From the Institute of Experimental and Clinical ... · From the Institute of Experimental and...
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From the Institute of Experimental and Clinical Pharmacology and Toxi- cology of the University of Lübeck Director: Prof. Dr. Markus Schwaninger Hippocampal corticosterone impairs memory consolidation during sleep but improves consolidation in the wake state Running title: hippocampal memory consolidation and corticosterone Inaugural dissertation to graduate a doctorate at the University of Lübeck - from the section of medicine - Submitted by Marie Karoline Gessert From Siegen Lübeck 2018
Transcript of From the Institute of Experimental and Clinical ... · From the Institute of Experimental and...
From the Institute of Experimental and Clinical Pharmacology and Toxi-
cology of the University of Lübeck
Director: Prof. Dr. Markus Schwaninger
Hippocampal corticosterone impairs memory consolidation during sleep
but improves consolidation in the wake state
Running title: hippocampal memory consolidation and corticosterone
Inaugural dissertation
to graduate a doctorate
at the University of Lübeck
- from the section of medicine -
Submitted by
Marie Karoline Gessert
From Siegen
Lübeck 2018
This work is supported by the “Deutsche Forschungsgemeinschaft” (SFB 654).
For my parents.
First reporter: Prof. Dr. rer. medic. Lisa Marshall
Second reporter: Prof. Dr. med. Christoph Helmchen
Day of the oral examination: 23 August 2019
Approved for print, Lübeck, 23 August 2019
Ph.D. board of the section of medicine
Table of contents
1 List of abbreviations ......................................................................................... 1
2 Introduction ...................................................................................................... 2
2.1 Background ............................................................................................... 2
2.2 Problem statement .................................................................................... 7
Methods .............................................................................................. 8
Hypotheses ......................................................................................... 9
2.3 Structure .................................................................................................... 9
3 Material and methods .................................................................................... 10
3.1 Animals and general procedure .............................................................. 10
3.2 Preparation of home cages ..................................................................... 11
3.3 Handling .................................................................................................. 12
3.4 Surgery and recovery .............................................................................. 12
3.5 Open field habituation ............................................................................. 14
3.6 Tasks ....................................................................................................... 14
Elevated plus maze test .................................................................... 14
Novel-object recognition and object-place recognition test ............... 16
Preparation, administration of substances and assessment of sleep 18
3.7 Brain removal and histological examination ............................................ 19
3.8 Data analysis ........................................................................................... 20
Testing for difference between conditions through variance analysis 20
Testing for difference between specific conditions............................ 22
Testing that performance is different from chance ............................ 23
4 Results ........................................................................................................... 24
4.1 Tasks ....................................................................................................... 24
Elevated plus maze .......................................................................... 24
Sample trials ..................................................................................... 24
Retention intervals ............................................................................ 24
Test trials .......................................................................................... 25
4.2 Histological examination .......................................................................... 27
5 Discussion ..................................................................................................... 29
5.1 Key findings ............................................................................................. 29
Hippocampus-dependent memory .................................................... 29
Hippocampus-independent memory ................................................. 29
Where and when do glucocorticoids affect consolidation? ............... 30
5.2 Related work ........................................................................................... 31
Dose-dependent effects of glucocorticoids ....................................... 31
Arousal-dependent effects of glucocorticoids ................................... 32
Connection between dose- und arousal-dependent effects of
glucocorticoids ............................................................................................... 33
5.3 Limitations ............................................................................................... 35
Improvements to the implants ........................................................... 35
Improvements to the apparatus for drug delivery.............................. 35
Improvements to the study design .................................................... 36
Improvements to the preparation of drugs ........................................ 38
Remarks by reviewers ...................................................................... 40
5.4 Future work ............................................................................................. 41
Sleep-stage dependent differences in memory consolidation ........... 41
Effects of glucocorticoids on sleep-stage dependent EEG-rhythms . 42
6 Summary ....................................................................................................... 45
7 Summary in German ...................................................................................... 47
7.1 Einleitung ................................................................................................ 47
7.2 Material und Methoden ............................................................................ 51
7.3 Daten-Analyse ......................................................................................... 53
7.4 Ergebnisteil ............................................................................................. 54
“elevated-plus-maze” Test ................................................................ 54
Sample trial ....................................................................................... 55
Behaltensintervall ............................................................................. 55
Test trial ............................................................................................ 55
Histologische Untersuchung ............................................................. 57
7.5 Diskussion ............................................................................................... 57
Einschränkungen .............................................................................. 60
Ausblick ............................................................................................ 63
7.6 Zusammenfassung .................................................................................. 64
8 Own contributions to the doctoral thesis ........................................................ 66
9 List of references ........................................................................................... 67
9.1 Journals and magazines ......................................................................... 67
9.2 Books ...................................................................................................... 75
9.3 Web pages .............................................................................................. 76
10 Appendix ........................................................................................................... I
10.1 List of tables ............................................................................................... I
10.2 List of figures .............................................................................................. I
10.3 Exemplary protocols .................................................................................. II
Handling .......................................................................................... II
Postoperative control ...................................................................... III
Habituation ..................................................................................... IV
Memory tasks .................................................................................. V
10.4 Histological results ................................................................................... VI
10.5 Stereotaxic coordinates of the rat brain ................................................... VII
10.6 Photos of the experimental room ........................................................... VIII
10.7 Poster for the „6. Lübecker Doktorandentag 2013“ .................................. XI
10.8 Further attachments ................................................................................ XII
11 Acknowledgment .......................................................................................... XIII
12 Curriculum vitae ............................................................................................ XIV
13 List of own publications .................................................................................. XV
14 Honesty declaration ...................................................................................... XVI
1
1 List of abbreviations
abbreviation meaning
ANOVA ANalysis Of VAriance
CA cornu ammonis
CORT corticosterone
dSub dorsal subiculum
EC entorhinal cortex
EEG electroencephalogram
EMG electromyography
FOS FBJ murine osteosarcoma viral onco-
gene
GABA gamma-aminobutyric acid
GC glucocorticoid(s)
GR glucocorticoid receptor
HBC hydroxypropyl-beta-cyclodextrine
LTD long-term depression
LTP long-term potentiation
MB mammillary body
MR mineralocorticoid receptor
NGFI-A nerve growth factor induced clone A
NMDA n-methyl-d-aspartic acid
PBS phosphate buffered saline
PGO ponto-geniculo-occipital
REM rapid eye movement (sleep)
SWS slow-wave sleep
VEH vehicle
2
2 Introduction
2.1 Background
With John O’Keefe, May-Britt and Edvard I. Moser winning the Nobel Prize in phys-
iology or medicine in 2014 for defining a spatial navigation system within the rat
brain (Burgess, 2014), cognitive neuroscience has become increasingly popular.
The key figure in this field of research is a structure lying deep within the medial
temporal lobe: the so-called “hippocampal formation” – named after its resemblance
to the seahorse. It consists of a group of neighboring regions including the dentate
gyrus, the subiculum, the presubiculum, the parasubiculum, the entorhinal cortex
and the hippocampus itself1. During the last 400 years it was associated with many
different functions, amongst others the sense of smell as being part of the evolution-
ary “rhinencephalon” and the orchestration of emotional expression via the “Papez
circuit” (Andersen et al., 2006).
Figure 1. Anatomy of the limbic system2.
1 The hippocampus again contains the fields CA1-CA3 as identified by Rafael Lorente de Nó, with CA standing for “cornu ammonis” after the Egyptian god Amun Kneph, who was carrying a ram with him; see Figure 1. 2 Adapted from: http://tayloredge.com/reference/Science/BiologySlides/LimbicSystem.gif, 27.10.2016.
3
Today its role in the formation of memory has been narrowed down to the encoding
and retrieval of long-term memory for facts or events, also referred to as declarative
or explicit memory. Roy et al., 2017, proposed distict neural circuits in the temporal
lobe system for these processes (see Figure 2): the encoding of episodic memories
in the hippocampus takes place in the CA1 → EC5 circuit, while for the retrieval the
dorsal subiculum (dSub) is interposed. The connection of the latter to the
mammillary bodies (MB) is thought to regulate memory-retrieval-based emotions,
while the connection to the entorhinal cortex (EC5) shall control the retrieval-based
instinctive fear responses. In combination, these circuits will allow the animals to act
more flexibly and to survive evolutionary change.
CA1 EC5
dSub
MB
Hippocampal Memory
Formation
Hippocampal Memory
Retrieval
Stress Hormone
Responses
Context-Dependent
Updating
Figure 2. Distinct neural circuits3. CA1 = cornu ammonis 1; dSub = dorsal subiculum; EC5 = medial entorhinal cortex 5; MB = mammillary body.
Nondeclarative or implicit memory such as the procedural memory, priming,
classical conditioning and non-associative learning, on the other hand, is
represented by other areas of the brain like the striatum, the motor- or neocortex,
the cerebellum, the amygdala or reflex pathways. The declarative memory as de-
scribed within the “declarative memory theory” by Squire (Squire et al., 1984; Squire
and Zola-Morgan, 1991; Squire, 1992; Squire, 2004) is furthermore divided into ep-
isodic and semantic memory (Andersen et al., 2006; see Figure 3). The term epi-
sodic memory stands for the recall of a certain event (what), which took place at a
3 Adapted from Roy et al. (2017): Distinct neural circuits for the formation and retrieval of episodic memories. Cell, 170(5), 1000-1012.
4
certain time (when) and a certain place (where). Because its discoverer Tulving
(Schacter and Tulving, 1994; Tulving, 1972; Tulving, 1983; Tulving et al., 2005)
declared that the individual in question must have a particular “autonoetic
consciousness” (Tulving, 1983), i.e., being able to identify his role in the event,
Griffiths et al. (1999) termed this memory type “episodic-like” for animals (Andersen
et al., 2006).
Human Memory
Sensory
Memory
(< 1 s)
Long-term
Memory
(life-time)
Short-term
Memory
(< 1 min)
Consolidation
(Hippocampus)Encoding
Explicit
Memory
(conscious)
Implicit
Memory
(unconscious)
Procedural
Memory
(skills, tasks)
Declarative
Memory
(facts, events)
Episodic
Memory(events, experiences)
Semantic
Memory
(facts, concepts)
Recall
Figure 3. Memory types, steps of memory formation and role of the hippocampus4.
Facing the process of memory formation itself, the scientific community agreed upon
three different steps that have to be passed to embed information permanently.
During the encoding phase, new information is collected in working memory (short-
term memory); during the consolidation phase, the collected information is
transferred from short-term- to long-term memory; during the recall phase,
information from long-term memory is recollected (Andersen et al., 2006; see Figure
3).
4 Based on http://www.human-memory.net/images/memory_types.jpg, 27.10.2016.
5
At the cellular level, these processes are said to be promoted by so-called “synaptic
plasticity”, a term first characterized by Martin et al. in 2000. His “synaptic plasticity
and memory” hypothesis implies that “activity-dependent synaptic plasticity is
induced at appropriate synapses during memory formation, and is both necessary
and sufficient for information storage underlying the type of memory mediated by
the brain area in which that plasticity is observed” (Andersen et al., 2006, p. 427).
LTP (“long-term potentiation”) and LTD (“long-term depression”) are models for two
kinds of synaptic change. As indicated by the name, LTP is a process in which an
increase in synaptic efficacy occurs, whereas in LTD a decrease is caused. Alt-
hough LTP cannot be equated with memory, there are analogies to be found. For
instance, both processes can be affected by sleep and stress.
While asleep, the individual repeatedly runs through two different stages of activity:
the non-REM sleep which is predominant in the first half of the night and
characterized by slow high-voltage EEG-waves (i.e., slow oscillations, spindles and
sharp wave ripples), muscular activity and dreamlessness – and the rapid-eye-
movement (REM) sleep which is predominant in the second half of the night and
characterized by rapid low-voltage EEG-waves (i.e., ponto-geniculo-occipital waves
and theta activity), muscular atonia and the occurrence of dreams.
Research investigating the effect of sleep on the consolidation of memories as-
signed specific functions to the aforenamed conditions. While slow-wave sleep
(which is part of the non-REM sleep) might support the reactivation and
redistribution of memories from temporary stores to long-term memory, REM sleep
is said to promote the synaptic consolidation of memories by disconnecting long-
term- from short-term memory storage (rev. in Diekelmann and Born, 2010 and
Ribeiro et al., 2004). Transferred to a more general model concerning creative prob-
lem solving proposed by Lewis et al., 2018 (“BiOtA” model: broader form of the in-
formation overlap to abstract framework), non-REM sleep serves the so called
“schema” learning by abstracting rules from corpuses of learned information. REM
sleep, on the other hand, supports the formation of novel, unexpected connections.
According to them, only the iterative interleaving of both sleep stages enables the
formation of complex knowledge frameworks and thus facilitates creative thought.
The link between the hippocampus and stress (first referred to as “stress-
hippocampus link” by Lupien and Lepage in 2001) is based on several observations,
starting by the discovery of abundant mineralocorticoid and glucocorticoid receptors
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in the animal and human hippocampus by McEwen et al. in 1986 and confirmed
later by many other scientists (Joëls, 2008; McEwen and Sapolsky, 1995; Reul and
de Kloet, 1985).
Behavioral studies furthermore attested an “inverted U-shaped function between the
level of acute stress and memory” summarized in the so-called “Yerkes-Dodson law”
(Robert and John, 1908). It implies that mild to moderate glucocorticoid levels im-
prove the effects of learning by facilitating the induction of long-term potentiation.
High levels, however, decline the effect below baseline by promoting the induction
of long-term depression in the CA1 region of the hippocampus (Andersen et al.,
2006; sample studies: Okuda et al., 2004; Roozendaal, 2000; Roozendaal et al.,
2002; Sandi and Rose, 1994). The cytological explanation for this activity-depend-
ent synaptic plasticity is based on the different affinities of the receptors mentioned
above. Mineralocorticoid receptors have a high affinity to bind glucocorticoids and
thus become first and predominantly occupied during low levels of stress hormones.
