PP1

Olfactory learning and memory in the greater short‑nosed fruit bat
Cynopterus sphinx: the infuence of conspecifcs distress calls
Koilmani Emmanuvel Rajan1
Received: 20 September 2020 / Revised: 13 July 2021 / Accepted: 4 August 2021 / Published online: 23 August 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
Abstract
This study was designed to test whether Cynopterus sphinx distress calls infuence olfactory learning and memory in con￾specifcs. Bats were exposed to distress calls/playbacks (PBs) of distress calls/modifed calls and were then trained to novel
odors. Bats exposed to distress calls/PBs made signifcantly fewer feeding attempts and bouts of PBs exposed to modifed
calls, which signifcantly induced the expression of c-Fos in the caudomedial neostriatum (NCM) and the amygdala com￾pared to bats exposed to modifed calls and trained controls. However, the expression of c-Fos in the hippocampus was not
signifcantly diferent between the experimental groups. Further, protein phosphatase-1 (PP-1) expression was signifcantly
lower, and the expression levels of E1A homologue of CREB-binding protein (CBP) (P300), brain-derived neurotrophic
factor (BDNF) and its tyrosine kinase B1 (TrkB1) receptor were signifcantly higher in the hippocampus of control/bats
exposed to modifed calls compared to distress calls/PBs of distress call-exposed bats. Exposure to the call possibly alters
the reciprocal interaction between the amygdala and the hippocampus, accordingly regulating the expression levels of PP1,
P300 and BDNF and its receptor TrkB1 following training to the novel odor. Thus, the learning and memory consolidation
processes were disrupted and showed fewer feeding attempts and bouts. This model may be helpful for understanding the
contributions of stressful social communications to human disorders.
Keywords Olfactory learning · Cynopterus sphinx · Distress calls · c-Fos · Brain-derived neurotrophic factor (BDNF)
Abbreviations
ALP Alkaline phosphatase
bAFM Broadband arched frequency modulation
BCIP 5-Bromo-4-chloro-3-indolylphosphate
disodium
BDNF Brain-derived neurotrophic factor
CREB-1 Cyclic AMP response element binding
protein-1
ERK-1/2 Extracellular-signal-regulated kinase-1/2
GAPDH Glyceraldehydes-3-phosphate dehydrogenase
IEG Immediate early gene
LTP Long-term potentiation
nAFM Narrow band arched frequency modulation
NBT Nitro-blue tetrazoliumchloride
NCM Caudomedial neostriatum
PBs Playbacks
PKA Protein kinase A
PP-1 Protein phosphatase-1
TrkB1 Tyrosine kinase B1
Introduction
Acoustic communication plays a signifcant role in informa￾tion exchange between conspecifcs (Fenton 2003; Gladziola
et al. 2012; Hörmann et al. 2020). Conspecifc communica￾tions are discrete acoustic structures that readily discrimi￾nate and transmit the intentional state of emitters to potential
receivers, termed “social calls”. Earlier studies reported that
social calls’ acoustic structures are context-specifc, such
as mother–pup reunion (Knörnschild et al. 2013), forag￾ing coordination (Wright et al. 2014), group recognition
(Budenz et al. 2009), mate attraction (Knörnschild et al.
2014) and distress (Huang et al. 2015). Distress calls are
produced during stressful situations such as extreme physi￾cal stress (Russ et al. 2005; Carter et al. 2015; Walter and
Schnitzler 2019) or when being threatened/attacked by a
predator (Lima and Ó Keefe 2013; Huang et al. 2015). The
* Koilmani Emmanuvel Rajan
[email protected]
1 Behavioural Neuroscience Laboratory, Department
of Animal Science, School of Life Sciences, Bharathidasan
University, Tiruchirappalli 620024, India
distress call structure is often in the range of low-frequency,
high-intensity broadband and is very similar among spe￾cies, manifesting as “repeated sequences” (Luo et al. 2013;
Hörmann et al. 2020). Earlier studies showed that the amyg￾dala plays a central role in neural circuits involved in social
vocalization and response (Naumann and Kanwal 2011;
Tressler et al. 2011; Gadziola et al. 2012). It is well known
that the amygdala receives intrinsically rewarding and aver￾sive input stimuli, which may be associated with acoustic
features within calls and context or experience (Ma et al.
2010; Sengupta et al. 2018).
Earlier studies demonstrated that olfactory information
is transferred from the olfactory bulb to the amygdala and
the hippocampus (Wilson et al. 2004). Depending on the
context, olfactory learning activates a signalling cascade
through protein kinase A (PKA), extracellular-signal-regu￾lated kinase-1/2 (ERK-1/2), and cyclic AMP response ele￾ment binding protein-1 (CREB-1) (Peng et al. 2010; Ganesh
et al. 2010a). Activated CREB requires CBP/CREB-bind￾ing protein (CBP) and its homologue E1A binding protein
(p300) to induce a network of genes that regulate learning
and memory. Studies have shown that p300 acetylates lysine
residues on histones and enhances memory formation (Mad￾dox et al. 2013). Conversely, serine/threonine (Ser/Thr) pro￾tein phosphatases (PPs: PP1, PP2A) dephosphorylate CREB
to suppress memory formation (Koshibu et al. 2009; Mauna
et al. 2011). Activated CREB infuences cellular processes
and enhances synaptic plasticity (Minatohara et al. 2016)
through the induction of C-fos. Training-dependent expres￾sion of C-fos in the amygdala and the hippocampus has been
reported as an indication of memory formation and leads to
the transcription of late-response genes (Luscher Dias et al.
