저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게
l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다:
l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다.
l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다.
저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다.
Disclaimer
저작자표시. 귀하는 원저작자를 표시하여야 합니다.
비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다.
변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.
이학석사 학위논문
강제적 수영에 의한 장기 공간 기억 향상에 관한 연구
Studies on long-term spatial memory enhancement by forced swim
2020년 2월
서울대학교 대학원
뇌과학협동과정
정 준 현
강제적 수영에 의한 장기 공간 기억 향상에 관한 연구
Studies on long-term spatial memory enhancement by forced swim
지도교수 강 봉 균
이 논문을 이학석사 학위논문으로 제출함
2019년 12월
서울대학교 대학원
뇌과학협동과정
정 준 현
정 준 현의 이학석사 학위논문을 인준함
2019년 12월
위 원 장 김 형 (인) 부위원장 강 봉 균 (인) 위 원 이 용 석 (인)
Studies on long-term spatial memory enhancement by forced
swim
Advisor. Professor Bong-Kiun Kaang
A dissertation submitted to the Graduate Faculty of Seoul National University in partial fulfillment of the requirement for the Degree of
Master of Science
December 2019
June-Hyun Jeong
Graduate School of Natural Sciences Seoul National University
Interdisciplinary Program in Neuroscience
Table of Contents
Page
List of Figures ……….2
Abstract ………...4
Introduction ………6
Materials and Methods ………9
Results ……….15
Figures ……….21
Discussion ………..39
References ………43
Abstract in Korean ……….49
List of Figures
Page
Figure 1. Stress-inducing forced swim paradigm was optimized to make sure long-term spatial memory enhancement occurs in the contextual fear conditioning test.
………21
Figure 2. The test group experiencing forced swim at the spacious cage shortly after encoding showed the highest freezing level 24 hr later.
………23
Figure 3. Forced swim paradigm performed shortly before or after encoding successfully improved spatial object recognition in the object location memory test.
………25
Figure 4. Contextual fear conditioning induced higher neuronal activity in CA1 than forced swim (collaborated with Dae-Hee Han and Jun- Yeong Beak).
………27
Figure 5. All test animals affected by contextual fear conditioning, forced swim, or forced swim + CFC showed similar neuronal activity in DG or CA3 (collaborated with Dae-Hee Han and Jun-Yeong Beak).
………29
Figure 6. Forced swim induced higher neuronal activity in LC than contextual fear conditioning (collaborated with Jun-Yeong Beak).
………31
Figure 7. PBS infusion in CA1 before encoding did not disrupt the forced swim effect on spatial memory in the contextual fear conditioning test.
………31
Figure 8. Long-term spatial memory facilitated by forced swim was more sensitive to the dopamine D1/D5 receptor antagonist (SCH) than the β-adrenergic receptor antagonist (Prop).
………33
Figure 9. Forced swim paradigm rescued long-term spatial memory in the contextual fear conditioning test despite the Prop infusion.
………35
Abstract
Studies on long-term spatial memory enhancement by forced swim
June-Hyun Jeong Interdisciplinary Program in Neuroscience The Graduate School Seoul National University
It is important to distinguish special moments from everyday memory and transform the former into long-term memory for survival. For this purpose, multiple neuromodulators are released from various brain regions in response to abnormal or uncertain events and employed to strengthen memory retention.
Here, we observed that wild-type mice experiencing forced swim shortly after encoding achieved long-term spatial memory enhancement in both contextual fear conditioning and object location recognition tests. Then, we found that the forced swim event induced
neuronal activity in dorsal hippocampus and locus coeruleus (LC) by staining c-Fos proteins with immunohistochemical techniques.
Finally, we determined that the memory retention facilitated by the swim had a significant susceptibility to the dopamine D1/D5 receptor antagonist rather than the β-adrenergic receptor antagonist when each drug was infused directly into the hippocampal CA1 before contextual fear conditioning. Based on the results, we suggest that dopamine is released from the LC to the hippocampus in response to the swim stimulus, which is able to boost initial memory consolidation.
Keyword : Forced swim, Hippocampus, Dopamine, Antagonist, Initial memory consolidation.
Student Number : 2018-21096
Introduction
In competitive environments, it is necessary to memorize valuable moments throughout a lifetime. The hippocampus, a part of the brain, plays a critical role in learning and memory, which covers memory acquisition, consolidation, and retrieval (Jarrard, 1993; Lorenzini, 1996). In order to provide long-lasting memory storage, the hippocampus receives multiple neuromodulators including acetylcholine, serotonin, norepinephrine, and dopamine from various brain regions because the hormones assist or mediate the process of synaptic plasticity which is closely associated with long-term memory (Palacios-Filardo and Mellor, 2019).
