European Journal of Pharmacology 807 (2017) 71–74

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Behavioural pharmacology

Effects of chronic bryostatin-1 on treatment-resistant depression in rats MARK

b a a,⁎
Daniel L. Alkon , Jarin Hongpaisan , Miao-Kun Sun
a
Blanchette Rockefeller Neurosciences Institute, 8 Medical Center Dr., Morgantown, WV, USA
b
NeuroDiagnostics LLC, Rockville, MD, USA

A R T I C L E I N F O A BS T RAC T

Chemical compounds studied in this article: Despite over a half-century's intensive research worldwide, the currently available antidepressants remain sub-
Imipramine (PubChem CID:8228) optimal. Therapeutic options for treatment-resistant depression, for instance, are rather limited. Here, we found
Bryostatin-1 (PubChem CID: 5280757) that rats exhibited a lasting treatment-resistant depressive immobility in response to open space swim test at a
Keywords: high intensity of induction. The induced depressive behavior is associated with a dramatic impairment in spatial
Bryostatin 1 learning and memory. Both the depressive immobility and impairment in spatial learning and memory are
Depression sensitive to a period of chronic treatment with bryopstatin-1, a relatively selective activator of protein kinase Cε.
Refractory depression Bryostatin-1-like analogues therefore might have therapeutic values for the treatment of treatment-resistant
Spatial learning and memory
depression.
Refractory depression

1. Introduction
Major depressive disorder is one of the most prevalent forms of
mental illness in humans, with prevalence rate of 16.2% in the United
States (Kessler and Bromet, 2013). The disorder is associated with high
morbidity and risk of premature mortality (Nestler et al., 2002; Ursano
et al., 2015) and is predicted to be the leading cause of disability in
Western countries by 2030 (Mathers and Loncar, 2006). Despite
intensive research worldwide since antidepressant drugs were intro-
duced into clinical practice over a half-century ago, there has been
almost no progress in developing new drugs through novel targets
(Mathew et al., 2008; Belzung, 2014). At least 30% of patients with
major depressive disorder do not show effective therapeutic benefits to
the available antidepressants (Ionescu et al., 2015). The non-respon-
sive cases were termed as treatment-resistant depression, or refractory
depression (Fava, 2003), and remain a tough clinical challenge today.
Recently, blocking glutamatergic system in the hippocampus (Lefebvre
et al., 2017), such as by using a low dose of ketamine, a potent non-
competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, has
been shown to produce rapid-onset antidepressant efficacy (Wang
et al., 2015; Bobo et al., 2016). The glutamatergic system in the brain
is widely involved in cognition and regulation of behaviors. The
potential problems with these antagonists as antidepressants include
the facts that their intended selectivity for targeting negative memory is
only partial at most and that the efficacy of ketamine tends to wear off
in time, in addition to other adverse effects (Wan et al., 2015). Long-
term impacts on memory operation and retrieval also need to be
carefully evaluated, especially when a continuation-phase administra-
tion of i.v. ketamine infusions is necessary (Vande Voort et al., 2016).
On the other hand, there is substantial evidence supporting acute
efficacy of electroconvulsive therapy in treatment-resistant depression.
Unfortunately, memory loss and the need for repetitive treatments
preclude its use as a long-term treatment option.
We have observed that when facing an open space, no-escape water
environment, depressive immobility would quickly dominate in ro-
dents' behavior after an initial (failed) intensive search for an escape
(Sun and Alkon, 2013). The impacts of the open space swim test on
behavior are age-, sex-, and intensity (duration)-dependent (Sun and
Alkon, 2008). In the present study, we directly evaluate whether
induction at high intensity (longer duration) would lead to depressive
behavior mimicking treatment-resistant depression and whether
chronic bryostatin-1 treatment, if induced, might be effective against
the treatment-resistant depressive immobility in animals. It has been
well established that protein kinase C is involved in the regulation and
control of many types of memories (Alkon et al., 2005). Bryostatin-1, a
relatively specific protein kinase C (PKC)ε activator, enhances the
expression and activity of brain-derived neurotrophic factor in the
hippocampus (Sun et al., 2015) and reverses the induced treatment-
resistant depressive immobility. Furthermore, our results show that the
induced depressive behavior mimics the human disease, exhibiting a

⁎
Corresponding author.
E-mail address: masun@brni.org (M.-K. Sun).