2016; Minatohara et al. 2016; Mukilan et al. 2018). Fur￾thermore, activation/inhibition of brain-derived neurotrophic
factor (BDNF) and its tyrosine kinase B1 (TrkB1) receptor
has been known to infuence long-term potentiation (LTP),
which is likely to contribute to synaptic plasticity, learning
and memory (de Deus et al. 2020).
The Indian greater short-nosed fruit bat Cynopterus
sphinx feeds on a variety of fruits, fowers and leaves, and
they can identify and learn about palatability based on
compounds originating from food sources (Elangovan et al.
2006). Earlier studies from my laboratory showed that C.
sphinx produces multiharmonic audible distress calls as
a narrowband arched frequency modulation (nAFM) and
a broadband arched frequency modulation (bAFM). The
specifc structural sequence of the call elicits a behavioural
response and alters the levels of stress hormones, neurotrans￾mitters and other molecules involved in the stress response
in conspecifcs (Ganesh et al. 2010b; Mariappan et al. 2013,
2016). The present study was designed to test whether olfac￾tory learning and memory were infuenced by a distress call
of conspecifcs. To test the hypothesis, individuals were
exposed to the distress call/PB of the distress call/modifed
call and were then trained to a novel odor to test their learn￾ing ability. Subsequently, the expression patterns of c-Fos
in the caudomedial neostriatum (NCM) region of the audi￾tory nuclei, the amygdala and the expression levels of PP1,
P300, BDNF and its receptor TrkB1 in the hippocampus
were examined.
Materials and methods
Animals
Male Cynopterus sphinx (forearm length 65±7 mm, body
mass 54.3±7.0 g) was captured using a mist net (9 m×2 m;
Avinet-Dryden, USA) in a guava orchard located 2 km away
from the Bharathidasan University Campus, Tiruchirappalli,
India (10° 16′ N; 78° 15′ E). Bat’s morphometric details were
recorded and tagged with plastic neck collars consisting of
light-refective colored tapes (Rajan and Marimuthu 1999)
and then transferred to the animal house facility. Bats were
maintained in the free-fight chamber (of 2.2×1.3×2.1 m)
under constant conditions (temperature 30 °C±3 °C, rela￾tive humidity 85±3%; light:dark 12 h:12 h) at animal house
facility and allowed to acclimatize for 5 days. Bats were feed
with the commonly available fruits [sapota (Achras sapota),
papaya (Carica papaya), banana (Musa paradisiaca) and
guava (Psidium guajava)] and water ad libitum. The health
status of animals was assessed by inspecting fur, bite wounds
and infections. Bats that were inactive and declining to eat
during the study were excluded and released at their capture
site.
Behavioural analysis
Bats (n=6 for each group) were randomly classifed into
four groups; (1) control (CON); (2) distress call group—bats
were exposed to the distress call of conspecifcs; (3) play￾back (PB) of distress call—bats were exposed to playback of
distress call and (4) modifed call—bats were exposed to PB
of modifed call. To test whether responses were specifc to
the distress call, the original distress call as well as the call
structure altered by changing the call sequence “modifed
call” (Mariappan et al. 2016) was used in this study. The
distress call was composed of a specifc sequence of two
types of syllables [narrow band arched frequency modula￾tion (nAFM: 123.33±1.5 ms; 5.75±0.1 kHz) and broad￾band arched frequency modulation (bAFM: 203.23±2.9 ms;
7.27±0.01 kHz)] alternatively. In the modifed call, the
sequence of syllables was changed to: nAFM, nAFM,
bAFM, and bAFM. Distress and modifed calls (Figs. 1, 2)
used in this experiment were reported earlier from my labo￾ratory (Mariappan et al. 2016).
Playback experiment
The experimental facility consists of two chambers: (1) free￾fight chamber (2.2 m × 1.3 m × 2.1 m), (2) experimental
chamber (2.1 m×2.4 m×2.4 m), and the window between
chambers facilitates to transfer the bats without disturbances.
An hour before the experiment bats were transferred from
the free-fight chamber to experimental chamber. Then,
distress calls or modifed calls were played back (20 s/
day) 15 min before the olfactory training (Mariappan et al.
2016). All the experiments were performed under red light
(0.09±0.02 lx) to minimize the visual cue (Shafe et al.
2014).