Although these neuromodulators often work together to modulate the long-term synaptic plasticity in the hippocampus, the role of each neuromodulator in the synaptic plasticity has been extensively studied for a long time. In particular, the locus coeruleus (LC) has been investigated by many researchers because the region is known as the sole source of norepinephrine in the brain (Sara, 2009). Plenty of data show that the amount of norepinephrine in the hippocampus is endogenously elevated by many emotional stimuli such as forced swim, foot shock, fox urine, same-sex intruder, and restraint stress
(Cahill et al., 1994; Hu et al., 2007; Segal and Bloom, 1976; Yuen et al., 2009). In addition, the related data demonstrate that the norepinephrine increase changes glutamatergic transmission, facilitates long-term synaptic plasticity, and enhances spatial memory retention via β-adrenergic receptors (Cahill et al., 1994;
Gelinas and Nguyen, 2005; Gray and Johnston, 1987; Hu et al., 2007;
Segal and Bloom, 1976).
Recent studies, however, show that noradrenergic neurons in the LC release both norepinephrine and dopamine simultaneously in response to environmental novelty or burst optogenetic activation of the LC (Kempadoo et al., 2016; Takeuchi et al., 2016). Further, the results suggest that the dopamine release from the LC to the dorsal hippocampus, not from the ventral tegmental area (VTA) which is the presumed dopamine source in the hippocampus, strengthens spatial memory. Ironically, the norepinephrine release does not seem to contribute to the memory because the intra-hippocampal injection of the β-adrenergic receptor antagonist could not impair it.
In order to alleviate the discrepancy in which of them from the LC is crucial for memory persistence, we set out to examine the hypothesis that dopamine rather than norepinephrine is a key hormone for the memory no matter what kinds of stimuli activate the LC. Since the novelty exposure stimulus has been already known to
retention (Lemon and Manahan-Vaughan, 2006; Li, 2003), we substituted the stimulus for forced swim stress which has been known to release norepinephrine and affect memory enhancement in the LC-NE system (Giustino, 2018). Then, we tested which antagonist (the dopamine D1/D5 receptor antagonist or the β- adrenergic receptor antagonist) is more critical or risky to the long- term memory facilitated by the forced swim in the contextual fear conditioning test.
Materials and methods
Animals
Adult male C57BL/6 mice (8~12 weeks of age) were used for all experiments. All mice were kept under a 12 hr light/dark cycle and given food and water ad libitum. All behavioral tests were performed during the light phase of the cycle. All procedures were overseen under the regulations from the Institutional Animal Care and Use Committee (IACUC).
Stereotaxic surgery
Anesthesia was induced by intraperitoneal injection of the ketamine/xylazine solution (26.6 % of ketamine, 4.5 % of xylazine in saline). The animals were positioned in the stereotactic frame (World Precision Instruments). The 24-gauge microinjection steel guide cannulas (4.0 mm length, 3.0 mm distance between cannulas) were implanted into the dorsal hippocampus. The cannula implantation coordinates were AP: -1.8 mm, ML: ±1.5 mm, and DV: -1.2 mm from bregma.
Behavioral pharmacology
The 31-gauge injection cannulas were connected to microsyringes (Hamilton Instruments) in a microinfusion pump (KD Scientific) through flexible plastic tubing. The tips of the infusion cannulas were protruded 0.5 mm below the tips of the guide cannulas. 0.5 μl of drug per side was infused at 0.2 μl/min by using the pump. After the microinfusion, the infusion cannulas were stayed for a further 1 min to aid drug absorption. The drug concentrations were 21.1 mM for β-adrenergic receptor antagonist propranolol (Sigma-Aldrich), 3.1 mM for D1/D5 receptor antagonist SCH23390 (Tocris). Phosphate- buffered saline was used as a vehicle. All drugs were stored at -20
oC before use.
Forced swim
Mice swum without the opportunity to escape in a transparent plastic rectangular cage (36 cm wide X 20 cm long X 28 cm high) or a visible Plexiglas small cylinder (8.5 cm diameter X 40 cm high).
Water depth was about 20 cm, which only can permit the mice to reach the bottom with the tail. Mice were forced to swim for 5 min at 20±1 oC or 25±1 oC water. All the swim procedures were performed
in the behavioral test rooms where mice were habituated for 5 days to minimalize the effects from environmental novelty. After finishing the swim, mice were carefully dried with a paper towel and transferred back to their home cages.