http://dx.doi.org/10.1016/j.ejphar.2017.05.001
Received 3 January 2017; Received in revised form 11 April 2017; Accepted 1 May 2017
Available online 02 May 2017
0014-2999/ © 2017 Elsevier B.V. All rights reserved.
D.L. Alkon et al.
dramatic impact on the ability of learning and memory. Based on the
results presented above, chronic bryostatin-1 might have therapeutic
value for the treatment of treatment-resistant depression.
2. Materials and methods
2.1. Animals and drug treatment
Adult male Wistar rats (175–200 g) were housed two per cage in a
temperature-controlled (20–24 °C) room for at least a week prior to
experimentation, allowed free access to food and water, and kept on a
12-h light/dark cycle.
Chronic effects of imipramine and bryostatin-1 on induced im-
mobility were evaluated, starting 24 h after the last trial of the 3 open
space swim test trials. Imipramine (Sigma), a prototypical tricyclic
antidepressant, was chronically used as a standard antidepressant
(15 mg/kg, i.p., daily for 5.5 weeks). Bryostatin-1 (Sigma) was admi-
nistered at 20 μg/m2 (tail i.v., 2 doses/week for 5.5 weeks). The dose of
bryostatin-1 was based on our preliminary dose-response studies that
smaller doses were ineffective against disorders-induced synaptic and
cognitive impairments. Non-treated groups received the same volume
of vehicle at the same frequency.
All procedures were conducted according to National Institutes of
Health Animal Care and Use Committee guidelines, and were approved
by the Ethical Committee of the Institute.
2.2. Open space swim test: induction of depressive behavior
Rats were placed individually in a round pool, which has a diameter of
152 cm and height of 60 cm and was filled with 40 cm H2O (24 ± 1 °C).
No escape was provided in these trials during the test. Rats were free to
swim (or not to swim) for 24 min and then removed and returned to their
home cages after drying. The observer(s) were obscured from sight of the
rats during the trials, but were able to observe the animals' behaviors on a
PC monitor during the trials. The same procedure (24 min session/day)
was followed 24 h later for 2 more days. The induction of depressive
immobility is age- and sex-dependent (Sun and Alkon, 2008). The session
of 24 min represents 2 fold of the duration (thus intensity; Sun and Alkon,
2008) needed to induce a significant depressive immobility in the male
rats at the age when they were tested. The swimming/drifting path was
recorded with a video-tracking system (Poly-Track Video Tracking
System, San Diego Instruments, Inc.). The distance moved (mobility)
includes all the distance moved during the entire 24 min, as caused by
active swimming/searching as well as slow drifts. We have previously
reported that the swim activity monitored accurately reflects duration of
mobility and that the maximum effect is induced on the third trial (Sun
and Alkon, 2003). For evaluating a long-term impact, the induction was
followed by ‘monitoring’ sessions (24 min) at a frequency of 1/week (Sun
and Alkon, 2004).
2.3. Spatial water maze learning and memory task
Effects of the induced depressive behavior on spatial learning and
memory were evaluated with the Morris water maze task, in a 152 cm-
diameter pool (24 ± 1 °C). The maze was divided into four quadrants.
Control rats were allowed to swim for 2 min in the pool without a
hidden platform, the same day when the other groups were on their last
monitoring session. Twenty-four h later, all rats were trained for 2
trials/day for 4 days to find a hidden platform (9 cm diameter). The
platform was centered in one of the quadrants and submerged about
2 cm below the water surface. At the start of all trials, rats were placed
in the water facing the maze wall, using different starting positions, and
allowed to swim until they found the platform, where they remained for
20 s. Any rat that failed to find the platform within 2 min was guided
there, with the maximum latency of 120 s scored. The swimming path
was recorded by a video-tracking system, which computed latency to
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European Journal of Pharmacology 807 (2017) 71–74
find the platform, distance swum, heading angle, and percentage of
time spent in the quadrants.
After the training trials, a probe trial of 60 s was given 24 h after the
last trial, with the platform removed, to assess memory for platform
location by the distance swum in the quadrants.
2.4. Visible platform test
After the probe trial of the water maze task, rats were tested on a
visible platform task (a non-spatial task; with the platform marked with
a pole that protruded 9 in. above the water surface but at a new
location) to evaluate their sensorimotor ability and motivation for an
escape. The latency to reach the visible platform was recorded and
compared between different groups.
2.5. Statistic analysis
Statistical analysis was performed using the analysis of variance (a
two-way ANOVA), followed by Newman-Keuls multiple comparisons
test, wherever appropriate. P < 0.05 was considered significant.
3. Results
3.1. Open space swim test induces depressive immobility in rats
The open space swim test (24 min/one trial/day for 3 days) induced
a significant immobility, which lasted for at least 1 week without
further sessions and weeks with separate open space swim test
monitoring episodes (1/week) after the initial 3 induction sessions
(Fig. 1). Typically, rats would initiate active swim in the first min and
gradually developed into intermittent periods of immobility, which
lasted longer and longer as the session continued. Without an effective
treatment, a rat in the immobility phase did not make any movements
other than those just sufficient to keep its head above the water surface
(Fig. 1 inset), a characteristic behavior of depression similar to those
reported in the forced swimming test, as we described previously (Sun
and Alkon, 2004). Imipramine, as the standard antidepressant to
determine the sensitivity to antidepressant treatment, and bryostatin-
1 were administered chronically to evaluate their impacts on the
induced immobility. The results after the initial 3 induction sessions
showed a significant group difference with the treatments (group data
points 4–8; F2,191=10.12, P < 0.001), whereas the initial OSST sessions
induced similar effects on the distance traveled among the groups (in
the first three sessions: F2,71=0.668, P > 0.05). The chronic treatment
with imipramine did not reduce the immobility as compared with the
vehicle group (F1,127=0.892, P > 0.05), indicating a depressive behavior
with the treatment-resistant profile. Bryostatin-1, on the other hand,
was effective in reducing the mobility (F1,127=12.033, P < 0.001;
compared with the vehicle group), although 2–3 weeks of treatment
were needed to produce the therapeutic effects, which lasted 2.5 weeks
after the last dose (the longest duration measured; Fig. 1).
3.2. Effects of induced depressive behavior on spatial learning
One day after the last monitoring session (Fig. 1), we tested the
ability of three groups of rats: vehicle-induction, induction-Bry, and
control (age-matched but without the induction), in spatial learning
and memory, using the hidden-platform water maze. As shown in
Fig. 2A, the speed and the extent of the learning were significantly
different among the groups (F2,191=15.431, P < 0.001), indicating that
spatial learning in the rats after depressive behavior induction was
significantly impaired, severer than the impairment associated with
shorter session-induced depressive immobility (Sun and Alkon, 2004).
Chronic bryostatin-1, however, essentially rescued rats' ability in
spatial learning (F1,127=0.912, P > 0.05; compared with the control
without the behavioral induction).
D.L. Alkon et al. European Journal of Pharmacology 807 (2017) 71–74