Olfactory training
Bats were individually trained to the novel odor [pieces of
chopped apple mixed with freshly prepared cinnamon pow￾der (0.8% wt/wt)] (Ratclife and ter Hofstede 2005) and their
activities were recorded using a computerized-activity moni￾tor (Electronic Engineering Corporation Inc., India). The
activity monitor consists of an infra-red (IR) receiver–trans￾mitter and a mass-sensitive platform (food tray). Their olfac￾tory learning was tested by proving control fruit (fresh pieces
of chopped apple) on one platform and fruit with novel odor
in the other platform. The constant distance (1.8 m) was
maintained between two platforms, as wells between the
platforms to perch and location of platforms were changed
randomly every day to prevent spatial learning. After accli￾matization, bats were individually trained to the novel odor
for 5 days (20 min/day), rested for 5 days and then tested
their memory for 5 days. Individual bat’s responses to novel
Fig. 1 Free-ranging C. sphinx distress call after becoming trapped in
a mist net. a Oscillogram of a continuous distress call, with narrow￾band arched frequency modulation (nAFM) and broadband arched
frequency modulation (bAFM) fused together and alternating con￾tinuously. b Spectrogram of continuous distress calls indicating har￾monics. c Relative power spectrum depicting peak frequencies of the
distress call
Fig. 2 The modifed distress call of C. sphinx shows variation in a the
oscillogram, with nAFM and nAFM fused together and then bAFM
and bAFM alternating continuously. b Spectrogram of modifed calls
indicating altered harmonics. c Relative power spectrum depicting
peak frequencies of the modifed distress call
odor in terms of: (1) fights out—short fights from the perch
(indicating bats are active); (2) attempts—approaches to the
food tray (novel odor), but returning without the piece of
fruit being picked up; (3) feeding bouts—landings on the
food tray and returning with the piece of fruit (Ganesh et al.
2010a, b; Mukilan et al. 2018).
Sample preparation, RNA isolation, and cDNA
synthesis
Animals (n=4 from each group) were euthanized and the
whole brain was dissected out. The caudomedial neostriatum
(NCM), amygdala and hippocampal region were dissected
as described elsewhere (Kalin et al. 1994), then bisected
for preparation of total RNA and protein. Total RNA was
isolated using TRIzol (Merck Specialties Pvt. Ltd., Mumbai,
India) and stored with RNase inhibitor (GeNei™; Merck
Specialties Pvt. Ltd.) at −80 °C. Total RNA (2.0 µg/sam￾ple) was reverse transcribed into cDNA (I Script Reverse
Transcription Kit; Bio-Rad Laboratories, Hercules, CA) in
accordance with the manufacturer’s instructions.
Quantitative real‑time PCR
The quantitative real-time PCR (qRT-PCR) reactions were
performed using the reaction mixure (20  µL; SSoAd￾vanced™ SYBR® green supermix; Bio-Rad Laboratories)
with specifc primers (10 µM) and cDNA (0.1 µg). The
specifc primers used were: TrkB1 (For 5′-CCAAGAGGC
TAAATCCAGTCC-3′ and Rev 5′-CCAGGTTACCAACAT
GCTAATA-3′); and GAPDH (glyceraldehydes-3-phosphate
dehydrogenase; For 5′-CGGGAAGCTCA CTGGCATGG-3′
and Rev 5′-CCTGCTTCACCACCTTCTTG-3′). The qRT￾PCR reactions were performed using the CFX-96 Touch™
Real-time PCR Detection System (Bio-Rad Laboratories
Inc.) following the conditions: initial denaturation (92 °C,
30 S); denaturation (92 °C, 5 S), annealing (TrkB1: 61 °C;
GAPDH: 58 °C for 5 S), extension (72 °C, 5 S), with 39
cycle repeats and melt-curve analysis (65–95 °C, 0.5 °C
increment for 0:05 S). Reactions were performed in tripli￾cates with three fold serial dilution of cDNA to verify the
consistency and then normalized with the internal control
GAPDH. The data are presented as mean fold change of
the relative expression (CFX Manager™ version 2 software,
CFX-96 Touch™ Real-time PCR Detection System; Bio￾Rad Laboratories Inc).
Western blotting
Samples were homogenised in ice-cold lysis bufer (150 mM
NaCl, 50 mM Tris–Hcl pH 7.5, 5 mM EDTA, 0.1% V/V
NP-40, 1.0  mM DTT, 0.2  mM sodium orthovanadate,
0.23 mM PMSF) with protease inhibitor cocktail (Sigma￾Aldrich, USA). Subsequently, homogenates were incubated
on ice for 30 min and then centrifuged (10,000×g) for
30 min at 4 °C. Finally, clear supernatants were collected by
centrifuged at 12,000×g for 15 min at 4 °C, and then stored
at −80 °C as aliquots. Protein concentration was estimated
using a Biophotometer (Eppendorf Inc., Germany). An equal
concentration of protein (60 μg) was mixed with a load￾ing bufer (100% glycerol, 125 mM Tris–HCL pH 6.8, 4%
SDS, 0.006% bromophenol blue, 2% mercaptoethanol) and
boiled for 5 min, then resolved on 10% polyacrylamide gel
(PAGE). Separated proteins were transferred electrophoreti￾cally onto the polyvinylidenedifuoride (PVDF) membrane
using Turbo-Mini PVDF Transfer Packs (Cat #1704156,
Bio-Rad Laboratories Inc, USA) with Trans-Blot® Turbo
Transfer System (Cat # 1704150; -Rad Laboratories Inc,
USA). The membranes were then pre-blocked with Tris￾bufered saline [(10 mM Tris-base pH-7.5, 150 mM Nacl
(TBS) containing non-fat dried milk (5.0%) and Tween-
20 (0.1%)] for 3 h at room temperature. Membranes were
incubated with the any one of the primary antibody, rabbit
monoclonal anti-C-fos (CST—Cat # 4384, 1:2000)/rabbit
monoclonal anti-phospho-C-fos (Ser 32) (CST—Cat # 5348,
1:2000)/rabbit monoclonal anti-PP-1α (CST—Cat # 2582,
1:2000)/rabbit polyclonal anti-p300(SC-584; 1:500)/rabbit
polyclonal mature anti-BDNF [SC-546 (N-20); 1:500] or
rabbit polyclonal anti-β-actin (SC-130656; 1:2000) antibody.