Contextual fear conditioning test
Mice were conditioned 2~3 weeks after the cannula implantation surgery for recovery but unoperated mice were not on hold before the conditioning. Then, all the mice were habituated to the experimenter’s hands and the behavioral test room for 5 min on 5 consecutive days. On the conditioning day, mice were conditioned with a 0.5 mA shock of 2 sec duration after 3 min exploration in the Coulbourn chamber (Coulbourn Instruments). After the shock, mice were stayed for a further 1 min and put back to their home cages.
After 24 hr, mice were exposed to the same chamber for 3 min, and freezing levels were recorded by the Freeze View program provided by Coulbourn Instruments.
Object location memory test
Mice were habituated to the experimenter’s hands for 5 min on 3 consecutive days and an opaque Plexiglas square chamber (25 cm X 25 cm) for 10 min on 6 consecutive days. On the training day, mice were exposed to the same chamber for 10 min in which two identical objects were positioned in the back corners of the arena. After 24 hr, mice were re-exposed to the environment for 5 min in which one of the two objects was displaced. The training and test sessions were recorded by a video camera (Canon). Nose touches to the objects or orientation toward the objects within 1 cm were scored manually because automated measurements falsely counted inattentive moments of walking around the objects. The behavioral analysis was performed blind.
Brain preparation and imaging
Brains were dissected out of the cranial cavity and fixed with 4 % paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4 oC for overnight. The brains were dehydrated with 30 % sucrose in PBS for 2 days at 4 oC and the dehydrated brains were frozen in a deep
freezer at least 2 hr. The brain slices (35 μm or 50 μm in thickness) depending on the purpose of imaging were prepared with a cryostat (Leica) and mounted on a glass slide with 50 % glycerol in PBS or VECTASHIELD medium including DAPI (Vector Laboratories). The brain slices were imaged by a fluorescence microscope (Nikon) or a confocal microscope (Leica).
Immunohistochemistry
Brain slices (35 μm in thickness) prepared as previously described were washed in PBS and incubated for 1 hr at room temperature with PBST-NGS (PBS containing 0.3 % Triton X-100 and 3 % normal goat serum). To stain c-Fos proteins, the slices were then incubated for overnight at 4 oC with guinea pig anti-cFos (1:1000, SYSY) in PBST-NGS. After incubation, those were washed three times in PBST and incubated again for 2 hr at room temperature with Alexa 488 goat anti-guinea pig (1:400, Invitrogen) in PBST- NGS. The sections were rinsed three times in PBST again and mounted on a glass slide with the VECTASHIELD medium including DAPI (Vector Laboratories).
Image analysis
Imaris software (Bitplane) was used to optimize confocal images and count c-Fos and DAPI positive cells. To count the cells, the cell layers of CA1, DG, and CA3 in a slice were outlined according to the DAPI signal. Because the DAPI signal did not indicate the cell layer of LC, all the LC regions were estimated or designated equally near the 4th ventricle. Then, proper equal cutoff thresholds of c-Fos and DAPI positive cell signals were applied to count the signals automatically in the regions of interest. All image analyses were performed blind.
Statistical analysis
Experimental data were analyzed statistically using GraphPad Prism 8 software. All data were shown as mean±SEM. Statistical significance was determined by unpaired t-tests. The statistical tests were two-tailed and the statistical significance was denoted by
****p < 0.0001, ***p < 0.0005, **p < 0.005, *p < 0.05.
Results
Forced swim procedures were optimized to make sure long-term spatial memory enhancement occurs in the contextual fear conditioning test.
Forced swim paradigm was first designed for analyzing a depression-like behavior and screening compounds with antidepressant activity (Porsolt et al., 1977). Later, in stress and emotional memory studies, the forced swim test paradigm was used as one of the acute stress to induce the increase of glutamatergic transmission in the prefrontal cortex (PFC) and promote working memory (Yuen et al., 2009). Besides, the swim dramatically increased norepinephrine release originated from the LC (Feng et al., 2018). Therefore, the forced swim stimulus would be a great example to determine whether dopamine and norepinephrine are always co-released from the LC and which one is a key hormone to promote memory retention.
Above all, we tested whether wild-type mice experiencing the forced swim shortly before or after contextual fear conditioning show higher freezing levels than control animals undergoing contextual fear conditioning without the swim (Fig. 1A). We set up one weak
shock (0.5 mA, 2 secs) for contextual fear conditioning to observe a dramatically increased freezing level by the forced swim stimulus.