Glucocorticoid receptors, however, have a lower affinity to bind glucocorticoids and
become increasingly occupied during rising levels of stress hormones, when miner-
alocorticoid receptors are starting to get saturated. Through cytological mechanisms
still unknown, the combination of fully saturated mineralocorticoid receptors and par-
tially occupied glucocorticoid receptors leads to a stable or even enhanced receptor
transmission (equal to long-term potentiation), whereas fully saturated glucocorti-
coid receptors cause attenuated excitatory input (equal to long-term depression;
Andersen et al., 2006). This theory is also referred to as “binary hormone response”
(Evans and Arriza, 1989) or “MR/GR balance” (Oitzl et al., 1995) theory.
In addition to these neurobiological effects, both receptors are said to interfere with
distinct steps of information processing. While the activation of mineralocorticoid
receptors modulates “selective attention”, causing appropriate behavioral re-
sponses to external stimuli (Oitzl and de Kloet, 1992; Sandi and Rose, 1994), glu-
cocorticoid receptors regulate the encoding and consolidation of memories (Conrad
et al., 1999; Oitzl and de Kloet, 1992). In the long run, chronic exposure to high
levels of stress eventually leads to structural changes in the hippocampus with
atrophy of CA1 and CA3 neurons in rats and primates (Kerr at al., 1991; Mizoguchi
et al., 1992; Sapolsky et al., 1985) and even affects the neurogenesis by inhibiting
postnatal and adult granule cell proliferation (Cameron and Gould, 1994; Gould et
al., 1997).
7
Besides the total amount of glucocorticoids during the consolidation process, also
their time of administration seems to be essential for the recall of memories. De
Quervain et al. (1998, 2009) found out that the more time between the encoding
phase and the administration of glucocorticoids, the worse the recall – especially if
the effects of the glucocorticoids last on during the recall process. More recent stud-
ies (Sazma et al., 2018) confirmed these observations – but only for rodents that
were habituated to the learning context. Those that did not get to know the test
setting before the encoding showed unchanged or even improved memories. The
authors assumed a so called “contextual binding”, propagating that the stressor itself
would serve as memorable event to enhance memories for other events that share
the same context.
When combining the two factors sleep and stress, studies revealed vigilance de-
pendent differences in the effect of glucocorticoids on the consolidation of memo-
ries. While the administration of glucocorticoids during sleep after the encoding pro-
cess seems to impair the recall of hippocampus-dependent memories, the admin-
istration during wakefulness enhances it (Plihal and Born, 1999; Plihal et al., 1999;
Wilhelm et al., 2011). This may be due to qualitatively distinct reactivation processes
during sleep and wakefulness. While “neuronal replay” of the information during
sleep leads to its stabilization (consolidation), “neuronal replay” during wakefulness
causes destabilization (Debiec et al., 2002; Diekelmann et al., 2011; Gupta et al.,
2010; Jezek et al., 2002; Nader and Hardt, 2009). An example for that is the different
replay of spike sequences in the two conditions. While it only occurs in forward order
during sleep (Lee and Wilson, 2002), forward and backward order is recorded during
wakefulness (Davidson et al., 2009; Foster and Wilson, 2006). More recent studies
(Chen and Wilson, 2017) refuted these observations and also reported neuronal
replay in forward and backward order during sleep. However, they described sleep
stage dependent differences concerning the dynamics of the replay (“normal” in time
during REM, “compressed” in time during SWS), indicating specific functions in
sleep yet to be discovered.
2.2 Problem statement
In the present study I attempt to put all these pieces together:
1. The leading role of the hippocampus in the consolidation process of declar-
ative memory.
8
2. The wake-sleep-dependent differences in the consolidation processes.
3. The modulation of the wake-sleep-dependent differences by glucocorticoids.
I aim to investigate the impact of glucocorticoids in the hippocampus on the consol-
idation process of declarative memory (Where do the glucocorticoids affect the
memory process?) and their onset of action (When do the glucocorticoids affect the
memory process – during wakefulness or sleep?). To prevent side-effects of gluco-
corticoids binding to receptors apart from the hippocampus (as it would be inevitable
for a systemic application in human studies) I use an animal model in which I implant
cannulas directly into the dorsal hippocampus. What makes my approach so unique
is that, up to now, comparable studies only focused on one type of memory (mostly
hippocampus-independent5) and only used systemically administered stress
hormones. Therefore, they were not able to differentiate between direct effects of
the glucocorticoids on specific brain structures and indirect effects caused by, for
example, a change in the neuromodulator concentration.
Methods
To be able to differentiate between the effects of the intrahippocampal infusion of
corticosterone and the consolidation during sleep on hippocampus-dependent and
–independent memories the rats had to undergo two different tests: the hippocam-
pus-dependent “object-place recognition test” and the hippocampus-independent
“novel-object recognition test”. In both tests, animals were confronted with two iden-
tical objects which were arranged at two of nine positions in a square box during the
sample trial. While one of the objects was exchanged for a new one during the test
trial in the hippocampus-independent “novel-object recognition test”, both objects
remained the same in the hippocampus-dependent “object-place recognition test”.
Here, however, one of the two identical objects was shifted to another place during
the test trial. Each memory test was executed under four conditions: intracerebral
application of corticosterone or vehicle (i.e., 0.9 % saline solution) during an 80
minutes retention interval in which the rats were allowed to sleep or were sleep-
deprived.
5 Okuda et al., 2004.
9
Hypotheses
Based on previous studies (Binder et al., 2012; Inostroza et al., 2013; Wilhelm et
al., 2011) and the facts mentioned above I postulate the following hypotheses:
2.2.2.1 Hippocampus-independent “novel-object recognition test”:
No differences in the test performances throughout all four conditions will occur.
2.2.2.2 Hippocampus-dependent “object-place recognition test”:
The following substance and sleep-state dependent differences in the test perfor-
mance will occur:
1. Administration of corticosterone:
a. Corticosterone administered during a waking period after the encoding
process will improve the consolidation (and hence the recall) of hippo-
campus-dependent memory.
b. Corticosterone administered during a sleeping period after the encod-
ing process will impair the consolidation (and hence the recall) of hip-
pocampus-dependent memory.
2. Administration of vehicle:
a. Sleeping vehicle-infused animals will perform better than sleep-de-
prived vehicle-infused and sleeping corticosterone-infused animals.
b. Sleep-deprived corticosterone-infused animals will perform better than
sleep-deprived vehicle-infused animals.
The test results are presented in chapter 4 and were published 2014 in the journal
Hippocampus (Kelemen et al., 2014).
2.3 Structure
The thesis consists of 14 chapters: following the introduction is the “Material and
methods” part in which the setup, the progress of the study and the analysis of the
data is explained. In chapter 4, the results of the different tasks and the histological
examination are presented. Chapter 5 contains a critical analysis of the test results
against the backdrop of competitive studies and points out limitations and future
work. After that, the work is summarized shortly in English (chapter 6) and exten-
sively in German (chapter 7). The last seven chapters are reserved for formalities
like, for example, references (chapter 9), lists of tables and figures (chapter 10) and
own publications (chapter 13).
10
3 Material and methods
3.1 Animals and general procedure
For the experiments, I used 24 adult male Long-Evans rats (Janvier, Le Genest-
Saint-Isle, France, 250-300 g). The number of animals was based on previous ex-
periments (Binder et al., 2012; Inostroza et al., 2013) and reflected the minimum
number of animals needed to prove significant effects. The experiments were
approved by the Schleswig-Holstein state authority6 and performed in accordance
with the NIH guidelines and the directive of the European Community Council
(86/609/ECC).
date activity
24.08. – 28.08.2011 preparation of home boxes, arrival, and
adaptation
29.08. – 04.09.2011 handling
31.08.2011 pilot surgery
01.09. – 07.09.2011 surgery, recovery
08.09. – 13.09.2011 habituation to the experimental room
14.09.2011 elevated plus maze (13.09.2011:
transport to the elevated plus maze
room and back)
16.09. – 03.10.2011 novel-object recognition and object-
place recognition tasks
20.09. – 12.10.2011 killing
November/December 2011 histology
Table 1. Timeline of the experiment.
Table 1 summarizes the timeline of the experiment. When the animals arrived, they
were put into the previously prepared home cages and left undisturbed for the next
five days to adapt to their surroundings (except for feeding and exchanging water
every two days). Afterward, they were handled twice a day for three consecutive
days to get accustomed to the experimenters and to facilitate the process of plug-
ging and unplugging for corticosterone application later on. Subsequently, two can-
nulas were implanted into the dorsal hippocampi of each animal under general an-
esthesia. The rats were given seven days of recovery before they were habituated
6 See “10.8 Further attachments” in the appendix, p. XII.
11
to the experimental room. To check each animal’s level of anxiety, they had to un-
dergo the so-called “elevated plus maze test” two days before the actual testing
started, as the effects of corticosterone interact with anxiety (Inostroza et al., 2011;
Pinheiro et al., 2007).
The experiments consisted of two different memory tasks (the hippocampus-
independent “novel-object recognition test” and the hippocampus-dependent
“object-place recognition test”), each of which was executed under four conditions:
an 80 minutes interval of sleep or sleep deprivation in between the sample and test
sessions, during which the animals received an intrahippocampal infusion
containing a solution of 10 ng corticosterone in 0.5 µl saline (0.9 %) or solely 0.5 µl
saline (0.9 %; “vehicle”), as soon as they showed first signs of sleep. In the wake
conditions, the timing of the infusion was matched to that of the sleep conditions.
The original procedure was to have each animal run through all of the eight experi-
mental conditions in a pseudorandom order, with at least three days in between
subsequent testing. The complete procedure was, however, only applied to those
rats whose implants stayed intact throughout the whole experiment. The ones where
a displacement of the cannulas could not be safely excluded were dropped out in
advance. After completing the experiments or when having lost their implants, the
animals were killed, and their brains were histologically examined to verify the cor-
rect position of the cannulas.
3.2 Preparation of home cages
Each animal was kept in a plastic cage filled with bedding and nesting material and
covered with a grid containing a hole for the water bottles. The cages were resting
on racks with visual contact with the neighboring rats. After the surgeries, the lids of
the cages were wrapped in plastic foil, so that the animals could not damage their
implants. The nibbled foliage was replaced. Each day after the experiments the rats
were fed, and every week the water bottles were refilled.
The lights in the home cage room were adjusted in a way that the luminosity in each
cage was about 50 lux (measured once at the beginning of the experiment). They
were connected to a timer, which turned them on at 6 a.m. and off at 6 p.m. each
day, subjecting animals to a constant sleep-wake cycle. To guarantee a maximum
percentage of SWS and a minimum concentration of corticosterone the tasks took
12
place between 8 a.m. and 1 p.m., with nobody entering the home cage room be-
tween 6 p.m. and 6 a.m.
3.3 Handling
Each animal was handled for three days before the surgery. The handling took place
twice a day (in the morning and the afternoon) for four minutes per animal. At first,
they were left untouched on the experimenter’s lap. Then, the contact was slowly
increased. The rats were touched on the bodies and heads and lifted with the fore-
limbs crossed in front of their chest to get them accustomed to the infusion proce-
dure. The activities and observations were written down in protocols7.
3.4 Surgery and recovery
Before being carried to the surgery room, the animals were weighed to calculate the
correct dose of drugs. Then, they received intraperitoneal infusions of atropine (par-
asympatholytic, 0.1 mg/kg) and medetomidine (sedative, 0.6 mg/kg). To implant the
cannulas8 the rats were anesthetized with isoflurane (induction: 1-2 % in 0.35l/min
O2, maintenance: 0.8-1.2 % in 0.35l/min O2), applied at first to an airtight plastic box.
When unconscious and immobile, they were retracted, placed on a heating pad to
maintain normal body temperature and connected to a mask providing the
anesthesia for the rest of the surgery. Their head was shaved and disinfected with
ethanol. Afterward, the head was fixed in a stereotactic frame. Then, the scalp was
cut along the midline with a scalpel and held aside with clips. After removing
overlaying tissues, the positions for the two cannulas (coordinates relative to
bregma: - 3.8 mm antero-posterior, ± 2.8 mm lateral, - 1.8 mm dorso-ventral) were
marked and the burr holes were drilled (see Figure 4). At the dorso-ventral coordi-
nates, the cannulas were implanted laterally tilted by 22 degrees respective to the
vertical axis with the help of a stereotactic guide9. After that, the four fixing screws
were screwed in manually and - together with the cannulas - covered with cold pol-
ymerizing dental resin10. In the end, the cannulas were capped with stylets (lengths:
11 mm) and the animals got 1 ml sodium chloride and atipamezole (antagonizes
medetomidine, 0.6 mg/kg) injected subcutaneously. Surgeries took about 60 to 90
minutes per animal and were conducted by Dr. Marion Inostroza.
7 See exemplary protocol in the appendix, p. II. 8 Stainless-steel guide cannula, Plastics One, USA, length: 11 mm, gauge: 23. 9 Cannula holder, Plastics One, USA. 10 Palapress, Heraeus Kulzer GmbH, Germany.
13
A pilot surgery was performed with animal T11, using blue ink to mark the position
of the cannulas.
Ventral
Hippocampus
Dorsal
Hippocampus
Fixing ScrewsHoles for
Cannulas
Incisor Bar Nose Bar
Ear Bar
Figure 4. The cannulas’ position in a stereotactic and anatomic model11.
The animals were observed after the surgery and kept warm with an infrared lamp
for the next few hours. They got softened crackers to eat and their weight was
checked daily for the next week. The wounds were treated with powder (azithromy-
cin) and ointment (betaisodona). To prevent the cannulas from becoming stuck and
to accustom the rats to the infusion process, the stylets were removed every day12.
11 Based on http://media.wiley.com/mrw_images/cp/cpns/articles/ns0901/image_n/ nns090102.gif, 27.10.2016 and http://kaylab.uchicago.edu/images/rat_brain.png, 27.10.2016. 12 See exemplary protocol in the appendix, p. III.
