.
Consistent with this possibility, pretreatment of hippocampal
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5986 • The Journal of Neuroscience, July 24, 2019 • 39(30):5986 – 6000

Neuromodulatory Action of Picomolar Extracellular A␤42

Introduction Materials and Methods

Next, we validated our in vitro and ex

oA␤42-induced conversion of early-LTP

the conversion from E-LTP to L-LTP

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