Further, we gave environmental changes for the forced swim such as swimming spaces (Fig. 1B) or water temperature to optimize the forced swim-contextual fear conditioning test paradigm. The test animals swum at the relatively spacious rectangular cage, but not the small cylinder, achieved elevated freezing levels compared with the control without the swim (Fig. 2A). Also, the test group experiencing the swim at 20 oC water temperature had a higher freezing level than the other test group swimming at 25 oC (Fig. 2B). Last, the test group swimming before contextual fear conditioning did not get a high freezing level compared to the test group with the swim after contextual fear conditioning (Fig. 2C). To sum up, the test group that underwent the forced swim in the relatively spacious rectangular cage at 20 oC water temperature briefly after contextual fear conditioning showed the highest freezing level 24 hr later among all groups (Fig. 2A, 2B, and 2C).
Forced swim paradigm performed shortly before or after encoding successfully improved spatial object recognition in the object location memory test.
We next examined whether the swim stimulus also causes long-
term spatial memory enhancement in the object location memory test like in the contextual fear conditioning test. We allowed mice to explore a square arena with two identical objects for a memory acquisition process. The forced swim was performed briefly before or after the encoding. After 24 hr, we then displaced one of the objects and analyzed the difference index, a normalized measure indicating the relative amount of time spent exploring the displaced one (Fig. 3A). We found that the test group experiencing the swim shortly before or after memory acquisition obtained a higher difference index than the control group without the stimulus (Fig.
3B).
Forced swim and contextual fear conditioning procedures activate both dorsal hippocampus and locus coeruleus.
To determine how the forced swim stimulus was able to enhance the long-term memory in both contextual fear conditioning and object location recognition tests, we stained c-Fos proteins with immunohistochemical techniques in brain sections including two candidates, dorsal hippocampus and locus coeruleus, after forced swim and/or contextual fear conditioning stimuli (Fig. 4A). Because c-fos expression occurs when neurons fire action potentials, the
response to various stimuli (Bullitt, 1990; Kubik et al., 2007).
Therefore, we could determine that both forced swim and contextual fear conditioning stimuli induced neuronal activity in the dorsal hippocampus and the locus coeruleus (Fig. 4B, 5A, 5B, 6A).
Interestingly, the neuronal activity by the forced swim stimulus was lower than the activity by the contextual fear conditioning in the CA1 (Fig. 4B). Conversely, the activity by the contextual fear conditioning was lower than the activity by the forced swim in the LC (Fig. 6A).
Otherwise, the stimuli combining both forced swim and contextual fear conditioning generally showed similar neuronal activity to the sole stimulus, either the forced swim or contextual fear conditioning (Fig. 4B, 5A, 5B, 6A).
PBS infusion in CA1 before encoding did not disrupt the forced swim effect on spatial memory in the contextual fear conditioning test.
Before testing whether the long-term memory enhanced by the forced swim is blocked by the dopamine D1/D5 receptor antagonist, we checked that two drug guide cannulas were implanted properly to the dorsal hippocampal CA1 (Fig. 4A). Also, we made sure that the memory enhancement by the forced swim was not interrupted by the the intra-hippocampal infusion of phosphate-buffered saline (PBS) as a vehicle before contextual fear conditioning (Fig. 4B, 4C). As a
result, the operated animals that were implanted with cannulas and infused with PBS in the CA1 showed a similar phenomenon to the unoperated group (Fig. 4C).
Long-term spatial memory facilitated by forced swim was more sensitive to the dopamine D1/D5 receptor antagonist (SCH) than the β-adrenergic receptor antagonist (Prop).
Next, to test which is a key modulator from the LC to the hippocampus to strengthen the memory retention, we bilaterally infused either the β-adrenergic receptor antagonist (Prop) or dopamine D1/D5 receptor antagonist (SCH) in the CA1 before contextual fear conditioning and observed significant memory impairment in the test group injected with SCH compared to the control group with PBS (Fig. 8A, 8B). The group with the Prop infusion also tended to weaken the memory compared with the control despite no significant difference (Fig. 8B). To sum up, the contextual fear memory enhanced by the forced swim had a significant susceptibility to SCH, not Prop, although Prop somewhat impaired the memory.
Forced swim paradigm rescued long-term spatial memory in the contextual fear conditioning test despite the Prop infusion.
Previous works have shown that both antagonists, SCH and Prop, impair long-term memory when the drugs are injected in the CA1 bilaterally (Frey et al., 1990; Ji et al., 2003; Ocarroll et al., 2006).