14
3.5 Open field habituation
Three batches of four animals per day ran through the experimental protocol de-
scribed below (i.e., each rat was allowed to explore the open field for five minutes
in the first and three minutes in the second run), except that there were no objects
presented and no substances injected. As the direction in which they were put into
the open field during the tasks was changed during the experimental testing to favor
the development of an allocentric strategy, it was done here, too. In the first session,
they were brought in from the south, in the second from the north and in the third
from the east. The activity in the open field and the boxes was recorded with video
cameras and written down in protocols13.
3.6 Tasks
Elevated plus maze test
The elevated plus maze is a widely used behavioral assay to measure the anxiety
behavior of rodents (Walf and Frye, 2007). Its setting consists of a “+”-shaped ap-
paratus with two opposing enclosed (45 x 10 cm, 40 cm high) and two opposing
open arms (45 x 10 cm) branching off at right angles from an equally open center
platform (10 x 10 cm). As the name already indicates, the construction itself is ele-
vated 50 cm above the floor (see Figure 5).
The model is based on the rats’ general aversion of open spaces, which is reflected
in a special behavior called “thigmotaxis”: the animals restrict their movements to
the enclosed area or the edges of adjacent spaces and avoid entering the open
arms. A low level of anxiety is therefore indicated by a high proportion of time spent
in the open arms (time in the open arms/total time in the open or enclosed arms)
and a high proportion of entries into the open arms (entries in the open arms/total
entries in the open or enclosed arms; Walf and Frye, 2007).
As the elevated plus maze is a test to check each animal’s level of anxiety and took
place in a different room, the animals were transported there in three groups one
day before the proper testing to get accustomed to the room and the transportation
process. After having spent fifteen minutes in the experimental room, they were
brought back to their home cages.
13 See exemplary protocol in the appendix, p. IV.
15
For the task, each animal was placed on the central platform facing an enclosed
wall. Subsequently, the rats were given five minutes to explore the maze, as Mont-
gomery revealed in 1955 that during this time, the most robust avoidance takes
place. Their activity was again recorded with video cameras and observations were
written down in protocols. Analyzing the individual behavior, an entry into one arm
was scored, when all four paws of the animal were inside.
Figure 5. Setting and proportions of the elevated plus maze test14.
14 Based on http://en.wikipedia.org/w/index.php?title=File:ElevatedPlusMaze.svg& page=1, 27.10.2016.
16
Novel-object recognition and object-place recognition test
Both memory tests were constructed in the same way: in a sample trial (encoding
phase) two identical objects were presented in a square arena composed of gray
PVC (80 x 80 cm wide, with 40 cm high walls). The animals were left to “actively
explore” the objects for two to five minutes. Their behavior (criteria: being within two
cm of an object, directing the nose towards the object and engaging in active ex-
ploratory behavior such as sniffing and licking, but no climbing) was scored with a
stopwatch. After having revealed fifteen seconds of exploratory behavior per object
after a minimum of two minutes or having passed the maximum of five minutes, the
rats were removed and put back into their retention boxes (35 x 35 cm wide, with 45
cm high walls). There they were allowed to sleep or kept awake by so-called “gentle
handling” (i.e., putting objects into the boxes, knocking at the walls, disturbing the
sleeping nests etc.) for an interval of 80 minutes (starting from the point of time,
when the last animal of the group had accomplished the sample trial). During this
period (also referred to as retention period or consolidation phase), the rats received
intracerebral infusions of corticosterone or vehicle. Therefore, they were connected
to a tubing system right after being removed from the test field. The infusion time
was either determined as the point of time at which the rat had fallen asleep in the
sleeping condition, or the meantime of the average sleep onset time of the animals
examined the days before in the awake condition. After 80 minutes the animals were
put back into the open field for the test trial (retrieval phase). Again, there were two
objects present in the arena, but this time their arrangement differed between the
two memory tests. In the hippocampus-independent “novel-object recognition test”
one of the objects was exchanged for a new one, while their position remained the
same as in the sample trial. In the hippocampus-dependent “object-place recogni-
tion test”, however, both objects remained the same, but the position of one of the
objects was changed compared to the sample trial (see Figure 6).
As opposed to the sample trial, all rats were allowed to explore the objects for three
minutes - irrespective of the total amount of time in which they were engaging in
“active behavior”. When all animals of the group had accomplished the second part
of the task, they were brought back to their home cages, the objects, the open field
and the retention boxes were cleaned with a 1:1 ethanol solution (a procedure which
was also done before the first task of the day and between each following task), the
17
tubing was refilled with corticosterone or vehicle and the second group of the day
was brought in.
All the sample and test trials and the retention intervals, during which the animals
were left alone to sleep, were recorded by a video camera and analyzed offline using
the ANY-maze tracking system15. Furthermore, general observations made during
the experiments were written down in protocols16.
sample trial test trial
test trialsample trial
Novel-object
recognition
Object-place
recognition
Injection of
corticosterone
Injection of
vehicleSleep Sleep-
deprivation
retention
interval
retention
interval
consolidation
consolidation
Figure 6. Experimental setup17.
The objects being used differed in height (10 - 15 cm), base diameter (8 - 10 cm),
color and shape. They were composed of glass to enable cleaning with ethanol and
heavy enough to prevent being moved by the rats. For each task, different sets of
objects were applied and each one was trial-unique. Pilot studies ensured that the
animals could discriminate between the various objects and did not show any pref-
erence.
To insulate the rats from acoustic stimuli from outside the room during the tasks, the
experimental room was prepared with a noise generator producing masking noise.
15 ANY-Maze, Stoelting Europe, Dublin, Ireland. 16 See exemplary protocol in the appendix, p. V. 17 Based on Dere et al. (2007): The pharmacology, neuroanatomy and neurogenetics of one-trial object recognition in rodents. Neuroscience and Biobehavioral Reviews 31, 673-704.
18
Additional to that, there were visual distal cues fixed to the walls (two rectangles at
the northern wall, two other rectangles at the eastern wall and a square at the
western wall), which stayed at the same place during the whole experiment and
could be used as orientational marks for the animals in the open field. This fact may
be especially important, as the direction in which the animals were placed into the
open field was changed for every task to make them allocentric and therefore hip-
pocampus-dependent (Langston and Wood, 2010).
Preparation, administration of substances and assessment of sleep
Each experimental day started with the preparation of corticosterone or vehicle and
the filling of the tubing.
For the solution, 5 mg of corticosterone18 were measured with a fine-scale, solved
in 16.6 ml of 0.9 % saline solution and mixed for two minutes with a vortex.
Calculation: As 1 g of powder contains 66.7 mg of corticosterone, 5 mg of
powder in 16.6 ml of 0.9 % saline solution corresponds to a ratio of 10 ng of
corticosterone in 0.5 µl of 0.9 % saline solution.
The amount of corticosterone used in the experiment (10 ng) is based on similar
studies using glucocorticoids for intrahippocampal infusions as well as infusions in
other comparable brain areas (Medina et al., 2007; Micheau et al., 1985; Lozano et
al., 2013). From the aforenamed works, the one of Medina et al. (2007) is the most
important. There they used doses from 5 to 60 ng (5, 10, 20, 30 and 60 ng), of which
a dose of 10 ng injected directly into the striatum caused the best results in the
inhibitory avoidance task. As I intended to examine the effects of glucocorticoids on
a small part of the brain, I chose to induce a strong supraphysiological increase of
the local corticosterone concentration rather than to mimic the effects of widespread
amounts seen under natural conditions.
After rinsing the tubing with 0.9 % saline solution, 10 ng of corticosterone in 0.5 µl
of saline or solely 0.5 µl of saline solution (0.9 %; “vehicle”) was sucked in manually,
leaving a bubble as space-holder and mark in between the substance to be applied
and the 0.9 % saline solution filling the rest of the tubing.
18 C174 SIGMA – Corticosterone: HBC complex, Sigma Aldrich, St. Louis, MO, USA; http://www.sigmaaldrich.com/catalog/product/sigma/C174?lang=de®ion=DE, 27.10.2016.
19
The substances were administered during the retention period, when the rats had
fallen asleep or after a given period of wakefulness (average sleep onset time by
the animals examined the days before). As rats do not always close their eyes when
falling asleep, the criterion to administer corticosterone or vehicle in the sleeping
condition was an absence of movement for at least 30 seconds (notably, when hav-
ing snuggled in a corner with the tail wrapped around the body). For bilateral sub-
stance administration, animals were connected to two infusion needles (30 gauge)
protruding 1.5 mm beyond the tip of the cannula, sticking on 1-m polyethylene tubing
(PE-20) and ending in two 10 µl microsyringes19 containing corticosterone or vehicle
right after being retracted from the open field. The syringes, in turn, were inserted in
an electric pump20 which administered 0.5 µl of infusion volume per hemisphere
over a period of 35 seconds21. To maximize diffusion and to prevent a backflow of
the drug into the cannulas, the syringes were removed 20 seconds after the appli-
cation and – together with the tubing – hung back over the boxes. To prevent them
from banging, they were wrapped in cellulose after checking their contents.
The total amount of time the rats slept during the retention period after the last ani-
mal was infused and the experimenters had left the room was scored by standard
visual procedures validated in previous studies (Inostroza et al., 2013; Pack et al.,
2007; Van Twyver et al., 1973). Comparisons with EEG-/EMG-based scoring pro-
vided an agreement of 92 %. Referring to the description given above, sleep was
assumed, when the animal displayed a specific sleep posture. However, instead of
waiting for the animals to be immobile for at least 30 seconds, it was scored already
after five seconds of motionlessness.
3.7 Brain removal and histological examination
After having run through all eight experiments or having lost their implants, animals
were killed and their brains were examined histologically. For the killing, they were
put under deep ketamine/xylazine anesthesia (initial dosage of 0.75 mg/kg ketamine
and 5 mg/kg xylazine injected intraperitoneally). Surgical preparation for perfusion
was conducted in the style of Gage et al. (2012) introducing a butterfly in the right
ventricle of the heart. Subsequent to using 30 ml of phosphate buffered saline (PBS:
0.1 M, pH = 7.3) mixed with 0.2 % of heparin, the animal was perfused with 200 ml
19 Hamilton, Reno, NV. 20 Sage Instruments, Boston, MA. 21 See “10.6 Photos of the experimental room” in the appendix, pp. VIII - X.
20
of PBS mixed with 4 % of paraformaldehyde. After removal, the brain was immersed
for at least five days in a 4 % formaldehyde solution and kept in the refrigerator.
For histology, it was cut into coronal sections of 50 µm by a vibratome, stained with
toluidine blue and examined under a light microscope to determine the location of
the infusion needle tips.
Perfusions were conducted by Dr. Sonja Binder, histology was performed by Dr.
Marion Inostroza.
3.8 Data analysis
In this section, I describe the statistical tests employed for the critical evaluation of
the hypotheses based on the experimental data. To compare the performance of
each rat in the two memory tasks in the four conditions, a discrimination ratio is
introduced. It is based on the assumption that the rats prefer to explore the new or
displaced objects rather than the old stationary ones. Therefore, the total time of
exploration for each object in the test trial is converted into a discrimination ratio.
For the novel-object recognition test, the discrimination ratio 𝑑𝑛 is:
𝑑𝑛 = (time spent exploring the novel object – time spent exploring the familiar object)
/ (time spent exploring the novel object + time spent exploring the familiar object).
And for the object-place recognition test:
𝑑𝑜 = (time spent exploring the displaced object – time spent exploring the non-dis-
placed object) / (time spent exploring the displaced object + time spent exploring
the non-displaced object).
To leverage the statistical tests described in the following, I follow the common prac-
tical assumption that measurements are normally distributed and variances be-
tween groups are equal (homoscedasticity).
Testing for difference between conditions through variance analysis
For analyses of the discrimination ratio, a two-way ANOVA with the factors SLEEP
𝐴 and STRESS 𝐵 was applied. Both factors have two levels: sleep deprivation vs.
normal sleep and corticosterone infusion vs. vehicle infusion. For each combination
of factor levels, the experiment is repeated 𝑘 times, resulting in four distinct average
discrimination ratios 𝑑𝑎𝑏 , 𝑎 ∈ {1,2}, 𝑏 ∈ {1,2}. The null hypothesis to be tested by
21
ANOVA is that the dependent variable 𝑑𝑟, resp. 𝑑𝑜, is not influenced by the two
factors. The hypotheses in particular are:
Factor A (influence of SLEEP):
- Null hypothesis H0 A : ∀ (p, q) αp = αq; with αp = effect of sleep on the memory
recall and αq = effect of sleep deprivation on the memory recall.
- Alternative hypothesis: H1A: ∃ (p, q) αp ≠ αq
→ A rejection of the null hypothesis would indicate an influence of SLEEP (i.e., the
conditions of sleep or sleep deprivation) on the memory recall.
Factor B (influence of STRESS):
- Null hypothesis: H0B: ∀ (p, q) βp = βq; with βp = effect of corticosterone on
the memory recall and βq = effect of vehicle on the memory recall.
- Alternative hypothesis: H1B: ∃ (p, q): βp ≠ βq
→ A rejection of the null hypothesis would indicate an influence of STRESS (i.e.,
the conditions of corticosterone or vehicle infusion) on the memory recall.
Interaction of the factors A and B (influence of SLEEP and STRESS):
- Null hypothesis: H0AB: ∀ (p, q, r, s) γpq = γrs; with γpq = effect of sleep and cor-
ticosterone on the memory recall γrs = effect of sleep deprivation and vehicle
on the memory recall.
- Alternative hypothesis: H1AB: ∃ (p, q, r, s): γpq ≠ γrs
→ A rejection of the null hypothesis would indicate an interaction between SLEEP
and STRESS on the memory recall.
The results of the two-way ANOVA follow an F-distribution. By comparing the F-
statistic 𝐹𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 of each factor and their combination with the quantile 𝐹𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 of the
F-distribution at the respective significance level, the three null hypotheses can be
rejected if:
𝐹𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 > 𝐹𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙22.