The membrane was washed with 1X TBS-T and bound anti￾bodies were detected by incubating for 4 h with goat anti￾rabbit (MERK Cat # 62110080011730; 1:2000) alkaline
phosphatase (ALP) conjugated antibody. The membrane was
washed with 1X TBS-T, and ALP activity was detected with
5-bromo-4-chloro-3-indolylphosphate disodium salt (BCIP)/
nitro-blue tetrazoliumchloride (NBT) (AP conjugate sub￾strate kit Cat # 1706432, Bio-Rad Laboratories Inc, USA).
The western blot images were acquired using Image Lab 2
software (Molecular Imager ChemiDoc XRS system, Bio￾Rad Laboratories, Inc, USA), then the trace quantity of each
band was measured and normalized with β-actin.
Statistical analysis
The signifcant diference was tested using two-way ANOVA
for behavioural data and one-way ANOVA for gene expres￾sion data and subsequently Bonferroni post hoc test was
performed [Sigma Stat software (Ver 22.0)]. Data were pre￾sented as a mean±standard error of the mean (SEM), and
plotted with KyPlot (Ver 5.0).
Results
Exposure to conspecifc distress calls can impair
olfactory learning and memory
The observed behavioural response provided evidence that
the distress call afects novel odor learning in C. sphinx
(Fig. 3 During olfactory learning, the C. sphinx behavioural
response to the novel odor difered signifcantly between
groups (F4,71=82.86; P<0.001) and between behavioural
responses (F2,71= 87.18; P < 0.001), as did the interac￾tion between group × behavioural response (F8,71=9.26;
P<0.001). Bonferroni post hoc comparisons revealed sig￾nifcant diferences between groups in feeding attempts
(P<0.001) and feeding bouts (P<0.001), but not in out-fies
(P=0.84) (Fig. 3a).
Similarly, during testing, bat responses to the novel odour
were signifcantly diferent between groups (F4,71=91.62;
P < 0.001) and between their behavioural responses
(F2,71=78.92; P<0.001), as was the interaction between
group×behavioural response (F8,71=11.42; P<0.001). A
post hoc test revealed signifcant diferences between groups
in feeding attempts (P<0.001) and feeding bouts (P<0.001)
but no diference between groups in out-fies (P=1.000)
(Fig. 3b). The observed behavioural data demonstrate that
the specifc structure of the distress call possibly suppresses
novel odour learning.
Exposure to conspecifc distress calls promotes c‑Fos
expression in caudomedial neostriatum (NCM)
Expression of c-Fos in the NCM region in the experimen￾tal groups was detected, as shown in Fig. 4a. There was a
signifcant diference in the c-Fos expression level between
groups (F3,15=246.12; P<0.001). In addition, post hoc
analysis revealed that the levels of c-Fos were signifcantly
higher in bats exposed to distress (P<0.001) and PB calls
(P<0.001) than in the control group, but there was no dif￾ference between the control and bats exposed to modifed
calls (P=0.87) (Fig. 4b). Similarly, the c-Fos expression
levels were signifcantly higher in bats exposed to distress
calls (P<0.001) and PB of distress calls (P<0.001) than
in the group exposed to modifed calls. The observed c-Fos
expression in the NCM of conspecifcs suggests that expo￾sure to distress/PB of distress possibly activates neuron in
the NCM region.
Exposure to conspecifc distress calls promotes c‑Fos
expression in the amygdala
As shown in Fig. 5a, the level of c-Fos expression in the
amygdala difered signifcantly between the experimental
groups (F3,15=120.88; P<0.001). The post hoc analysis
showed that the c-Fos expression levels were signifcantly
higher in bats exposed to distress calls (P<0.001) and PB of
distress calls (P<0.001), but there was no signifcant difer￾ence between the control and bats exposed to modifed calls
(P=0.246). Interestingly, when compared with the modifed
call, the level of c-Fos expression was signifcantly higher
Fig. 3 The specifc structure of distress calls infuences the olfac￾tory learning and memory of C. sphinx conspecifcs. Behavioural
responses of C. sphinx to the novel odour during a training and b
retention tests. Data are shown as the mean±SEM, *indicates a sig￾nifcant diference (***P 0.001) with respect to comparisons within
groups (a Con vs Distress call; b Con vs PB distress call; c modifed
call vs PB distress call; d modifed call vs distress call)
in the distress call- (P<0.001)- and the PB of distress call
(P<0.001)-exposed groups (Fig. 5b). The observed expression
pattern of c-Fos in the amygdala region suggests that the call
structure specifcally activates the amygdala.