Surprisingly, unlike SCH, the group infused with Prop and forced to swim somewhat seemed to be resistant to the drug. The group obtained a similar freezing level to the control group infused with PBS but no forced to swim (Fig. 8B). To confirm that the forced swim degrades the drug efficacy, we added a new test group infused with Prop but without swimming and determined that the Prop infusion caused a significant memory impairment when the forced swim process was omitted (Fig. 9A and 9B). These results suggest that dopamine rather than norepinephrine released by the forced swim improves the long-term memory because SCH rather than Prop sufficiently occluded the forced swim effect.
Figures
A
B
Figure 1. Stress-inducing forced swim paradigm was optimized to make sure long-term spatial memory enhancement occurs in the contextual fear conditioning test.
(A) Behavioral test paradigm (illustrated by using https://app.biorender.com).
(B) Real pictures of two different swimming places where the forced swim was performed.
A
B
C
Figure 2. The test group experiencing forced swim at the spacious cage shortly after encoding showed the highest freezing level 24 hr later.
(A) Test animals swum at the big cage achieved significantly elevated freezing levels compared with control animals that did not experience the swim stimulus (unpaired t-test; *p < 0.05). Whereas, the other test animals swum at the small cylinder did not obtain enhanced freezing levels compared with the control animals.
(B) Like the test animals swum at 20 oC water temperature, the test mice forced to swim at 25 oC water temperature also seemed to have higher freezing levels than the control but no significant difference.
(unpaired t-test; *p < 0.05, p = 0.14)
(C) Although the post-encoding swim group obtained a significantly elevated freezing level (unpaired t-test; *p < 0.05), the pre- encoding swim group did not get an increased freezing level compared with the control animals that did not get the swim stimulus.
B A
Figure 3. Forced swim paradigm performed shortly before or after encoding successfully improved spatial object recognition in the object location memory test.
(A) Behavioral test paradigm (illustrated by using https://app.biorender.com).
(B) Test mice experiencing forced swim briefly before or after encoding showed significantly enhanced spatial memory in the object location memory test compared with control animals without the forced swim stimulus (unpaired t-test; *p < 0.05 or **p < 0.005).
A
B
Figure 4. Contextual fear conditioning induced higher neuronal activity in CA1 than forced swim (collaborated with Dae-Hee Han and Jun- Yeong Beak).
(A) Stimulus-induced neuronal activity paradigm.
(B) Representative images of CA1 in 4 different groups, a: Home cage, b: Forced swim, c: CFC, d: Forced swim + CFC (left panel).
The CFC group and the forced swim + CFC group had a similar percentage of c-Fos positive cells over DAPI positive cells. However, the forced swim group obtained the ratio significantly lower than the CFC group did (unpaired t-test; **p < 0.005). Otherwise, the ratios were significantly high in all test groups compared with the home cage group regarded as a control group. (unpaired t-test; ****p <
0.0001, **p < 0.005).
A
B
Figure 5. All test animals affected by contextual fear conditioning, forced swim, or forced swim + CFC showed similar neuronal activity in DG or CA3 (collaborated with Dae-Hee Han and Jun-Yeong Beak).
(A) Representative images of DG in 4 different groups, a: Home cage, b: Forced swim, c: CFC, d: Forced swim + CFC (left panel). All test groups not including the home cage group showed a similar percentage of c-Fos positive cells over DAPI positive cells. The ratios were significantly high in the test groups compared with the home cage group. (unpaired t-test; ***p < 0.0005, **p < 0.005).
(B) Representative images of CA3 in 4 different groups, a: Home cage, b: Forced swim, c: CFC, d: Forced swim + CFC (left panel).
Like the DG region, all test groups not including the home cage group showed a similar percentage of c-Fos positive cells over DAPI positive cells. The ratios were significantly high in the test groups compared with the home cage group. (unpaired t-test; ****p <
0.0001, ***p < 0.0005).
A
B
Figure 6. Forced swim induced higher neuronal activity in LC than contextual fear conditioning (collaborated with Jun-Yeong Beak).
(A) Representative images of LC in 4 different groups, a: Home cage, b: Forced swim, c: CFC, d: Forced swim + CFC.
(B) The forced swim stimulus tended to have more c-Fos positive cells than the contextual fear conditioning did (unpaired t-test; p = 0.09). Further, the forced swim + CFC induced significantly higher neuronal activity than the CFC alone (unpaired t-test; **p < 0.005).
Otherwise, all the test animals undergoing the stimuli obtained significantly high ratios compared with the home cage group.
(unpaired t-test; ***p < 0.0005, **p < 0.005).