22 A comprehensive description of the two-way ANOVA test as employed in this work is given in Köhler et al., 2012.
22
Testing for difference between specific conditions
Unfortunately, the two-way ANOVA test does have one major problem: positive re-
sults are difficult to interpret. A rejection of the null hypothesis means that there must
be at least one group among the k groups with a mean that differs significantly from
the other groups’ means at the chosen significance levels. However, the test gives
no indication, which group(s) this might be.
To solve this problem, different “a posteriori” or “post-hoc” multiple comparison tests
have been developed, of which the Student-Newman-Keuls test is the most powerful
because it applies subsequent testing. Its disadvantage is that neither the exact
value of probability of making a type I error nor the confidence intervals around the
differences between means can be computed (Abdi and Williams, 2010).
The Newman-Keuls test performs a pairwise comparison of group means 𝑑𝑔𝑟𝑜𝑢𝑝 to
determine, if they are different. Group means are ranked in ascending order and the
largest and smallest are compared for as long, as there are significant differences
between them. When the null hypothesis for a pair of means cannot be rejected –
i.e., the two means are not significantly different –, the test stops immediately and
for all other means of a smaller difference, the null hypothesis cannot be rejected
either.
The test for two specific means 𝑑𝑥 and 𝑑𝑦
is based on the studentized range distri-
bution and its 𝑞 statistic:
𝑞 =𝑑𝑥 −𝑑𝑦
√𝑀𝑆𝐸
2∗(
1
𝑛𝑥+
1
𝑛𝑦) ; with MSE = mean square error similar to ANOVA and nx, ny =
sample/group sizes.
The 𝑞 value is compared to the quantile 𝑞𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 of the q-distribution. If 𝑞 > 𝑞𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙,
the null hypothesis is rejected and the two groups’ means are considered signifi-
cantly different.
The original Student-Newman-Keuls test is intended for the analysis of balanced
design groups. The above variant of this scheme is suited for unequal sample sizes
and implemented in the software program SPSS23.
23 http://de.wikipedia.org/wiki/Post-hoc-Test#Student-Newman-Keuls-Test, 27.10.2016.
23
Testing that performance is different from chance
Finally, each group’s performance in the test trial is compared to the chance level.
For this purpose, the one-sample t-test for unpaired sample sizes24 is used. It com-
pares the experimentally collected mean of a group sample 𝑑𝑔𝑟𝑜𝑢𝑝 with the mean
over all groups ��.
The test statistic is calculated according to the following formula25:
𝑡𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 = |𝑑𝑔𝑟𝑜𝑢𝑝 −��|
𝑠∗ √𝑛; with n = sample/group size, d = arithmetic mean over all
groups, s = empirical standard deviation, dgroup = group mean.
The value for tobserved is then compared to the corresponding quantile tcritical of the t-
distribution. The null hypothesis is rejected, if 𝑡𝑜𝑏𝑠𝑒𝑟𝑣𝑒𝑑 > 𝑡𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙, which means that
the group’s mean of the test performance is different from the population’s mean of
the test performance, i.e., above chance level.
The software packages Statistica26 and SPSS IBM27 are employed for the statistical
evaluation.
24 http://de.wikipedia.org/wiki/Einstichproben-t-Test, 27.10.2016. 25 adapted from Köhler et al., 2012. 26 StatSoft Inc, OK, USA. 27 Armonk, NY, USA.
24
4 Results
4.1 Tasks
Elevated plus maze
All animals except number 11 (which was used for pilot surgery) were tested in the
elevated plus maze before the memory test.
With an average proportion of 45.0 ± 3.1 % of time spent in the open arms and 24.0
± 2.1 % of entrances into the open vs. closed arms, the rats showed normal levels
of anxiety compared to prior studies (Hogg, 1996). Also, there was no correlation
between the individual results of the elevated plus maze test and the performance
in any of the memory tests.
Sample trials
The duration and exploration time of the sample trials were comparable for both
tests in all four conditions (one sample t-test with a p value > 0.24 for all compari-
sons).
Retention intervals
Neither sleep onset nor sleep duration differed between the corticosterone- and ve-
hicle-injected sleeping conditions for either task (here, also the one sample t-test
was used). In the novel-object recognition task, sleep onset of the vehicle-injected
animals was 40.67 ± 4.99 min vs. 35.50 ± 3.87 min for the corticosterone-injected
animals (p = 0.42). The first group slept for 24.76 ± 3.12 min, while the latter slept
29.32 ± 3.53 min (p = 0.34). In the object-place recognition task, the vehicle-injected
animals fell asleep after 39.29 ± 6.19 min and the corticosterone-injected after 38.27
± 3.51 min (p = 0.89). Sleep duration in the first group was 28.13 ± 3.12 min vs.
24.12 ± 4.12 min in the second group (p = 0.50). The findings were comparable with
results from previous studies using similar procedures (Inostroza et al., 2013).
The infusion of corticosterone occurred on average at 38.80 ± 2.16 min in the sleep
conditions and 40.35 ± 1.33 min into the retention interval in the wake conditions (p
= 0.42).
25
The animals mostly remained asleep during the infusion. In only six cases transient
arousal occurred during the infusion procedure (three rats in the object-place-recog-
nition/corticosterone condition; one rat in the object-place recognition/vehicle condi-
tion and two rats in the novel-object recognition/vehicle condition).
Test trials
4.1.4.1 Novel-object recognition
The novel-object recognition test was passed through for 39 times in total28. Two-
way ANOVA test showed that neither the factor SLEEP (F (1,35) = 0.61, p = 0.44;
sleep/sleep-deprivation) nor the factor STRESS (F (1,35) = 0.09, p = 0.77; corti-
costerone/vehicle) affected the animals’ performance in the novel-object recognition
test in a significant way and that there was no significant interaction between the
factors (F (1,35) = 0.08, p = 0.78; SLEEP x STRESS). Furthermore, according to
the one sample t-test, the performance of all groups was above chance level (every
p < 0.01) (see Figure 7).
4.1.4.2 Object-place recognition
The object-place recognition test was passed through for 33 times in total29. Two-
way ANOVA test also indicated no significant effect of the factor SLEEP (F (1,29) =
0.07, p = 0.80; sleep/sleep-deprivation) or the factor STRESS (F (1,29) = 0.01, p =
0.92; corticosterone/vehicle) on the animals’ performance in the object-place recog-
nition test, when considered reclusively. However, a significant interaction was re-
vealed (F (1,29) = 11.09, p = 0.002; SLEEP x STRESS). The post-hoc analysis of
the results by the Student-Newman-Keuls test related the four conditions as follows.
In the sleeping conditions, the administration of corticosterone led to impaired test
performances in the object-place recognition task compared to the infusion of
vehicle (t(15) = 2.32, p = 0.035; sleep + corticosterone/sleep + vehicle), with the
vehicle-infused animals showing significant memory for the task (p = 0.007), while
the performance of the corticosterone-infused animals did not differ from chance (p
= 0.10).
28 With the minimum number of animals in the vehicle-and-sleep-deprivation condition (7), followed by the vehicle-and-sleep (9) and the corticosterone-and-sleep condition (10). The maximum number of rats was found in the corticosterone-and-sleep-deprivation condition (13). 29 With the minimum number of animals in the vehicle-and-sleep condition (6), followed by the vehi-cle-and-sleep-deprivation (7) and the corticosterone-and-sleep-deprivation condition (9) and ending up with the corticosterone-and-sleep condition (11).
26
Concerning the sleep-deprived conditions, the results were reversed. Here, the in-
fusion of corticosterone caused an increase in test performance in the object-place
recognition task compared to the infusion of vehicle (t(14) = 2.39, p = 0.032; sleep-
deprivation + corticosterone/sleep-deprivation + vehicle) with the vehicle-infused
animals not performing above chance (p = 0.18) and the corticosterone-infused an-
imals showing significant memory (p = 0.001).
In summary, the highest performances in the object-place-recognition test were
registered for the vehicle-and-sleep condition and the corticosterone-and-sleep-
deprivation condition, while the animals in the vehicle-and-sleep-deprivation condi-
tion and the corticosterone-and-sleep condition performed poorest (see Figure 7).
Figure 7. Results of the test trials30. The numbers associated with the bars indicate the number of ani-mals, which underwent the test. The star (*) marks the groups, that were significantly different from the other groups by Newman-Keuls posthoc test and t-test (each with p < 0.05). The (+) indicates that the test performances were above chance level (p < 0.05). The error bars indicate standard deviations. Sleep/Vehicle = infusion of vehicle during sleep; Wake/Depri = infusion of vehicle during sleep-depriva-tion; Sleep/Cort = infusion of corticosterone during sleep; Wake/Cort = infusion of corticosterone during sleep-deprivation.
30 Adapted from Kelemen, E., Bahrendt, M., Born, J., & Inostroza, M. (2014): Hippocampal corti-costerone impairs memory consolidation during sleep but improves consolidation in the wake state. Hippocampus, 24(5), 510-515.
27
4.2 Histological examination
From the total number of 24 animals, 20 underwent histological examination.
The excluded ones were either used for the pilot-surgery (number 11) or lost their
implants before the first test trial (number 9, 13, 22).
Among the included animals, the position of two cannulas could not be determined
at all (number 10 and 2) and the position of two other animals’ cannulas was only
detectable on one side (number 14 and 19) because of problems with the perfusion
of the rats’ brains. The cannulas of the remaining sixteen animals all hit parts of the
hippocampus with the following distribution31.
Most of the cannulas lay in the lacunosum moleculare of the hippocampus (fourteen
cannulas ~ 41,2 %), followed by the equally frequented oriens layer and radiatum
layer (each with five cannulas ~14,7 %) and the likewise balanced pyramidal cell
layer and molecular layer of the dentate gyrus (each with four cannulas ~ 11,8 %).
Only two cannulas were found in the field CA1/CA3 of the hippocampus (~ 5,9 %).
The partition of the positions reflects the arrangement of the structures in the hippo-
campus32, with the lacunosum moleculare in its center and the other layers grouped
around.
Figure 8. Results of the histological examination.
31 See Figures 8 and 9 and “10.4 Histological results” in the appendix, p. VI. 32 See “10.5 Stereotaxic coordinates of the rat brain” in the appendix, p. VII.
lacunosum moleculare
oriens layer
radiatum layer
pyramidal cell layer
molecular layer of dentategyrus
field CA1/CA3
28
Figure 9. Example photomicrograph illustrating the placement of the needle tips in one rat. The large arrows point to the cannula tips, the small ones to the infusion needle tips.
29
5 Discussion
5.1 Key findings
When comparing the results of the test trials with the hypotheses I raised at the
beginning of the experiment, I find all my assumptions confirmed.
Hippocampus-dependent memory
- Administration of corticosterone: The animals in the sleep-deprived corticosterone-
condition performed significantly better than the sleeping ones, whose results, in
turn, did not differ from chance.
- Administration of vehicle: The animals in the sleeping vehicle-condition performed
significantly better than the sleep-deprived ones, whose results, in turn, did not differ
from chance.
- Combination of SLEEP and STRESS factor: The sleeping vehicle-infused animals
performed significantly better than the sleep-deprived corticosterone-infused ones.
The sleep-deprived corticosterone-infused animals, on the other hand, performed
significantly better than the sleep-deprived vehicle-infused ones. Regarding the
question, if the animals in the corticosterone and sleep-deprivation condition per-
formed better than the rats in the vehicle and sleep condition, I could neither deter-
mine a significant effect of the SLEEP nor of the STRESS factor alone. The same
goes for the question if the animals in the corticosterone and sleep condition per-
formed worse than the rats in the vehicle and sleep-deprivation condition. It is only
their interaction which caused significant differences.
Hippocampus-independent memory
Concerning the influence of glucocorticoids and sleep on the consolidation and re-
call of hippocampus-independent memory as tested in the “novel-object recognition
test”, I also observed neither a significant effect of the SLEEP nor the STRESS fac-
tor alone. However, in contrast to the “object-place recognition test”, there was no
significant interaction between the two factors and no significant difference between
the performances of the four conditions (Bussey et al., 2000; Mumby et al., 2002).
Nevertheless, all animals performed above chance level, indicating that the test is
based on a learning process which however does not rely on different corticosterone
levels or sleeping conditions.
30
Where and when do glucocorticoids affect consolidation?
By applying these results to the question where and when glucocorticoids interfere
with the consolidation process of memories initially asked, the following theses can
now be stated:
1. The hippocampus with its many glucocorticoid receptors (Joëls, 2008;
McEwen et al., 1986; McEwen and Sapolsky, 1995; Reul and de Kloet, 1985)
plays a leading role in the consolidation of declarative memory (Carr et al.,
2011; Eichenbaum, 1993). This thesis is emphasized by the fact that we can
take direct influence in the consolidation process of hippocampus-dependent
memory via the administration of glucocorticoids through cannulas implanted
selectively into the dorsal hippocampus33. Furthermore, the administration of
vehicle - regardless of the test - does not influence the test performance when
considered alone, indicating that there has to be a selective binding site for
corticosterone in the hippocampus (Lupien and Lepage, 2001).
2. The consolidation of hippocampus-dependent memories differs dependent
upon vigilance-state34. This thesis is emphasized by the fact that the animals
in the vehicle-injected conditions that could sleep performed significantly bet-
ter than those which were sleep-deprived. At the same time, there was no
difference in the results of the test trials between the sleeping and the sleep-
deprived animals in the hippocampus-independent “novel-object recognition
test”.
3. The consolidation of hippocampus-dependent memories is modulated by
glucocorticoids. Similar to the conclusions to which Plihal and Born (1999),
Plihal et al. (1999) and Wilhelm et al. (2011) came, when studying the effects
of a systemic administration of glucocorticoids on the memory recall in
humans, I assess that the administration of corticosterone during
wakefulness enhances the consolidation and recall of hippocampus-
dependent memories, while its administration during sleep impairs it. How-
ever, the intrahippocampal infusion of corticosterone did not have any effect
on the animals’ performance in the hippocampus-independent “novel-object
33 The post-hoc histological examination shows that only three cannulas are not in the right position: one is not detectable at all and two are only detectable for one side. 34 While the reactivation of memories during wakefulness destabilizes them due to additional pro-cessing of external stimuli, they are stabilized by the reactivation during sleep (Debiec et al., 2002; Diekelmann et al., 2011; Gupta et al., 2010; Jezek et al., 2002; Nader and Hardt, 2009).