Exposure to conspecifc distress calls induces c‑Fos
expression in the hippocampus
Further analysis showed that the level of c-Fos expression
(Fig. 6a) in the hippocampus was not signifcantly diferent
between the groups (F3,15=3.12; P=0.076). The results of
the post hoc analysis demonstrated that there was no sig￾nifcant diference in c-Fos between the control and bats
exposed to modifed calls (P=0.781). In comparison, there
was no signifcant diference after the bats were exposed to
distress calls (P=0.12) or PB of distress calls (P=0.23)
compared with the trained group. Similarly, estimated c-Fos
expression levels in bats exposed to a distress call (P= 0.46)
or the PB of a distress call (P= 0.48) were not signifcantly
diferent from bats exposed to modifed calls (Fig. 6b). This
observation demonstrates that a specifc call structure dif￾ferentially induces c-Fos expression in the hippocampus;
however, the observed diferences in expression were not
signifcantly diferent.
Exposure to conspecifc distress calls selectively
regulates protein phosphatase‑1 (PP‑1) and p300
expression in the hippocampus
A contrasting pattern of PP-1 and p300 expression was
observed between the groups (Fig. 7a). The estimated level
of PP1 reached significance with respect to the groups
(F3,15  =285.48; P<0.01). The post hoc analysis also dem￾onstrated that PP-1 expression was signifcantly higher after
the bats were exposed to distress calls (P<0.001) and PBs
of distress calls (P<0.001) than after the bats were exposed
to the trained control group. In comparison, in the modifed
call-exposed group, the PP-1 expression levels were signif￾cantly higher in both the distress call (P<0.001)- and the
PB of the distress call (P<0.001)-exposed groups, but there
Fig. 4 The specifc structure of the distress call promotes c-Fos
expression in the caudomedial neostriatum (NCM) of C. sphinx.
Bats were allowed to listen to the distress call/PB of the distress call/
modifed call and were trained to a novel odour after 15 min. a Rep￾resentative western blots showing the expression patterns of c-Fos in
the NCM regions of experimental groups. Estimated levels of b c-Fos
expression showing signifcant diferences (*P<0.05; ***P<0.001)
between groups (a Con vs Distress call; b Con vs PB distress call; c
distress call vs modifed call; d PB distress calls vs modifed call; e
Con vs Modifed call)
Fig. 5 Conspecifc distress calls with specifc structures induce the
expression of c-Fos in the amygdala of C. sphinx. Bats were allowed
to listen to the distress call/PB of the distress call/modifed call and
were trained to a novel odour after 15 min. a Representative western
blots showing the expression patterns of c-Fos in the amygdala in the
experimental groups. Estimated levels of b c-Fos expression showing
signifcant diferences (*P<0.05; ***P<0.001) between groups (a
Con vs distress call; b Con vs PB distress call; c distress call vs modi￾fed call; d PB distress calls vs modifed call; e Con vs modifed call)
was no signifcant diference between the control and modi￾fed call-exposed groups (P=0.084) (Fig. 7b). In parallel,
the estimated expression levels of p300 were signifcantly
diferent between groups (F3,15=132.46; P<0.001). Inter￾estingly, post hoc analysis showed no signifcant difer￾ence between the control and modifed call-exposed groups
(P=0.652). In comparison, p300 levels were signifcantly
lower in the distress call (P<0.001) and the PB of the dis￾tress call (P<0.001) groups compared to the control group.
Similarly, when compared with the modifed distress call
group, the p300 levels were signifcantly lower in the distress
call (P<0.001) and the PB of the distress call (P<0.001)
groups (Fig. 7c). Evidently, PP1 and p300 were diferentially
altered in the hippocampus of conspecifcs by the specifc
structure of the distress call.
Exposure to conspecifc distress calls suppresses
the novel odour training‑induced expression
of brain‑derived neurotrophic factor (BDNF)
and its receptor tyrosine kinase (Trk) B1
in the hippocampus
Further, the BDNF expression (Fig. 8a) levels were sig￾nifcantly diferent between the groups (F3,15  = 305.57;
P<0.001). Post hoc analysis revealed that the BDNF levels
in the distress call (P<0.001)- and the PB of the distress
call (P<0.001)-exposed groups were signifcantly lower
than that of the control group. Furthermore, the observed
diferences were signifcantly lower in bats exposed to dis￾tress calls (P<0.001) and the PB of distress calls (P<0.001)
compared to the modifed call-exposed group. However, the
expression was not signifcantly diferent between the con￾trol and modifed call-exposed groups (P= 0.068) (Fig. 8b).