Fig. 1. Chronic bryostatin-1 treatment produces a lasting therapeutic effect on the induced treatment-resistant depressive immobility. The mobility was induced in a 24-min OSST
session (1/day) for 3 consecutive days, followed by ‘monitoring’ sessions (1/week). The agents were administered one day after the last induction session for 5.5 weeks. It took weeks of
treatments with bryostatin-1 for observing the full therapeutic effects, while the imipramine treatment for same period had no effects. The swimming distance in the first trial session
(arbitrary units in OSST: 23,279.21 ± 773.56 [approximately 179 ± 5.9 m]; OSST/Imi: 23,526.31 ± 725.04; and OSST/Bry: 23,671.24 ± 698.25) differed insignificantly among the
groups (F2,23=0.0731, P > 0.05) and was calculated as 100% in each rat. Data are represented as the mean ± S.E.M. Bry: bryostatin-1; Imi: imipramine; OSST: open space swim test.
Inset: a photo taken during induced non-searching immobility (with eyes closed).

induction with chronic bryostatin-1 treatment spent more time search-

ing in the target quadrant (F2,23=6.719, P < 0.001; Fig. 2B). The results
reveal that chronic brystatin-1 recovered the rats’ ability in spatial
memory recall after the behavioral induction (F1,15=0.918, P > 0.05;
compared with the control).

3.3. Effects of induced depressive behavior on a visible platform test

Measurements of mobility and escape activity also depend on basic

Fig. 2. Effects of induced depressive behavior on spatial learning and memory in rats. A:
Impairment of spatial learning by induced depressive behavior. The water maze learning
started 1 day after the last monitoring session. The learning impairment was sensitive to
bryostatin-1 treatment, administered during the 5.5 weeks after the initial induction
phase. No drug was administered during the days when the rats were performing the
learning and memory task. Each point represents the mean value ( ± S.E.M.) from 8 rats
per group. B: Results of the memory probe test, 24 h after the last water maze learning
trial, showing the target quadrant ratio (target quadrant distance over the distance
average in the remaining quadrants). “*” (P < 0.05) and “NS” (P > 0.05) mark the level of
significant differences when compared with the “Control” group. C: The induced
depressive behavior and bryostatin-1 administration did not affect the ability of the rats
to sense and reach the visible platform, a non-spatial test. Data are represented as the
mean+S.E.M. “NS” indicates an insignificant difference when compared with the “OSST”
group. Bry: bryostatin-1; Imi: imipramine; OSST: open space swim test.
Memory retention was evaluated after the training. Quadrant tests
24 h after the last training trial revealed that the rats after the
depressive behavior induction did not show significant preference for
the target quadrant, where the platform was previously placed during
the training. The control rats and the rats after depressive behavior
functions of the tested subjects: their sensorimotor activity and
motivation for an escape. Therefore, the sensorimotor activity and
escape motivation of rats were tested in a visible platform test, in which
no learning is involved. The escape latency to a visible platform,
however, did not differ among the groups tested (after the water maze
spatial learning: F2,23=0.362; P > 0.05, Fig. 2C), indicating that the
depressive behavior induction or treatment did not significantly affect
their sensorimotor ability or escape motivation. Thus, the behavioral
changes observed cannot be attributed to changes in non-specific
sensorimotor ability and in basic motivation for an escape. During
the experimental periods, no rats showed any apparent sign of
discomfort, hypervigilance or easy startle.
4. Discussion
The primary objective of this study was to investigate whether the
open space swim test when delivered at high intensity would induce
treatment-resistant depression and whether such depressive behavior,
if observed, would be sensitive to chronic treatment with PKCε
activators. Treatment-resistant depression is defined as a lack of
symptomatic response to adequate first-line pharmacological therapy
(Trivedi et al., 2006). The essential criterion used in our study is a lack
of behavioral response to a clinically effective antidepressant treat-
ment, especially when treated chronically (Willner and Belzung, 2015).
The resistance is thus considered as “absolute” since the imipramine is
used at an adequate dose and for an adequate duration (Berman et al.,
1997). When tested at lower intensity (a shorter period of induction),
the type of induced immobility was associated with anhedonia (Sun