31
recognition test”. Evidence suggests that the converse effects of the gluco-
corticoids may be due to the diminution of sharp wave and ripple events
usually accompanying hippocampal memory reactivation by high levels of
stress hormones (Weiss et al., 2008). Along with the wake-sleep-dependent
differences in the consolidation of hippocampus-dependent memories,
reduced memory reactivation during a waking period would help to diminish
the labilization of respective memory traces (thus making them less
vulnerable to non-specific interference), while reduced memory reactivation
during a sleeping period would help to diminish their stabilization (Wilhelm et
al., 2011). Concerning the initially mentioned “contextual binding” theory
(Sazma et al., 2018), another possible explanation for the vigilance-
dependent effects of corticosterone could be a change of context during
sleep. While the administration of stress hormones during a wake state
occurs in a similar context as the learning materials, the mental context
during sleep conditions is notably different. Therefore, a rise in stress
hormones during sleep strengthens the consolidation of events that occur in
the new context, but not for events that occurred before the context shift and
the resulting network reset. Sazma et al. deduce that a coordinated activity
of the hippocampus and HPA-axis is needed for glucocorticoids to enhance
the consolidation of memories.
5.2 Related work
Dose-dependent effects of glucocorticoids
Concerning the dose-dependency of the glucocorticoids’ effects as reported by
Okuda et al. (2004), Roozendaal et al. (2000; 2002) and Sandi and Rose (1994),
the present study was not able to differentiate between the absolute levels of the
drug in the corticosterone conditions (all animals received the same amount of
corticosterone). The four conditions of each test can, however, be arranged
according to the wake-sleep-dependent changes of the natural corticosterone
concentration (Penalva et al., 2003). Because rats are night-active animals, the
lowest levels of corticosterone are found at the beginning of the light period
(sleeping period); there is an abrupt rise in the corticosterone concentration towards
the end of the light period, reaching a peak level at the beginning of the dark period
(waking period).
32
Regarding the influence of sleep deprivation on the natural corticosterone levels,
there are inconsistent reports. Zant et al. (2011) describe no increase in the corti-
costerone concentration during the first three hours of sleep deprivation, whereas
Penalva et al. (2003) depict a marked increase after one hour and Meerlo et al.
(2002) after six hours. Transferring these observations to the four test conditions
(and keeping in mind that the experiments were executed during the first half of the
animals’ night), the following order can be set up: the lowest concentration of
corticosterone is present in the sleeping vehicle condition, a medium concentration
can be found in the sleep-deprived vehicle- and the sleeping corticosterone
condition, while the sleep-deprived corticosterone condition must have had the
highest levels of corticosterone. The graphical account of the relationships produces
a U-shaped curve (see Figure 7) similar to the one Okuda et al. (2004) achieve in
their studies.
They used an “object-recognition task” comparable to the one used in this study, but
examined the effects of corticosterone and vehicle injected systemically three
minutes after the sample trial on the performance of a test trial one hour and 24
hours later in two different conditions: the animals in the first condition were not
habituated to the training apparatus, while the ones in the second condition got to
explore it twice a day for one week prior to the experiments. Thus, they expected
different levels of arousal to be present in the two conditions35. The results regarding
performance after a retention interval of one hour revealed the already mentioned
U-shaped curve for the condition without habituation to the training apparatus36,
while there was no difference in performance of the animals that were habituated to
the experimental setup before the tasks. Okuda et al. (2004) deduce that arousal is
necessary for the modulating effect of glucocorticoids on the consolidation of hippo-
campus-dependent memories.
Arousal-dependent effects of glucocorticoids
Okuda’s thesis is further supported by the observation that stress hormones do not
uniformly modulate the consolidation of all kinds of information, but rather selectively
affect the memory of emotionally laden information (de Quervain et al., 2009). The
35 That is, a high level in the condition without habituation to the experimental setup and a low level in the condition with habituation to the experimental setup. 36 The highest discrimination index was found in the vehicle-injected condition, decreasing indexes in the conditions that received infusions with 0.3 and 1.0 mg/kg of corticosterone and an increasing index in the condition which got 3.0 mg/kg of corticosterone.
33
explanation for the forenamed phenomenon is given by neuroanatomical studies
analyzing the amygdala as the center of threat detection and triggered conditioned
and unconditioned fear responses. They revealed that emotionally arousing
experiences induce a release of norepinephrine in the basolateral amygdala (Galvez
et al., 1996; McIntyre et al., 2002; Quirarte et al., 1998), while lesions or
pharmacological inactivation of the amygdala (as through a blockade of β-
adrenoreceptors) prevent glucocorticoid effects on memory consolidation as well as
on memory recall (Quirarte et al., 1997; Roozendaal et al., 1999; Roozendaal et al.,
2002).
Connection between dose- und arousal-dependent effects of glucocor-
ticoids
But where is now the link between Okuda’s results concerning different levels of
arousal (i.e., different levels of norepinephrine) and the present results regarding
different wake-sleep-states?
Note, that I tried to avoid emotional arousal in the study design by habituating the
animals to the transport, the experimental room and its setup, to be able to investi-
gate the effect of corticosterone apart from influences by other hormones such as
norepinephrine.
Nevertheless, there are circadian changes in the natural norepinephrine concentra-
tion similar to the changes described for corticosterone which cannot be controlled
(Kalén et al., 1989). While there are high levels of norepinephrine present during
wakefulness, its concentration drops during sleep. Concordantly, Tononi et al.
(1994) found that wakefulness – whether spontaneous or induced – leads to an
increased expression of immediate early gene products such as FOS and NGFI-A
as markers of functional activity in noradrenergic neurons of the locus coeruleus.
Transferring this information to the present model, the lowest levels of norepineph-
rine would be present in the sleeping vehicle and the sleeping corticosterone condi-
tion, whereas the highest levels would occur in the sleep-deprived animals. These
natural changes of the norepinephrine concentration could explain the differences
in the modulating effect of corticosterone on the consolidation of hippocampus-
dependent memory despite the absence of artificially induced emotional arousal.
Because the concentration of norepinephrine is low during sleep (low emotional
arousal) and a high concentration (high emotional arousal) is needed for
34
glucocorticoids to take influence on re-encoding processes (McReynolds et al.,
2010; Roozendaal et al., 2006a; Roozendaal et al., 2006b), the infusion of
corticosterone had no effect on the sleeping animals. In sleep-deprived animals,
where the level of norepinephrine is naturally higher, the performance in the object-
place recognition test was, however, enhanced.
This hypothesis is further backed up by the recently introduced “tagging” theorem:
it implies that high levels of cortisol and norepinephrine during the encoding process
promote the effective “tagging” of memories that had elicited responses in the hip-
pocampus and amygdala. By initiating molecular cascades that upscale the protein
synthesis they promote long-term plasticity and therefore serve as a “mnemonic fil-
ter” (de Jesús and Fishbein, 2018; Payne and Kensinger, 2018; Ritchey et al., 2017;
Sazma et al., 2018).
In the present study, I did not focus on different effects of corticosterone as a
function of a varying time of administration. While in the sleeping conditions the
infusions were given one minute after the animals had fallen asleep (at 38.80 ± 2.16
min), they were administered after 40.35 ± 1.33 min in the sleep-deprived conditions
(the individual times differ from 16 to 58 minutes depending on the mean time of
sleep onset of the animals examined the days before). Nevertheless, de Quervain’s
and his team’s discovery (1998, 2009) that the more time between the encoding
phase and the administration of corticosterone the worse the recall could also be
interpreted based on different levels of norepinephrine. Granted that running
through an experiment always causes some kind of arousal and that the latter itself
leads to an increase of the norepinephrine concentration in the body as well as in
the brain, one would expect the following constellation: high levels of norepinephrine
for the time directly after the experiment, decreasing during the interval where there
was no task to be attended. Glucocorticoids administered shortly after a sample trial
would, therefore, meet up with high concentrations of norepinephrine and enhance
the consolidation of hippocampus-dependent memory, whereas an infusion after
some time of stress-recovery would coincide with low levels of norepinephrine pre-
venting the glucocorticoids’ positive impact.
35
5.3 Limitations
A limitation of the study certainly is the loss of animals with intact implants resulting
in unbalanced treatment conditions with small numbers. To verify the effects and to
improve the validity of the statistics, a rerun of the experiments with at least 24 ani-
mals as claimed by Ennaceur et al. (1997) should be conducted.
The aim to prevent the rats from losing their cannulas before completing the exper-
iment requires improvements to the implants, on the one hand, and to the apparatus
for the drug delivery, on the other hand. Suggestions are given in the following sub-
sections.
Improvements to the implants
Concerning the implants, I used four fixing screws (two above and two below the
holes for the cannulas) to provide additional attachment for the dental resin. An aug-
mentation of the number and/or of the size and a shift of their position to the corners
of the skull could perhaps enhance the adhesion. Furthermore, there may be other
kinds of fixing material (like cement for example) which stick better to the bone sur-
face than dental resin (which has a very smooth surface and therefore a low fric-
tional resistance) and are lighter. The latter feature would possibly avoid the decli-
nation of the animal’s head due to the “extra” weight of the implant which itself in-
creases the tension of the connected tubing. Finally, I observed liquorrhea with
some animals after the implantation of the cannulas indicating a puncture of the
ventricles. The fluid formed cavities between the skull’s surface and the lower side
of the implants in some animals causing further instability. The problem could be
solved by separately sealing the holes of the cannulas after the implantation or by
changing their positions to prevent penetration of the lateral cerebral ventricles.
Improvements to the apparatus for drug delivery
Regarding the apparatus for drug delivery, the weight of the tubing could also be
decreased similarly to the fixing material. I tried to achieve a reduction by stripping
the tubing off their metal coats during the experiment37. Unfortunately, some animals
started to nibble through the exposed polyethylene causing its contents to spread
uncontrollably in the boxes as well as in the brains. The best solution would hence
be tubing with reinforcements against gnawing. However, they should be lightweight
37 See photo 3 in “10.6 Photos of the experimental room” in the appendix, p. X.
36
(i.e., titanium or carbon covers). Additionally, their suspension could be improved by
using a rotating disk to which the tubing leading to the cannulas is connected on the
lower side and the tubing leading to the syringes and the pump on the upper side.
This set-up would serve three purposes:
1. It would decrease the weight on the animals’ heads (as the tubing is fixed to
the platform).
2. It would prevent the entangling of the suspending cords (contributing to the
pull on the implants by shortening their total length).
3. It would reduce the transfer of the animals’ individual movements to the
neighboring rats via the clothesline and the adjacent cords (preventing them
from being stirred up in the sleep conditions).
Finally, the sleep environment could be improved by allocating a separate pump to
each retention box (avoiding the replugging of the syringes after the administration
of drugs for the following animals), by muting its operating noise and by operating it
by remote control.
Improvements to the study design
In addition to the technical improvements of an experimental rerun, also modifica-
tions to the future study design itself should be made. First, a fixed time for the
administration of corticosterone or vehicle in the sleep-deprived conditions should
be determined before starting with the tasks. That would ameliorate the
comparability of the drugs’ effects on the performances in these conditions and
contribute to the observation that the time of the infusion interferes with the
experimental outcome (the more time between the encoding phase and the
administration of glucocorticoids the worse the recall; de Quervain et al., 1998,
2009).
Then, it would be desirable to have a simultaneous recording of EEG- and EMG-
waves to be able to identify the exact time of sleep-onset and the percentage of the
different sleep states in the sleeping conditions. The forenamed combination would
also help to differentiate between relaxed wakefulness and sleep in the sleep-de-
prived animals. That, in turn, should reduce the liberation of additional stress
hormones due to external stimulation and thus account for stabilization of the inde-
pendent variables.
37
The third improvement concerns the observation made by Sazma et al., 2018 that
post-encoding stress has different effects on memory consolidation depending on
whether the animals are habituated to the learning context prior to enconding, or
not. If they are habituated, post-encoding stress leads to an impairment of memory,
if not, the results of the animals do not differ from chance or are even improved. The
authors conclude a so called “contextual binding” meaning that the stressor itself
serves as a memorable event enhancing memories for other events that share the
same context. It would be interesting to see, if I were able to reproduce the results
of my study even in groups that were not habituated to the learning context and
which role sleep played (Sazma et al. suggest a “change of context” in sleep, leading
to opposing effects of stress hormones during wakefulness and sleep).
Furthermore, Oyanedel et al. showed in 2018 that the effects of sleep on the con-
solidation of episodic memory are stronger in a slightly different task paradigm, the
so called “what-where-which” test. It consists of two sample phases and one test
phase. In sample phase one, the animals are confronted with two different objects
in context A. In sample phase two, the same two objects with swapped locations are
presented in a new context B. For the test phase, two identical copies of one of the
objects from the sample phases are presented either in context A or B. At that time,
one of the objects is located at the same spot as during the sample phase, while the
other one is presented in a new location and context. Here, binding in episodic
memory is expressed by an enhanced exploration of the displaced object in the
novel context. The authors compared both test forms, the “what-where-when” test
which we used, too, and the “what-where-which” test with regard to different effects
of sleep on the consolidation of episodic memory. Their explanation for the better
results of the “what-where-which” task paradigm is that the latter neglects the tem-
poral aspect which seems to be covered separately from the visuospatial context
and therefore emerges later on in the test phase. A rerun of my study with the “what-
where-which” test could perhaps produce even clearer data.
Last but not least, recent studies (Sawangjit et al., 2018) suggest that common hip-
pocampal mechanisms enhance consolidation in both hippocampus-dependent and
-independent memory systems through reactivation of contextual features. The au-
thors deduce that all memories have to be encoded within a spatiotemporal context
- that is, as episodic memories. Therefore, the hippocampus-dependent episodic
memory system is designated a super-ordinate position to organize long-term
38
memory in general and the classification into hippocampus-dependent and -inde-
pent memories is brought into question.