A
B
C
Figure 7. PBS infusion in CA1 before encoding did not disrupt the forced swim effect on spatial memory in the contextual fear conditioning test.
(A) Representative coronal section of the dorsal hippocampus.
Dashed lines indicate the sites of implanted drug guide cannulas.
(B) Behavioral test paradigm (illustrated by using https://app.biorender.com).
(C) Unoperated mice without drug infusion and guide cannula implanted animals injected with PBS as a vehicle showed similar phenomena in freezing levels. The test mice infused with PBS and forced to swim also achieved significantly enhanced memory retention compared with the control animals infused with PBS but no forced swim (unpaired t-test; *p < 0.05).
A
B
Figure 8. Long-term spatial memory facilitated by forced swim was more sensitive to the dopamine D1/D5 receptor antagonist (SCH) than the β-adrenergic receptor antagonist (Prop).
(A) Behavioral test paradigm (illustrated by using https://app.biorender.com).
(B) The dopamine D1/D5 receptor antagonist (SCH) significantly impaired the memory retention influenced by the forced swim (unpaired t-test; ***p < 0.0005). The β-adrenergic receptor antagonist (Prop) also disrupted the memory but no significant difference (unpaired t-test; p = 0.07). The difference of the freezing levels between the test animals infused with SCH and the others with Prop was also significant (unpaired t-test; *p < 0.05). Dashed line indicates the average freezing level of all groups before conditioning.
A
B
Figure 9. Forced swim paradigm rescued long-term spatial memory in the contextual fear conditioning test despite the Prop infusion.
(A) Behavioral test paradigm (illustrated by using https://app.biorender.com).
(B) The forced swim stimulus significantly increased freezing levels despite the Prop infusion (unpaired t-test; *p < 0.05). The freezing levels of the PBS infusion groups with and without the forced swim were higher than the freezing levels of the Prop infusion groups with and without the forced swim respectively (unpaired t-test; p = 0.07).
Dashed line indicates the average freezing level of all groups before conditioning.
Discussion
It has been generally considered that norepinephrine is released by unpleasant states such as emotionally stressful events, and dopamine is discharged by salience related to reward or novelty in the neuromodulatory system for a long time. Furthermore, it has been simply informed that according to which kind of stimulus happens, norepinephrine originated from the LC or dopamine from the VTA plays a key role in long-term plasticity and memory persistence in the hippocampus. However, neural circuits associated with the neuromodulatory system are complex because many neuromodulators are propagated throughout the brain at the same time in response to a certain stimulus and affect mood, attention, and motivation (Palacios-Filardo and Mellor, 2019; Sara, 2009).
Previous dopamine studies suggest that environmental novelty elevates dopamine in the hippocampus and strengthens initial memory consolidation through the hippocampal-VTA loop (Bethus et al., 2010; Lisman and Grace, 2005). On the other hand, the recent works postulate that the LC neurons are more responsive to environmental novelty and co-release norepinephrine and dopamine in the hippocampus (Kempadoo et al., 2016; Takeuchi et al., 2016).
They demonstrated that dopaminergic projection from the VTA to
the hippocampus was very sparse in both dopamine transporter (DAT)-Cre and TH-Cre transgenic mice compared with dopaminergic terminal axons in the hippocampus from the LC.
Further, their data showed that artificial photoactivation of LC-TH+ neurons, as well as the natural novelty exposure stimulus, enhanced memory persistence which was not impaired by Prop but SCH.
In our study, we determined that the forced swim stimulus, one of the emotional stress, enhanced the long-term spatial memory in both contextual fear conditioning and object location recognition tests.
Then, we confirmed that the forced swim triggered neuronal activity in the dorsal hippocampus and the LC by the immunohistochemical staining of c-Fos proteins. In the end, we demonstrated that dopamine rather than norepinephrine promoted the long-term memory because the intra-hippocampal infusion of SCH only sufficiently occluded the forced swim effect in the optimized forced swim-conditioning fear conditioning test protocols.
When we established the forced swim protocols, we tried to avoid the effects coming from environmental novelty because we would like to test if the LC activated by a stressful event alone still co-releases both neuromodulators, and dopamine mainly affects initial memory consolidation. For this purpose, we habituated mice for 5 days in the experimental surrounding where the forced swim protocols were performed and forced the mice to swim in the transparent cage where
mice were able to explore the familiar environment during swimming.