73
D.L. Alkon et al.
and Alkon, 2013), a core symptom of depression, and was however,
sensitive to acute imipramine, iproniazid, or mianserin (Sun and
Alkon, 2003), or chronic imipramine administration (Sun and Alkon,
2004), consistent with a behavioral pattern of the depressive immobi-
lity. It remains to be studied whether it would be sensitive to low dose
of ketamine (Willner and Belzung, 2015). Nevertheless, the increase in
swimming distance in the treated group is unlikely the result of a
gradually increased physical resistance, since it was not observed in the
absence of the treatment. Fatigue is unlikely to be involved in the
immobility in the test, since unloaded rats can swim for many hours
without showing physical fatigue (Dawson and Horvat, 1970).
Evidence has been provided that an effective anti-depressive treatment
relieves the rats from the OSST-induced impairments in spatial
learning and memory (Sun and Alkon, 2004), suggesting a direct
involvement of depressive behavior in the impaired learning and
memory. We have also observed that the type of the induced
depressive-like immobility was not sensitive to buspirone, an anxiolytic
(Sun and Alkon, 2004), indicating the specific nature of the responses.
The co-occurrence and association of depression and memory
impairment in humans is a striking feature of the two disorders.
Memory, especially explicit memory, is consistently impaired in
depressed patients (Ellwart et al., 2003). It is interesting that the open
space swim test intensity used in this study impaired rats’ ability to
learn and memorize the water maze task. The impairment was more
severe than the one evoked with the test at lower intensity (Sun and
Alkon, 2004). We have previously found that the impairment is not just
associated with the swimming or water environment, since similar
memory impairment was also induced in the multi-trial passive
avoidance task (Sun and Alkon, 2004). In the present study of impacts
of induced depressive behavior on learning and memory, bryostatin-1
was administered weeks before the learning and memory tasks, so that
the differences in learning and memory could not be due to an acute
effect of the agent. Our results show that the induced depressive
behavior induced with the swim sessions indeed has a dramatic
influence on subsequent learning and memory.
Many features of human major depression, such as hopeless
immobility, despair behavior, and self-injurious behavior, occur across
other species. Animal models are indispensable in clarifying patho-
physiology that underlies depression, depression-cognition interaction,
and in searching for new antidepressants. The validity of our model for
depression is indicated by emergence of non-searching immobility,
which is considered one core symptom of depressive behavior (Porsolt
et al., 1997; Cryan et al., 2002). At lower intensity (11 min for rats at an
age of 2 months), the swim induction induces depressive immobility,
which is sensitive to all three major classes of effective antidepressants,
including imipramine, and a selective serotonin reuptake inhibitor
(Sun and Alkon, 2003). We have also determined that the swim
distance reliably reflects the active swimming status of the animals
during the trial period in the test (Sun and Alkon, 2003) and with state
of anhedonia (Sun and Alkon, 2013).
The strengths of our study include the first, to our knowledge,
evaluation of the sensitivity of rats to depressogenic events at higher
intensity, which actually transformed a treatment-sensitive type of
depressive behavior to a treatment-resistant one. However, it remains
to be studied whether OSST induces higher stress intensity than other
models (e.g. learned helplessness, repeated immobilization, etc.) used
to induce the treatment-resistant depression. Furthermore, our results
suggest that chronic treatment with bryostatin-1-like agents might
have therapeutic value for the treatment-resistant depression, provid-
ing a lasting therapeutic effect on the disorder.
Authorship contributions
Participated in research design: Alkon and Sun.
Conducted experiments: Hongpaisan and Sun.
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European Journal of Pharmacology 807 (2017) 71–74
Performed data analysis: Sun.
Wrote or contributed to the writing of the manuscript: Alkon,
Hongpaisan, and Sun.
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