Improvements to the preparation of drugs
One last suggestion concerns the preparation of the drugs. Because corticosterone
is insoluble in water, I used a preformed water-soluble complex of corticosterone
and 2-hydroxypropyl-β-cyclodextrin38. However, I did not include the cyclodextrin in
the vehicle. Studies investigating the utility of cyclodextrin as a vehicle for the central
nervous administration of drugs (Yaksh et al., 1991) though describe no effect upon
the nociceptive or motor function after intrathecal infusion or on EEG and general
behavior after intracerebral infusion in concentrations as high as 20-40 % w/v (the
present product contains 7 % w/v of corticosterone in 2-hydroxypropyl-beta-
cyclodextrin according to the certificate of analysis39). However, they observe an
increase in the respective durations of the effects regarding the intrathecally deliv-
ered drugs. The authors speculate that cyclodextrin could alter the rate of appear-
ance of free drug and, thereby, its clearance. Furthermore, they hypothesize that
the agent may have reduced the rate at which the peptides were metabolized in the
spinal space (as the incorporated drug may be at least partially sheltered from en-
zymatic hydrolysis). Nevertheless, the effect cannot be reported for the intracerebral
administration of drugs – probably also because Yaksh et al. did not apply quantita-
tive testing like the hotplate and the tail flick test to the second group. Note, that the
intracerebrally delivered drugs did not have direct contact to the hippocampus
(Yaksh et al. used a ventricular cannula (24 ga SS TW) placed 0.5 mm lateral to the
bregma and 3 mm from the dura) and that the scientists did not utilize hormones but
capsaicin and opioids to be solved in cyclodextrin.
It would, therefore, be important to know whether the intracerebral administration of
cyclodextrin has an effect of its own on brain cells in general and hippocampal cells
in particular and whether the combination with corticosterone changes the clearance
of the hormone and thus enhances its effect compared to naturally circulating stress
hormones. I contacted the producer Sigma Aldrich for further information about the
38 C174 SIGMA – Corticosterone: HBC complex, Sigma Aldrich, St. Louis, MO, USA; http://www.sigmaaldrich.com/catalog/product/sigma/C174?lang=de®ion=DE, 27.10.2016. 39 http://www.sigmaaldrich.com/Graphics/COfAInfo/SigmaSAPQM/COFA/C1/C174/C174-BULK _________041M4606V_.pdf, 27.10.2016.
39
substances’ implications on rat brains but was instructed that they did not conduct
studies with their products.
Despite the disadvantages mentioned above, there are not many other possibilities
to dissolve corticosterone. Yaksh et al. compared the characteristics of cyclodextrin
as a vehicle to dimethylsulfoxide and ethanol. In contrast to cyclodextrin, the in-
trathecal administration of dimethylsulfoxide directly influences the cell function by
reducing the levels of substance P and calcitonin gene-related peptide as well as
the micturition reflex and the resting blood pressure. Moreover, dimethylsulfoxide
solutions are typically prone to precipitation of the drug when diluted with saline.
Ethanol, on the other hand, is known to impair memory both after acute and chronic
administration by stimulating hippocampal gamma-aminobutyric-acid (GABA)-A-re-
ceptors and thus inhibiting N-methyl-D-aspartate (NMDA)-receptor-dependent
synaptic long-term potentiation and long-term depression in the hippocampus (Dere
at al., 2007). While long-term potentiation seems to promote spatial learning
(Hölscher, 1999), long-term depression is suggested to be critical for one trial object
recognition (Kemp and Manahan-Vaughan, 2004). Although infusions of ethanol in
a dose of 2.4 g/kg after the sample trial did not impair one-trial object recognition in
C75BL/6J mice after a 24-h retention interval (Ryabinin et al., 2002), there are no
studies that examine the effect on performances after an 80 minutes retention
interval (when the ethanol concentration in the brain presumably is still above zero).
Additionally, ethanol does have a sedative effect which could mingle with the wake-
sleep-dependent effects of glucocorticoids I aim to examine.
Furthermore, there are suggestions to dissolve corticosterone in oil-based vehicles
(i.e., sesame oil). These also drop out for the present study design because their
adhesive power deters them from being delivered through cannulas with a diameter
of 0.5 mm. Additionally, there is no research at all concerning the effect of
intracerebrally administered oil neither on the function of the brain in general nor the
encoding and consolidation of hippocampus-dependent memories.
Finally, the C174 SIGMA – Corticosterone: HBC complex I used was prepared as
free-flowing powder in a package of 100 mg. I had to measure 5 mg manually each
day with a fine-scale which turned out to be somewhat tricky because of the pow-
der’s susceptibility to humidity (note, that the experiments were conducted during
midsummer) and the inaccuracy of the scale. Thus, I cannot exclude differences in
the concentration of the solution. The preparation process would have been made
40
easier by employing a more accurate fine-scale or using readily packed-up doses
of 5 mg Corticosterone: HBC complex. Furthermore, I could have prepared a stock
solution for all corticosterone-days with the 100 mg package (I did not do so because
I did not know, if the corticosterone would precipitate or the solution would change
in other ways during the course of the experiment) or asked the manufacturer of the
powder to do so for me, too.
Remarks by reviewers
When my work was accepted for publication in the journal Hippocampus, reviewers
remarked that I did no control infusions of corticosterone in other brain regions and
though could not prove that the described effects were due to an interaction with
glucocorticoid receptors in the hippocampus itself. Furthermore, I neither measured
the local spreading of the substance in the brain nor the distribution via the blood
circulation. To be able to certify a single interaction of stress hormones in the hip-
pocampus it would, therefore, be necessary to identify and exclude other brain areas
also containing glucocorticoid receptors and to prevent the dispersion of the sub-
stance throughout the brain. The latter will probably be almost impossible, as there
is no way to isolate the hippocampus from the rest of the brain in a living organism.
A solution for the problem could, for example, be an adaption of the corticosterone
dose to a portion which still is sufficient to occupy enough local hippocampal recep-
tors to cause significant effects in memory testing, but does not produce any inter-
fering side-effects of adjacent brain structures. Additionally, one could try to connect
it to bigger or hippocampus-specific (i.e., antibodies) molecules or to block the glu-
cocorticoid receptors of the other brain areas. With regard to corticosterone levels
in the blood, one should bear in mind that the substance has to be absorbed into
the blood and to cross the blood-brain-barrier before entering the systemic blood
circulation in a quantifiable concentration. Andersen et al. (2006) describes in his
book the use of osmotic minipumps to enable a steady concentration of drugs in
specific brain areas and the use of autoradiography to mark the local spread of a
radiolabeled drug (Attwell et al., 2001; Steele and Morris, 1999).
41
5.4 Future work
Sleep-stage dependent differences in memory consolidation
Apart from the improvements named above, it would be desirable to have a
simultaneous recording of EEG- and EMG during the retention interval, as evidence
suggests differences concerning the functions and the type of information being
encoded between the various sleep stages resulting in converse effects of circulat-
ing transmitters. The dual-process hypothesis promoted by Maquet (2001) accredits
facilitation of declarative, hippocampus-dependent memory to slow-wave sleep
(SWS) and facilitation of non-declarative, hippocampus-independent memory to
rapid-eye-movement (REM) sleep. The sequential hypothesis, on the other hand,
states that the optimum benefits of sleep on the consolidation of declarative and
non-declarative memory occurs, when SWS is followed by REM sleep (Gais et al.,
2000; Giuditta et al., 1995). Diekelmann and Born, who compared both theses in a
paper in 2010 and favored the second one, explained the process as follows. The
active system consolidation, which takes place during the SWS and integrates newly
encoded memories with pre-existing long-term memories inducing conformational
changes in the respective representations, is complemented by synaptic
consolidation stabilizing the transformed memories during REM sleep. Different
levels of neuronal activity accompany both processes. While there is a widespread
synchronization of neuronal activity induced by slow oscillations during SWS, REM
sleep is characterized by de-synchronization possibly reflecting a disengagement of
memory systems sheltering the synapses from external stimuli. The authors’ prefer-
ence is supported by more recent studies in which only the iterative interleaving of
non-REM and REM sleep is said to boost the formation of complex knowledge
framework and thus allow creative thought (Lewis et al., 2018). Lewis et al. connect
non-REM sleep with the encoding of new memories and the abstraction of rules (so
called “schema” learning), while REM sleep is associated with the replay of memo-
ries already encoded and the formation of novel, unexpected connections.
Consistent with the study design, performance of the animals in the hippocampus-
dependent object-place recognition test should increase with the total amount of
sleep, the percentage of SWS and the cycles of SWS followed by REM sleep. Dur-
ing the hippocampus-independent novel-object recognition test, however, it should
be enhanced with the percentage of REM sleep (in addition to the total amount of
sleep and the cycles of SWS followed by REM sleep). It would be interesting to see,
42
whether a deprivation of SWS or REM sleep, respectively, affects the consolidation
of hippocampus-dependent or rather hippocampus-independent memories as
stated by the dual-process hypothesis, or if there is an improvement in performance
the more sleep-cycles the subjects complete as to be deduced from the sequential
hypothesis.
Effects of glucocorticoids on sleep-stage dependent EEG-rhythms
A concomitant EEG-recording could further illuminate the function of glucocorticoids
in the consolidation process of hippocampus-dependent memories. As already
indicated above, SWS and REM sleep are characterized by specific rhythms in brain
electric activity. SWS, for example, is dominated by so-called slow oscillations which
have their origin in the neocortex and regulate the dialogue between the cortex and
subcortical structures by inducing up- and down-states of neuronal activity (i.e.,
depolarization and hyperpolarization; Destexhe et al., 2007; Luczak et al., 2007;
Steriade, 2006). Thalamocortical spindles and hippocampal sharp-wave ripples typ-
ically occur with a specific temporal relationship to each other and the slow oscilla-
tion (Isomura et al., 2006; Mölle et al., 2002; Mölle et al., 2006; Sirota et al., 2003;
Steriade et al., 2006). The spindles themselves seem to prime cortical networks for
the long-term storage of memory representations (Diekelmann and Born, 2010),
while the sharp-wave ripples accompany the neuronal replay of the previously
encoded information (Buzsáki, 1989; Nádasdy et al., 1999; Peyrache et al., 2009;
Wilson and McNaughton, 1994).
During REM sleep, on the other hand, ponto-geniculo-occipital (PGO) waves origi-
nated in the pontine brain stem and theta activity predominating the hippocampus
are found (Diekelmann and Born, 2010). Their function is somewhat unclear – PGO-
waves have been associated with increased activity of plasticity-related immediate
early genes and brain-derived neurotrophic factor in the dorsal hippocampus (Datta,
2006), whereas theta oscillations similar to sharp-wave ripples seem to play a role
in neuronal replay (Louie and Wilson, 2001; Poe et al., 200040).
Evidence suggests that neuromodulators such as acetylcholine and glucocorticoids
act in parallel and serve as a switch between the sleep-stage-dependent processes
40 Hippocampal place cells encoding a familiar route are reactivated preferentially during the throughs of theta oscillations, while place cells encoding novel routes are reactivated during the peaks.
43
of memory reactivation and redistribution to the neocortex (during SWS) and syn-
aptic consolidation (during REM sleep). While low concentrations during SWS are
suggested to enable a flow of information from the hippocampus to the neocortex
by reducing the tonic inhibition of hippocampal CA3 and CA1 feedback cells
(Hasselmo, 1999; Hasselmo and McGaughy, 2004; Lewis et al., 2018; Marshall and
Born, 2007), high concentrations during REM sleep might isolate the neocortical
synapses from further information which could interact with the synaptic
consolidation. One might speculate that an intrahippocampal infusion of
corticosterone during sleep would, therefore, cause a decrease of the hippocampal
sharp-wave ripples during SWS and an increase of hippocampal theta oscillations
during REM sleep. This could be investigated by administrating corticosterone sep-
arately during SWS and REM sleep.
Furthermore, it remains to be ascertained whether externally delivered corti-
costerone also affects neocortical rhythms such as spindle and slow oscillatory ac-
tivity. Previous studies provide evidence that the latter seem to play a significant
role in the consolidation of hippocampus-dependent memory (Oyanedel et al.,
2014), but there is a lack of knowledge whether glucocorticoids affect these rhythms
in any way. The proposal that high cholinergic activity during REM sleep might
promote synaptic consolidation by supporting plasticity-related immediate early
gene activity (Teber et al., 2004) and the maintenance of long-term potentiation
(Lopes Aguiar et al., 2008) suggests a possible interaction with the downscaling
function of slow oscillations as promoted by the synaptic homeostasis hypothesis.
By eliminating weak connections and preserving the relative strength of the remain-
ing connections, the signal-to-noise ratio is improved, and synapses can be reused
for future encoding (Dash et al., 2009; Vyazovskiy et al., 2008).
On the other hand, studies assert that although there is a global decrease in the
expression of markers indicating synaptic potentiation after a period of sleep, they
are increasingly expressed in specific regions at the same time (Ribeiro et al., 1999,
2004, 2007).
Additionally, behavioral experiments indicate a more significant benefit from sleep
for weakly than for strongly encoded memories (Drosopoulos et al., 2007; Kuriyama
et al., 2004). Diekelmann and Born (2010) therefore conclude that synaptic
downscaling is only one feature of sleep-dependent consolidation facilitating the en-
coding of new information through clearing saturated synapses for reuse. Further
44
research has to be done to uncover the synaptic processes behind the encoding
and consolidation of memory.
45
6 Summary
The present study examines the effect of sleep and stress on the consolidation of
episodic, i.e., hippocampus-dependent memory.