Consistent with a previous study which showed inputs driving place field spiking in the CA1 increased in amplitude during novel environment exploration (Cohen, 2017), the CA1 c-Fos staining data proved that the contextual fear conditioning stimulus that was conducted in a novel chamber induced higher neuronal activity than the forced swim stimulus. In contrast, the forced swim led to higher neuronal activity in the LC than the contextual fear conditioning, indicating that the forced swim was stronger stress to mice than the contextual fear conditioning because a previous study found that the amount of c-Fos positive cells in the brainstem including the LC was dependent on the strength of a noxious stimulation (Bullitt, 1990).
Therefore, the c-Fos staining data did not only show that neuronal activity occurs in the dorsal hippocampus and LC but also demonstrated that the optimized forced swim protocols would be a great model of a strong stressful stimulus with a small novelty effect.
Although we made the changes in natural stimuli, behavioral tasks, and timing of drug infusion, our results still underpinned or supported the recent studies. Together, we suggest that emotional stress, as well as environmental novelty, co-releases both dopamine and norepinephrine from the LC to the hippocampus, and dopamine rather than norepinephrine released by the stress has a pivotal role in long-
to identify molecular and neural circuit mechanisms for the long- term memory by the forced swim stimulus should be explored to support the statement. For example, our current data did not show that dopamine was released from the LC rather than the VTA in order to strengthen memory retention when the forced swim procedures were performed. Therefore, we are planning two future experiments.
One is to check whether the swim is able to induce neuronal activity in the VTA using the c-Fos staining technique. The other is to test whether the stimulus can still enhance long-term spatial memory in the contextual fear conditioning test when the dopaminergic cell groups in the LC or the VTA region are blocked by either chemogenetic or optogenetic method.
References
Bethus, I., Tse, D., and Morris, R. G. M. (2010). Dopamine and Memory: Modulation of the Persistence of Memory for Novel Hippocampal NMDA Receptor-Dependent Paired Associates. Journal of Neuroscience, 30(5), 1610–1618.
Bullitt, E. (1990). Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. The Journal of Comparative Neurology, 296(4), 517-530.
Cahill, L., Prins, B., Weber, M., and Mcgaugh, J. L. (1994). β- Adrenergic activation and memory for emotional events. Nature, 371(6499), 702–704.
Cohen, J. D., Bolstad, M., and Lee, A. K. (2017). Experience- dependent shaping of hippocampal CA1 intracellular activity in novel and familiar environments. ELife, 6.
Feng, J., Zhang, C., Lischinsky, J., Jing, M., Zhou, J., Wang, H., … Li,
specific in vivo detection of norepinephrine. Neuron, 102(4), 745- 761
Frey, U., Schroeder, H., and Matthies, H. (1990). Dopaminergic antagonists prevent long-term maintenance of posttetanic LTP in the CA1 region of rat hippocampal slices. Brain Research, 522(1), 69–75.
Jarrard, L. E. (1993). On the Role of the Hippocampus in Learning and Memory in the Rat. Behavioral and Neural Biology, 60, 9–26.
Ji, J.-Z., Wang, X.-M., and Li, B.-M. (2003). Deficit in long-term contextual fear memory induced by blockade of β-adrenoceptors in hippocampal CA1 region. European Journal of Neuroscience, 17(9), 1947–1952.
Gelinas, J. N., and Nguyen, P. V. (2005). Beta -Adrenergic Receptor Activation Facilitates Induction of a Protein Synthesis-Dependent Late Phase of Long-Term Potentiation. Journal of Neuroscience, 25(13), 3294–3303.
Giustino, T. F., and Maren, S. (2018). Noradrenergic Modulation of Fear Conditioning and Extinction. Frontiers in Behavioral Neuroscience, 12.
Gray, R., and Johnston, D. (1987). Noradrenaline and β- adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons. Nature, 327(6123), 620–
622.
Hu, H., Real, E., Takamiya, K., Kang, M.-G., Ledoux, J., Huganir, R.
L., and Malinow, R. (2007). Emotion Enhances Learning via Norepinephrine Regulation of AMPA-Receptor Trafficking. Cell, 131(1), 160–173.
Kempadoo, K. A., Mosharov, E. V., Choi, S. J., Sulzer, D., and Kandel, E. R. (2016). Dopamine release from the locus coeruleus to the dorsal hippocampus promotes spatial learning and memory. Proceedings of the National Academy of Sciences, 113(51), 14835–14840.
Kubik, S., Miyashita, T., and Guzowski, J. F. (2007). Using immediate-early genes to map hippocampal subregional functions. Learning & Memory, 14(11), 758–770.