Expression of its receptor TrkB1, which responds to the acti￾vation of BDNF, was also signifcantly diferent between
Fig. 6 The specifc structure of the distress call induces the expres￾sion of c-Fos in the hippocampus of C. sphinx. Bats were allowed
to listen to the distress call/PB of the distress call/modifed calls and
were trained to a novel odour after 15 min. a Representative western
blots showing the expression patterns of c-Fos in the hippocampus
of the experimental groups. Estimated levels of b c-Fos expression
showing no signifcant diference in any comparison between groups
Fig. 7 Conspecifc distress calls with specifc structures diferen￾tially regulate the expression levels of PP1/P300 in the hippocam￾pus of C. sphinx. Bats were allowed to listen to the distress call/PB
of the distress call/modifed call and were trained to a novel odour
after 15 min. a Representative western blots showing the expression
patterns of PP1 and P300 in the hippocampus of the experimen￾tal groups. Estimated levels of b PP1 and c P300 showing contrast￾ing patterns of expression and signifcant diferences (***P<0.001)
between groups (a Con vs Distress call; b Con vs PB distress call; c
distress call vs modifed call; d PB distress calls vs modifed call; e
Con vs modifed call)
674 Journal of Comparative Physiology A (2021) 207:667–679
1 3
the groups (F3,15 =2 86.17; P<0.001). Interestingly, post
hoc analysis revealed signifcantly lower expression in indi￾viduals exposed to distress calls (P<0.001) and PB distress
calls (P<0.001) than in the control group. Similarly, the
expression of TrkB1 levels in bats exposed to distress calls
(P<0.001) and PB distress calls (P<0.001) were signif￾cantly lower than that in the modifed call-exposed group.
However, the diference in expression between the control
and modified call-exposed groups reached significance
(P<0.001) (Fig. 8c). The expression pattern of BDNF in the
experimental groups showed that diferent call structures dif￾ferently activated the expression of BDNF and its receptor.
Discussion
Earlier studies from my laboratory demonstrated that C.
sphinx produces distress calls, which trigger call-specifc
changes in the behavioural, physiological/autonomic state
of the receiver (Ganesh et al. 2010b; Mariappan et al. 2013,
2016). The distress call of C. sphinx could be a unique syl￾labi possibly emitted with a distinct temporal pattern (Bohn
et al. 2008), could be context-specifc and could encode an
emotional state (Pfalzer and Kusch 2003; Carter et al. 2015;
Walter and Schnitzler 2019). This study was designed to test
whether exposure to conspecifc distress calls afects olfac￾tory learning in C. sphinx.
Conspecifc distress call and olfactory learning
After the bats were exposed to the conspecifc distress call/
PB of the distress call, they made few feeding attempts and
feeding bouts compared to the control/bats exposed to the
modifed call. Distress calls are known to induce neural
responses in the auditory cortex (Martin et al. 2017), amyg￾dala (Gadziola et al. 2016) and hypothalamic–pituitary–adre￾nal (HPA) axis (Mariappan et al. 2013), and share functional
similarities with a human’s fearful scream (Hechavarria et al.
2020). Neuronal circuits processing social calls may shape
the receptive feld selectivity, plasticity and temporal fring
pattern (Naumann and Kanwal 2011; Peterson and Wen￾strup 2012), possibly due to the contribution of amygdala
circuits in aversive or appetitive learning (Namburi et al.
2015; Beyeler et al. 2016). The reconciliation of amygdala
activity also depends on the experience, i.e., aversive sound
and angry prosody (Andics et al. 2010; Leitman et al. 2010),
and raises the possibility that distress call-induced activa￾tion may be linked with threatened stimuli-mediated acti￾vation of the amygdala associated with aggression or fear
(Peterson and Wenstrup 2012; Michael 2019), anxiety-like
disorder in humans or social communications with negative
emotions (Erkin and Wagen 2007; Sengupta et al. 2018).
Therefore, the individuals exposed to distress calls/PBs of
distress calls showed fewer feeding attempts and bouts to the
novel odour during training and testing. The call sequence
of the modifed call may be within the acoustic range of C.
sphinx and activate diferent populations of neurons in the
amygdala (Gadziola et al. 2012) but may not encode stress￾ful information (Mariappan et al. 2016). Thus, bats exposed
to modifed calls learned to the novel odour and responded
during retention.