It originates from the bias that glucocorticoids, on the one hand, seem to consistently
enhance the encoding of memories, while their effects on memory consolidation
remain inconsistent41. Possible explanations involve vigilance42 and, in particular,
sleep-stage43 dependent effects. The critical preliminary study was conducted by
Wilhelm et al. in 2011. They were able to show facilitatory effects of glucocorticoids
on memory consolidation when administered during wakefulness and inhibitory
effects, when administered during sleep. However, they employed a human model
in which corticosterone was administered systemically, so they could neither deter-
mine the exact time at which the hormone crossed the blood-brain-barrier nor its
specific intracerebral concentration nor the region of its effect.
To determine where and when glucocorticoids would interfere with the memory con-
solidation, I used an animal model with rats that got intracerebral infusions of corti-
costerone via cannulas in their dorsal hippocampi44 at a predetermined point in time.
In a so-called “within-subject design”, the animals had to undergo two tasks (hippo-
campus-independent novel-object recognition task and hippocampus-dependent
object-place recognition task) under four conditions (infusion of corticosterone or
vehicle during an 80 minutes retention interval, while the rats were allowed to sleep
or were sleep-deprived).
I hypothesized that the performance of the rats in the hippocampus-dependent task
would differ dependent upon the substance and vigilance state, while there would
be no effects on the hippocampus-independent task. The results proved the hypoth-
esis as correct. While the retrieval of memories in the hippocampus-independent
novel-object recognition task was neither affected by the factor SLEEP (F (1,35) =
0.61, p = 0.44) nor by the factor STRESS (F (1,35) = 0.09, p = 0.77) nor by their
interaction (F (1,35) = 0.08, p = 0.78), a significant interaction in the hippocampus-
dependent object-place recognition task was found (F (1,29) = 11.09, p = 0.002).
41 de Quervain et al., 1998; de Quervain et al., 2009; Kirschbaum et al., 1996; Maheu et al., 2004; Rimmele et al., 2012; Wolf, 2009. 42 de Kloet et al., 1999; de Quervain et al., 2009; McIntyre et al., 2012; Schwabe et al., 2012. 43 Born and Wagner, 2004; Plihal and Born, 1999; Plihal et al., 1999. 44 As the hippocampus has abundant glucocorticoid receptors (Joёls, 2008) and hippocampus-de-pendent memories seem to be very sensitive to stress hormones (Lupien and Lepage, 2001).
46
The administration of corticosterone during a sleeping interval impaired consolida-
tion compared to vehicle-infused condition (t (15) = 2.32, p = 0.035), whereas corti-
costerone enhanced performance, when applied during a sleep-deprived interval
compared to the vehicle-infused condition (t (14) = 2.39, p = 0.032).
In conclusion, this study supports the concept that (1.) the hippocampus with its
abundant glucocorticoid receptors (Carr et al., 2011; Eichenbaum 1993) plays a key
role in the consolidation of hippocampus-dependent memories; and (2.) the effects
of corticosterone differ vigilance- and stress-dependently (see Figure 10).
This work hence poses the new research question which mechanisms account for
the opposing effects of stress hormones on the consolidation of hippocampus-de-
pendent memories.
sample trial test trialretention
interval
consolidation
Direct interaction of
corticosterone with
glucocorticoid-receptors in
the hippocampus
Result
sleep-deprivation
+ corticosterone
= memory
sample trial test trialretention
interval
consolidation Result
sleep
+ corticosterone
= memory
Figure 10. Summary of the results.
47
7 Summary in German
7.1 Einleitung
Während der letzten vierhundert Jahre wurde der Hippocampus in Verbindung mit
vielen unterschiedlichen Funktionen gebracht, u.a. sagte man ihm eine Mitwirkung
am Geruchssinn als Teil der Riechhirnformation sowie der Verarbeitung und des
Ausdrucks von Emotionen über den Papez’schen Neuronenkreis nach (Andersen
et al., 2006).
Heutzutage sehen Forscher die Hauptaufgabe des Hippocampus in der Abspeiche-
rung und Abfrage der Langzeitgedächtnisinhalte für Fakten und Ereignisse, auch
als deklaratives oder explizites Gedächtnis bezeichnet. Demgegenüber sind für das
nicht-deklarative oder implizite Gedächtnis (z.B. prozedurales Gedächtnis, Priming,
klassische Konditionierung und nicht-assoziatives Lernen) vor allem Strukturen wie
das Striatum, der Motor- oder Neokortex, das Kleinhirn, die Amygdala oder Re-
flexwege zuständig.
Das deklarative Gedächtnis wird wiederum in das episodische und das semantische
Gedächtnis aufgeteilt (Squire et al., 1984; Squire und Zola-Morgan, 1991; Squire,
1992; Squire, 2004; Andersen et al., 2006; siehe Abbildung 3). Der Begriff episo-
disch steht dabei für die Verknüpfung eines bestimmten Ereignisses (was) mit ei-
nem bestimmten Zeitpunkt (wann) und einem bestimmten Ort (wo).
Aber wie läuft der Prozess der Gedächtnisbildung nun ab? Es werden mindestens
drei verschieden Stufen, die zur Verankerung einer Information im Gehirn notwen-
dig sind, unterschieden. In der ersten Stufe, dem Enkodierungsprozess, werden
neue Daten in das Arbeitsgedächtnis (Kurzzeitgedächtnis) aufgenommen und ge-
speichert. In der zweiten Stufe, dem Konsolidierungsprozess, werden die neuen In-
formationen vom Kurz- in das Langzeitgedächtnis überführt. In der dritten Stufe,
dem Abrufungsprozess, werden die Daten dann aus dem Langzeitgedächtnis ab-
gerufen (Andersen et al., 2006; siehe Abbildung 3).
Hierbei auftretende synaptische Umbauprozesse werden unter dem Begriff “synap-
tische Plastizität” bzw. „zelluläre Plastizität“ zusammengefasst (Martin et al., 2000).
Die Verbindung zwischen Zellen kann hierbei gestärkt (long-term potentiation) oder
geschwächt (long-term depression) werden. Diese Vorgänge werden durch unter-
schiedliche Umweltfaktoren beeinflusst. Zwei davon sind Schlaf und Stress.
48
Jüngste Forschungsergebnisse belegen, dass Schlaf die Konsolidierung und damit
den Abruf von Langzeitgedächtnisinhalten fördert. Dabei werden den verschiede-
nen Schlafstadien REM (rapid-eye-movement) und SWS (slow-wave sleep) unter-
schiedliche Funktionen nachgesagt. Während des REM-Schlafes soll die synapti-
sche Konsolidierung von Gedächtnisinhalten durch die Trennung von Langzeit- und
Kurzzeitgedächtnisspeichern gefördert werden. SWS wiederum unterstützt die Re-
aktivierung und Neuverteilung von Daten des Kurzzeitgedächtnisses auf das Lang-
zeitgedächtnis (Diekelmann und Born, 2010). Neuste Studien (Lewis et al., 2018)
belegen diese Theorie. In ihrem so genannten „BiOtA“-Modell (broader form of the
information overlap to abstract framework, zu deutsch: breitere Form der Informa-
tionsüberschneidung in einem abstrakten Netzwerk) assoziieren die Autoren mit
dem non-REM-Schlaf sogenanntes „Schemalernen“, d.h. Abstrahieren von Informa-
tionen aus vorher Gelerntem. Während des REM-Schlafes soll dann die Neuver-
knüpfung von Informationen stattfinden. Lewis et al. postulieren, dass nur durch das
Zusammenspiel beider Schlafformen die Bildung von komplexen Gedächtnisstruk-
turen und damit kreativem Denken ermöglicht wird.
Die Annahme, dass Stress ebenfalls die Gedächtnisbildung beeinflusst, stützt sich
auf mehrere Beobachtungen. Zunächst beschrieben verschiedene Wissenschaftler
unabhängig voneinander eine Vielzahl von Mineralo- und Glukokortikoidrezeptoren
in der Hippocampusformation der Ratte und des Menschen (Joëls, 2008; McEwen
et al., 1986; McEwen und Sapolsky, 1995; Reul und de Kloet, 1985). Bei oben ge-
nannten Stoffen handelt es sich um Hormone, die von der Nebenniere während
Stresssituationen ausgeschüttet werden. Später wurde in Verhaltensstudien ein
vom jeweiligen Stresshormonspiegel abhängiger Lerneffekt belegt, der in der soge-
nannten “Yerkes-Dodgsen-Regel” Einzug erhielt (Robert und John, 1908). Diese
besagt, dass niedrige bis normale Glukokortikoid-Spiegel die Lerneffekte durch För-
derung der Bildung von “long-term potentiation” unterstützen, hohe bis sehr hohe
Stresshormonspiegel jedoch das Gegenteil (“long-term depression”) bewirken (An-
dersen et al., 2006; Beispielstudien: Okuda et al., 2004; Roozendaal, 2000; Roo-
zendaal et al., 2002; Sandi und Rose, 1994).
Studien, die beide Umweltfaktoren (Schlaf und Stress) kombinieren, belegen Vi-
gilanz abhängige Effekte der Stresshormone auf die Gedächtniskonsolidierung:
Während die Applikation von Glukokortikoiden im Schlaf nach der Enkodierung den
49
Abruf Hippocampus abhängiger Gedächtnisinhalte verschlechtert, scheint die Ap-
plikation während eines Wachintervalls nach der Enkodierung diesen zu verbessern
(Plihal und Born, 1999; Plihal et al., 1999; Wilhelm et al., 2011).
In der vorliegenden Studie wurde versucht, alle o.g. Fakten – die Vigilanz abhängi-
gen Unterschiede des Konsolidierungsprozesses und deren Modulation durch
Stresshormone – bei der Hippocampus abhängigen Gedächtniskonsolidierung zu
verknüpfen. Es galt herausfinden, an welchem Ort und zu welchem Zeitpunkt die
Glukokortikoide mit dem Konsolidierungsprozess der Gedächtnisinhalte interferie-
ren. Um systemische Nebeneffekte zu vermeiden, wurde hierfür ein Tiermodell ge-
nutzt, bei dem das Glukokortikoidderivat Corticosteron direkt über vorher dort ste-
reotaktisch implantierte Kanülen in den dorsalen Hippocampus injiziert wurde.
Basierend auf vorangegangen Studien der Arbeitsgruppe (Binder et al., 2012; Ino-
stroza et al., 2013; Wilhelm et al., 2011) lautete die Hypothese, dass Glukokor-
tikoide, die in einem Wachintervall nach der Enkodierung appliziert werden, die Kon-
solidierung Hippocampus abhängiger Gedächtnisinhalte verbessern würden, wäh-
rend die Gabe im Schlaf nach der Enkodierung diesen Prozess verschlechtern
würde.
Dazu wurde ein Studienmodell entwickelt, in dem der Effekt von Schlaf bzw. Wach-
heit nach der Applikation von Corticosteron bzw. Placebo auf Hippocampus abhän-
gige und –unabhängige Gedächtnisprozesse untersucht werden konnte. Zur Ver-
wendung kamen hierbei zwei unterschiedliche Gedächtnistests: der Hippocampus
abhängige “object-place recognition” Test (zu deutsch: Objekt-Ort-Erkennung) und
der Hippocampus unabhängige “novel-object recognition” Test (zu deutsch: Neue-
Objekt-Erkennung).
Beide Tests bestanden jeweils aus zwei Versuchsteilen, dem “sample trial” und dem
“test trial”. Ersterer war für beide Versuche gleich: Den Ratten wurden zwei identi-
sche Objekte (Glasgegenstände, die mit unterschiedlich gefärbten Materialien ge-
füllt waren und sich in Höhe, Durchmesser, Farbe und Form unterschieden; vorab
durchgeführte Testdurchläufe hatten ergeben, dass die Ratten keine Präferenz für
bestimmte Objekte aufbauten) auf zwei von neun unterschiedlichen Positionen ge-
zeigt. Diese durften die Tiere für jeweils zwei bis fünf Minuten untersuchen und wur-
den währenddessen mit der Videokamera gefilmt. Sobald sie sich 15 Sekunden mit
jedem der zwei Objekte beschäftigt (Kriterium: sich in Reichweite von 2 cm zum
50
Objekt befinden, die Nase zum Objekt richten, sich aktiv mit dem Objekt beschäfti-
gen, z.B. schnüffeln, lecken, aber nicht klettern) und mindestens zwei Minuten oder
die vollen fünf Minuten im Experimentalfeld verbracht hatten, wurden die Ratten
vom Testfeld entfernt und in die Schlafboxen im Experimentalraum gesetzt.
Anschließend folgte ein 80-minütiges Behaltensintervall, in dem die Ratten entwe-
der schlafen durften oder durch sogenanntes “gentle handling” (zu deutsch: vorsich-
tiger Umgang; Einbringen von Objekten in die Schlafboxen, Klopfen an die Wände
der Boxen, Durcheinanderbringung der Schlafnester) wach gehalten wurden und 10
ng Corticosteron in 0.5 µl 0.9 %iger Kochsalzlösung oder nur 0.5 µl 0.9%ige Koch-
salzlösung (Placebo) nach einem definierten Zeitabschnitt per Pumpe appliziert be-
kamen. Auch dieser Schritt wurde mit der Kamera festgehalten, ebenso wie der “test
trial”, der sich zwischen den beiden Versuchen unterschied.
Während in dem Hippocampus unabhängigen “novel-object recognition” Test einer
der beiden Gegenstände gegen einen neuen ausgetauscht wurde, ohne dass sich
deren Positionen änderte, wurde im Hippocampus abhängigen “object-place recog-
nition” Test die Position eines der beiden Gegenstände verändert, ohne dass einer
der beiden Gegenstände ausgetauscht wurde. Die Ratten durften diesmal unabhän-
gig von ihrem Verhalten den Gegenständen gegenüber drei Minuten das Testfeld
erkunden. Zur Verifizierung, ob die vor der Durchführung der Versuche implantier-
ten Kanülen auch wirklich im dorsalen Hippocampus lagen, wurden die Gehirne der
Versuchstiere anschließend fixiert und histologisch untersucht.