Lemon, N., and Manahan-Vaughan, D. (2006). Dopamine D1/D5 Receptors Gate the Acquisition of Novel Information through Hippocampal Long-Term Potentiation and Long-Term Depression. Journal of Neuroscience, 26(29), 7723–7729.
Li, S., Cullen, W. K., Anwyl, R., and Rowan, M. J. (2003). Dopamine- dependent facilitation of LTP induction in hippocampal CA1 by exposure to spatial novelty. Nature Neuroscience, 6(5), 526–531.
Lisman, J. E., and Grace, A. A. (2005). The Hippocampal-VTA Loop:
Controlling the Entry of Information into Long-Term Memory. Neuron, 46(5), 703–713.
Lorenzini, C. A., Baldi, E., Bucherelli, C., Sacchetti, B., and Tassoni, G. (1996). Role of dorsal hippocampus in acquisition, consolidation and retrieval of rats passive avoidance response: a tetrodotoxin functional inactivation study. Brain Research, 730(1-2), 32–39.
Ocarroll, C. M., Martin, S. J., Sandin, J., Frenguelli, B., and Morris, R.
G. (2006). Dopaminergic modulation of the persistence of one-trial hippocampus-dependent memory. Learning & Memory, 13(6), 760–
769.
Palacios-Filardo, J., and Mellor, J. R. (2019). Neuromodulation of hippocampal long-term synaptic plasticity. Current Opinion in Neurobiology, 54, 37–43.
Porsolt, R. D., Pichon, M. L., and Jalfre, M. (1977). Depression: a new animal model sensitive to antidepressant treatments. Nature, 266(5604), 730–732.
Sara, S. J. (2009). The locus coeruleus and noradrenergic modulation of cognition. Nature Reviews Neuroscience, 10(3), 211–223.
Segal, M., and Bloom, F. E. (1976). The action of norepinephrine in the rat hippocampus. IV. The effects of locus coeruleus stimulation on evoked hippocampal unit activity. Brain Research, 107(3), 513–
525.
Takeuchi, T., Duszkiewicz, A. J., Sonneborn, A., Spooner, P. A., Yamasaki, M., Watanabe, M., … Morris, R. G. M. (2016). Locus coeruleus and dopaminergic consolidation of everyday memory. Nature, 537(7620), 357–362.
Tanaka, M. (1999). Emotional Stress and Characteristics of Brain Noradrenaline Release in the Rat. Industrial Health, 37(2), 143–156.
Tully, K., and Bolshakov, V. Y. (2010). Emotional enhancement of memory: how norepinephrine enables synaptic plasticity. Molecular Brain, 3(1), 15.
Yu, A. J., and Dayan, P. (2005). Uncertainty, Neuromodulation, and Attention. Neuron, 46(4), 681–692.
Yuen, E. Y., Liu, W., Karatsoreos, I. N., Feng, J., Mcewen, B. S., and Yan, Z. (2009). Acute stress enhances glutamatergic transmission in prefrontal cortex and facilitates working memory. Proceedings of the National Academy of Sciences, 106(33), 14075–14079.
국 문 초 록
일상 기억들 중에서 특별한 순간들을 구별하고 장기 기억으로 변환시키는 것은 생존을 위해 중요하다. 이를 위해, 비정상적이거나 또는 불확실한 사건들에 휘말렸을 때 다수의 신경조절물질들은 다양한 뇌 영역들로부터 방출되고 기억을 강화하는데 사용된다.
여기서, 우리는 일반 야생형 쥐들에게 공간 기억에 관한 경험을 가지게 하고 얼마 지나지 않아 강제적 수영을 시켰을 때 공포 상황 조건화 실험과 물체 위치 인지 실험에서 공간 기억이 향상되는 것을 관찰했다. 그리고 나서, 우리는 강제적 수영이 배측 해마와 청반에서 신경 활성을 유도하는 것을 c-Fos 단백질의 면역조직화학염색을 통해 확인했다. 마지막으로, 우리는 강제적 수영에 의해 향상된 기억이 해마의 CA1에 직접적으로 베타-아드레날린 수용체 길항제 혹은 도파민 D1/D5 수용체 길항제를 주입했을 때 도파민 수용체 길항제에 상당히 민감하게 반응하는 것을 알아냈다. 결론적으로, 우리는 강제적 수영 자극에 의해 도파민은 청반으로부터 해마로 방출되고 초기 기억 강화에 도움을 줄 것이라고 제안한다.
주요어 : 강제적 수영, 해마, 도파민, 길항제, 초기 기억 강화.
학 번 : 2018-21096