Distress call‑specifc syllable sequence induces
expression of c‑Fos in auditory circuit
The activity-dependent expression of c-Fos is the most com￾monly used marker to measure neuronal activity, and c-Fos
immunoreactivity is elevated in distinct brain regions paired
with vocalization or receiving vocal signals compared with
silent bats (Ganesh et al. 2010b; Schwart and Smotherman
2011). In this study, a higher level of c-Fos expression was
noted in the NCM of bats exposed to distress/PB distress
Fig. 8 C. Sphinx distress call with a specifc structure diferentially
regulates the expression of BDNF and its receptor TrkB1 in the hip￾pocampus of conspecifcs. Bats were allowed to listen to the dis￾tress call/PB of the distress call/modifed call and were trained to a
novel odour after 15  min. a Representative western blots showing
the expression patterns of BDNF in the hippocampus of the experi￾mental groups. Estimated levels of b BDNF and c the mRNA levels
of TrkB1, showing signifcant diferences (***P<0.001) between
groups (a Con vs distress call; b Con vs PB distress call; c distress
call vs modifed call; d PB distress calls vs modifed call; e Con vs
modifed call)
calls compared to modifed calls. This can be interpreted
as showing that the specifc structure of the distress call/
PB of the distress call might promote the induction of c-Fos
in the NCM region, as in other animal models (Monbureau
et al. 2015), and the expression of c-Fos has been shown to
play key roles in synaptic plasticity, learning and memory
(Luscher Dias et al. 2016; Mukilan et al. 2018; Kanemoto
et al. 2020). Further exposure to distress calls/PBs of distress
calls increased c-Fos expression compared to the modifed
call group. This is possibly due to the specifc feature of
a distress call that activates auditory cortex neurons and
demonstrates experience-dependent plasticity and learning
in other bat species (de Hoz et al. 2018; Hörpel and Fir￾zlaf 2019), other animal models and humans (Kanwal and
Rauschecker 2007; Behler and Uppenkamp 2020). Earlier
studies in other animal models and in humans demonstrated
that in the amygdala receiving auditory input (Sander and
Scheich 2001), a stressful auditory signal induces the expres￾sion of c-Fos in the amygdala and develops fear memory
associated with the auditory signal (Nauman and Kanwal
2011; Chaaya et al. 2019). It is well known that amygdala
neurons respond to social calls selectively, and their activity
is positively correlated with social interactions (Katayama
et al. 2009), including fear-related signals in bats (Nauman
and Kanwal 2011) and humans (Whalen et al. 2004). In
other models, multimodal brain cluster analysis suggest that
movement (fight, vocalization, body movement) associated
motor control circuits and auditory associated motor control
circuits are under general cerebral motor system. However,
the activation of movement associated motor circuits did
not induce IEGs expression in the hippocampus, and higher
auditory brain regions (Nelson 1996; Feenders et al. 2008).
Therefore, observed elevated level of c-Fos expression in
NCM and amygdala is possibly due to auditory input, which
activates emotional reactivity, fear learning and memory
circuits embedded in the amygdala (Namburi et al. 2015;
Beyeler et al. 2016) and humans (Wiethof et al. 2009).
Distress call syllable sequences are specifcally shifts
expression of c‑Foss, protein phosphatase‑1 (PP‑1)
and p300 in amygdala, and hippocampus
Odor-associated stimuli are connected through multiple
regions of the brain, and input from sensory neurons in
the olfactory bulb transmits olfactory information to other
brain regions, including the olfactory bulb, amygdala and
hippocampus (Wilson et al. 2004; Sosulski et al. 2011).
The amygdaloid complex is known to connect two sensory
systems (i.e., auditory and olfactory) that are sensitive to
stress, which can lead to impairments in learning and mem￾ory (Soudry et al. 2011; Kiyokawa et al. 2012). Distress
calls induce neuronal activation in the amygdala, result￾ing in an increase in neuronal excitability that may recall
the fear memory of C. sphinx associated with distress calls
(Chattarji et al. 2015) or may suppress exploratory behaviour
to the novel odor, possibly by inhibiting amygdala output
(Colas-Zelin et al. 2012). Thus, the bats exposed to distress
calls/PBs of distress calls showed fewer feeding attempts
and bouts towards novel odors. However, the modifed call
acoustic properties are not similar to the conspecifc distress
call, are unfamiliar to the bats (Mariappan et al. 2016), and
may not provide specifc information to the receiver (Fallow
et al. 2013). Supporting earlier observations, in this study,
individuals exposed to the modifed calls learned the novel
odor and responded during retention.
The reciprocal interaction between the amygdala and the
cortex/hippocampus has been known to alter behavioural
metaplasticity by aversive/threatening stimuli (Schmidt et al.
2013; Saha et al. 2020). Therefore, the transcription of tar￾get genes was examined in the hippocampus. The training￾induced expression of p300 was signifcantly lower in the
hippocampus of bats exposed to distress calls/PBs of dis￾tress calls. However, p300 expression levels were elevated
in control/bats exposed to modifed calls after the bats were
trained with novel odor, which suggests that p300 was acti￾vated by novel odour stimulus (Oliveira et al. 2011). This
observation suggests that novel odor-induced stimuli play
an agnostic role in p300 expression in the hippocampus
and may further activate the transcription of target genes
(Oliveira et al. 2011). Conversely, transcription of CREB￾targeted genes is suppressed by protein phosphatases (PP1α,
PP2A) and phosphorylation of CREB (Koshibu et al. 2009;
Mauna et al. 2011), thereby reducing synaptic plasticity. In
line with earlier reports, the level of PP1α in the hippocam￾pus was signifcantly low in bats exposed to modifed calls
and similar to control bats, but not in bats exposed to distress
calls/PBs of distress calls (Koshibu et al. 2009; Mauna et al.
2011). These results suggest that reduction of the PPs and
elevated expression levels of CBP/P300 could be linked with
bat learning and responses to the novel odor.
Distress call syllable‑specifc sequences regulates
olfactory learning
Activity-dependent expression of BDNF and its tyrosine
receptor kinase B1 (TrkB1) has been known to regulate syn￾aptic plasticity, LTP and memory formation (Sakata et al.
2013; Zhong et al. 2016). In this study, the level of BDNF
was signifcantly lower in bats exposed to distress calls/PBs
of distress calls than in control bats/bats exposed to modi￾fed calls. The observed results suggest that exposure to the
distress call/PB of the distress call activates the amygdala
and possibly modifes amygdala activity and plasticity in
other brain regions, including the hippocampus (Schmidt
et al. 2013), which may alter metaplasticity. Thus, exposed
bats showed few feeding attempts and bouts to novel odour
during training and testing. Supporting this observation, ear￾lier studies in other models reported that exposure to stress
reduces BDNF expression in the hippocampus (Makhathini
et al. 2017). Elevated levels of BDNF in the hippocampus
after training with the novel odor in the control/modifed
call-exposed group could be correlated with a greater num￾ber of responses to the novel odor (Sakata et al. 2013; Tong
et al. 2018). In addition, it has been demonstrated that ele￾vated levels of BDNF in the hippocampus act through its
high-afnity receptor TrkB1 to trigger synaptic plasticity/
long-term memory (Nasrallah et al. 2019). The expression
level of TrkB1 was signifcantly higher in the trained control
and the bats exposed to modifed calls than in the distress
call/PB of the distress call-exposed groups. The observed
behaviours of the control/modifed call-exposed groups
are in line with earlier reports stating that overexpression/
activation of TrkB1 in the hippocampus enhances learning
and memory (Karpova et al. 2014; Nasrallah et al. 2019).
Reduced levels of TrkB1 possibly disrupt synaptic plastic￾ity; therefore, individuals exhibit very low feeding bouts to
novel odors, which is similar to other animal models that
have shown impaired learning and memory (Badowska￾Szalewska et al. 2010; Ren et al. 2015). These results suggest
that BDNF and TrkB1 expression may be tightly controlled
by input from the amygdala, which may act as a master
switch in learning and memory in diferent environmental
stimuli and biological contexts.
Conclusion
Linking with other studies, distress calls are functionally
similar to a human’s fearful screams, and the specifc struc￾ture of the distress call activates the NCM and the amygdala
of the receiver. The acoustic structure of the distress call/PB
of the distress call may activate neuronal circuits involved
in aggressive, fearful and afliative behaviour embedded in
the amygdala, which signifcantly induces c-Fos expression
compared to bats exposed to modifed calls. Interestingly,
the reciprocal interaction between the amygdala and the
cortex/hippocampus could be diferentially regulated by the
distress call/PB of the distress call/modifed call, which fur￾ther selectively drives the expression of PP1, CBP/P300 and
BDNF and its receptor TrkB1 following training to the novel
odor. Therefore, the exposure of C. sphinx to distress calls/
PBs of distress calls infuences conspecifc olfactory learn￾ing and memory. Furthermore, this model can be used to
understand the fear/threatened stimuli related to social com￾munication-induced learning and memory disorders (Fig. 9).
Fig. 9 Schematic representation
of the intracellular signalling
molecules (Egr-1—early growth
response 1 gene; TOE 1—target
of Egr 1; 5-Hydroxytryptamine
(5-HT-serotonin); ACTH—
adrenocorticotropic hormone;
CORT—corticosterone; DA—
dopamine; DAT—dopamine
transporter; GR—glucocorti￾coid receptor; SRC-1—ster￾oid receptor co-activator;
D1DR—dopamine receptor;
Nurr-1—nuclear receptor￾related factor-1; TH—tyrosine
hydroxylase; PP1α—protein
phosphatase 1; BDNF—brain￾derived neurotrophic factor;
TrkB—tropomyosin receptor
kinase B) altered by exposure to
the distress call or the modifed
distress call. Observed changes
in this study marked with *
green arrows indicate the efects
of modifed calls, and red
arrows indicate the efects of
distress calls
Acknowledgements KER thank the anonymous reviewer for their sug￾gestions that improved this manuscript. This Project is fnancially sup￾ported by Tamil Nadu State Council for Higher Education (TANSCHE)
and Rashtriya Uchchatar Shiksha Abhiyan (RUSA) 2.0-Biological Sci￾ences. The Department of Animal Science is supported by Depart￾ment of Science and Technology (DST)-Fund for Improvement of S&T
Infrastructure (FIST) and DST-Promotion of University Research and
Scientifc Excellence (PURSE). Experimental protocol used in this
study was approved by Bharathidasan University Wild Animal Ethics
Committee (03/AS/BUWAE/2008), which was in compliance with the
laws in India. Experiments were designed to minimize the number of
animals used.
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