Meine Hypothesen bezüglich der Ergebnisse der Versuchsreihen lauteten wie folgt:
Im Hippocampus unabhängigen “novel-object recognition” Test wird weder eine Be-
einflussung der Performanz durch den Vigilanz-, noch durch den Susbtanzfaktor
allein, noch durch deren Kombination erwartet. Alle Tiere sollten unter allen Kondi-
tionen in etwa gleich abschneiden. Im Hippocampus abhängigen “object-place
recognition” Test wird hingegen eine Staffelung der Gruppen nach ihrem Testab-
schneiden wie folgt angenommen: Wenn die Ratten während des Schlafintervalls
Placebo erhalten, sollten sie besser abschneiden, als wenn sie während des
Wachintervalls Placebo oder während des Schlafintervalls Corticosteron erhalten.
Andererseits sollten die Tiere, die während des Wachintervalls Corticosteron erhal-
ten, besser abschneiden, als die, die während des Wachintervalls Placebo erhalten.
51
7.2 Material und Methoden
Für die Experimente wurden 24 männliche Ratten der Rasse “Long Evans” (Janvier,
Le Genest-Saint-Isle, Frankreich, 250-300 g) verwendet, deren Anzahl auf den Er-
gebnissen früherer Studien beruhte und das Minimum der benötigten Tiere zur Ge-
währleistung signifikanter Testergebnisse darstellte (Binder et al., 2012; Inostroza
et al., 2013). Die Studie wurde vorab vom Ministerium für Natur und Umwelt des
Landes Schleswig-Holstein gebilligt45 und im Einklang mit den NIH Richtlinien und
der Direktive 86/609/ECC des europäischen Gerichtshofs durchgeführt.
Nach Ankunft der Ratten im Labor wurden sie zunächst für fünf Tage ohne Men-
schenkontakt (außer Auffüllen der Futtertröge und Wasserflaschen) in ihren Schlaf-
boxen im Schlafsaal belassen, um sich an die Umgebung zu gewöhnen. In eben
diesem Raum wurde vorab eine Zeitschaltuhr installiert, die jeweils um 6 Uhr mor-
gens das Licht an- und um 18 Uhr abends das Licht ausschaltete. Die Experimente
wurden später zwischen 8 und 13 Uhr durchgeführt, um ein Maximum an SWS und
ein Minimum an Stresshormonen zu garantieren.
Im Anschluss an die räumliche Eingewöhnungsphase fand eine menschliche Ein-
gewöhnungsphase an die Experimentatoren, das sogenannte “handling”, statt. Da-
für wurden die Ratten jeweils zweimal am Tag für vier Minuten zunächst nur gestrei-
chelt, später auch hochgehoben und in die Position gebracht, die für das Einstecken
der Kanülen zur Applikation des Corticosteron eingenommen werden musste. Das
Verhalten jedes einzelnen Tieres wurde dabei in Protokollen festgehalten46.
Auf das “handling” folgte die Operation der Ratten. Dabei wurde jedem Tier stere-
otaktisch in Narkose eine Kanüle in die dorsalen Hippocampi implantiert und an-
schließend mit Zusatzschrauben in der Schädeldecke und Zahnzement auf dem
Kopf fixiert. Mit einem Versuchstier wurde vorab einen Probelauf inklusive histolo-
gischer Aufbereitung des Gehirnes und Verifizierung der richtigen Positionierung
der Kanülen durchgeführt.
Jedes Tier durfte sich nach der Operation mindestens eine Woche lang erholen,
bevor es sich zusammen mit den anderen dem “elevated plus maze”-Test unterzie-
hen musste. Dieses Verfahren dient der Feststellung der Ängstlichkeit eines Ver-
suchstieres. Dabei wird jede Ratte in die Mitte eines kreuzförmigen Labyrinths mit
45 Siehe Anhang, S. XII. 46 Siehe Anhang, S. II.
52
jeweils vier gleich langen Armen, von denen jeweils zwei geschlossen und zwei
geöffnet sind, gesetzt und darf dieses für fünf Minuten erkunden. Mittels Videoana-
lyse wird anschließend die Länge der Zeit, die das Tier in den geschlossenen und
den offenen Armen verbringt, bestimmt. Die jeweilige Gesamtverweildauer im ge-
schlossenen und offenen System gibt später Aufschluss darüber, ob es sich bei
dem Versuchstier eher um ein Ängstliches oder ein Neugieriges handelt und ermög-
licht je nach Studiendesign ein mögliches Ausschließen von ungeeigneten Tieren
vor dem eigentlichen Experiment. Um die Testergebnisse nicht zusätzlich durch die
Ausschüttung von Stresshormonen während des Transportprozesses und des Um-
gebungswechsels zu verfälschen, wurden alle Experimentaltiere bereits einen Tag
vorher in den Untersuchungsraum gebracht und dort für 15 Minuten allein gelassen.
Als letzten Schritt vor der eigentlichen Durchführung der Studie fand die Gewöh-
nung der Ratten an den Experimentalraum, die sogenannte “open field habituation”
statt. Dafür wurde jeweils drei Gruppen von vier Tieren pro Tag die Möglichkeit ge-
geben, die Schlafboxen im Experimentalraum und das Experimentalfeld kennen zu
lernen. Die Zeit, die die Ratten auf dem Experimentalfeld verbringen durften, orien-
tierte sich dabei an der Länge der später während der eigentlichen Studie gewählten
Zeitabschnitte (jeweils fünf und drei Minuten entsprechend den Zeiten des “sample
trial” und “test trail”). Im Unterschied zu den Experimenten wurden den Tieren zu
diesem Zeitpunkt jedoch keine Objekte präsentiert und sie bekamen auch keine
Substanzen injiziert47. Zur Vorbeugung der Entwicklung von Erkundungsmustern,
die eine potentielle Objektpräferenz begünstigen könnten, wurden die Ratten genau
wie in der eigentlichen Studie von drei unterschiedlichen Seiten auf das Experimen-
talfeld gesetzt. Des Weiteren wurde der Raum mittels eines Geräuschgenerators
von externen akustischen Reizen abgeschirmt und Orientierungshilfen an den Wän-
den des Experimentalraums befestigt.
Die Experimentaltage starteten jeweils mit der Vorbereitung der Infusionslösung.
Dafür wurden 5 mg Corticosteron mit einer Feinwaage abgewogen und mit 16.6 ml
0.9 %iger Kochsalzlösung in einem Vortex gemischt. Anschließend wurden die
Schläuche, die später mit den Kanülen der Ratten verbunden werden sollten, mit
0.9 %iger Kochsalzlösung gespült und mit einer kleinen Luftblase am zum Ver-
suchstier offenen Ende versehen. Hinter die Luftblase wurde dann 0.5 µl 0.9 %iger
Kochsalzlösung mit 10 ng Corticosteron oder pur (Placebo) eingesaugt und die
47 Siehe Anhang, S. IV.
53
Schläuche bis zur Applikation beiseite gehängt48. Die Infusion von Corticosteron
bzw. Placebo fand – wie bereits oben beschrieben – während des 80-minütigen
Behaltensintervalls zwischen dem “sample trial” und dem “test trial” statt. In der
Schlafbedingung wurde dafür der Zeitpunkt gewählt, in der das jeweilige Tier für
mindestens 30 Sekunden keine Aktivität gezeigt hatte. Um die Ratten nicht durch
das Einstecken der Schläuche wieder zu wecken, wurden die Tiere direkt nach dem
“sample trial” bereits mit der Pumpe verbunden und 20 Sekunden nach der Infusion
wieder abgekabelt. Bei der Schlafentzugsbedingung wurde als Applikationszeit-
punkt jeweils der Mittelwert aus den Zeiten, die die Ratten der Vortage bis zum Ein-
schlafen in der Experimentalbox gebraucht hatten, verwendet. Das Verhalten der
Ratten auf dem Testfeld und in den Experimentalboxen wurde wieder per Video für
die spätere Auswertung aufgezeichnet und handschriftlich protokolliert49.
Nachdem alle Tiere alle Experimente durchlaufen hatten (oder vorzeitig durch Dis-
konnektion der Kanülen ausgeschieden waren), fand die Vorbereitung für die histo-
logische Aufarbeitung statt. Dazu wurden die Ratten in Narkose gelegt und mittels
Formalininfusion in das schlagende Herz eine Fixierung aller Organe – inklusive des
Gehirns – herbeigeführt (Gage et al., 2012). Letzteres wurde für die Untersuchung
in 4 %iger Formalinlösung im Kühlschrank aufbewahrt und anschließend mittels
Vibratom in 50 µm große Scheiben geschnitten, mit Toluidine-Blau gefärbt und unter
dem Lichtmikroskop untersucht.
7.3 Daten-Analyse
Für die Datenanalyse wurde ein Vergleichswert (zu englisch: discrimination ratio)
entwickelt, mit dem das Abschneiden der einzelnen Ratten in den unterschiedlichen
Settings miteinander verglichen werden konnte. Dieser basierte auf der Annahme,
dass die Versuchstiere das neue bzw. verschobene Objekt jeweils bevorzugt be-
handeln würden und berechnet sich für die zwei Tests wie folgt:
“Novel-object recognition” Test:
Discrimination ratio = (Explorationszeit für das unbekannte Objekt – Explora-
tionszeit für das bekannte Objekt) / (Explorationszeit für das unbekannte Ob-
jekt + Explorationszeit für das bekannte Objekt).
48 Zur Durchführung der Experimente siehe Einleitung und Anhang, S. V. 49 Siehe Anhang, S. V.
54
“Object-place recognition” Test:
Discrimination ratio = (Explorationszeit für das verschobene Objekt – Explo-
rationszeit für das stationäre Objekt) / (Explorationszeit für das verschobene
Objekt + Explorationszeit für das stationäre Objekt).
Die Werte der “discrimination ratio” wurden dann für eine zweifache Varianzanalyse
(zu englisch: two-way ANOVA - ANalysis Of VAriance) verwendet. Diese vergleicht
die Effekte zweier unabhängiger Faktoren (SCHLAF und STRESS) auf eine abhän-
gige Variable (Testperformanz). Dabei wird sowohl der Einfluss jedes Faktors für
sich allein genommen, als auch deren Wechselwirkung auf das Outcome der Tests
berechnet.
Bei der Interpretation der Zahlen ist allerdings Vorsicht geboten, denn der Test kann
bei positive Ergebnissen (also einer Ablehnung der Nullhypothese) nicht unterschei-
den, welche Untergruppe im Einzelnen dazu geführt hat. Um dieses Problem zu
lösen, wurden verschiedene “a posteriori”- oder “posthoc”-Tests erfunden, von de-
nen der Student-Newman-Keuls-Test wohl der bekannteste ist. In Letzterem wer-
den die Mittelwerte jeder Testgruppe paarweise miteinander verglichen und dann
sequentiell der vom Namensgeber entwickelten “Studentischen Variationsbreite q”
gegenüber gestellt.
Schließlich wurde der Einstichproben t-Test verwendet, um feststellen zu können,
ob es sich bei einer Testperformanz einer der Untergruppen um ein zufälliges Er-
eignis handelt. Der Test prüft dabei anhand des Mittelwerts einer Stichprobe, ob der
Mittelwert einer Grundgesamtheit sich von einem vorgegebenen Sollwert unter-
scheidet.
7.4 Ergebnisteil
“elevated-plus-maze” Test
Dieser Test wurde von allen Ratten bis auf diejenige, die für den Testlauf des Ope-
rationsverfahrens verwendet wurde, durchlaufen. Die Ergebnisse belegten für jedes
Tier ein durchschnittliches Level an Ängstlichkeit, sodass keines von den Experi-
menten ausgeschlossen werden musste (45.0 ± 3.1 % Aufenthalt in den offenen
Armen und 24.0 ± 2.1 % Betreten der offenen Arme; Hogg, 1996). Des Weiteren
schien kein Zusammenhang zwischen dem individuellen Abschneiden im “elevated-
55
plus-maze” Test und der späteren Performanz einer jeden Ratte im Gedächtnistests
zu bestehen (die Bonferroni-Methode ergab keine signifikanten Unterschiede).
Sample trial
Die Dauer und Erkundungszeit der “sample trials” war für beide Tests in allen vier
Bedingungen in etwa vergleichbar (p-Wert mittels Einstichproben t-Test > 0.24 für
alle Vergleiche).
Behaltensintervall
Bezüglich des Einschlafzeitpunkts und der Schlafdauer in den einzelnen Bedingun-
gen der zwei Gedächtnistests bestanden ebenfalls keine nennenswerten Unter-
schiede: Im “novel-object recognition” Test schliefen die Tiere in der Placebo-Be-
dingung nach 40.67 ± 4.99 min ein, die mit Corticosteron Versehenen nach 35.50 ±
3,87 min (p-Wert mittels Einstichproben t-Test = 0.42). Erstere Gruppe schlief an-
schließend insgesamt für 24.76 ± 3.12 min, letztere für 29.32 ± 3,53 min (p = 0.34).
Im “object-place recognition” Test verhielt es sich ähnlich: Die mit Placebo versehe-
nen Ratten schliefen nach 39.29 ± 6.19 min ein, die mit Corticosteron versehenen
nach 38.27 ± 4.12 min (p = 0.89). In der Placebo-Bedingung schliefen die Tiere
dann für 28.13 ± 3.12 min, in der Corticosteron-Bedingung für 24.12 ± 4.12 min (p
= 0.5). Diese Ergebnisse sind vergleichbar mit Werten aus vorangegangenen Stu-
dien (Inostroza et al., 2013). Der Zeitpunkt der Corticosteron-Infusion in den Bedin-
gungen Wachheit und Schlaf unterschied sich auch nicht signifikant voneinander:
Während die Applikation in der Schlaf-Bedingung nach 38.80 ± 2.16 min stattfand,
lag sie in der Wach-Bedingung bei 40.35 ± 1.33 min (p = 0.42).
Test trial
7.4.4.1 “novel-object recognition” Test
Der “novel-object recognition” Test wurde insgesamt 39-mal durchlaufen50. Die Aus-
wertung des zweifaktoriellen ANOVA-Tests ergab dabei weder eine signifikante Be-
einflussung der Testperformanz durch den Faktor SCHLAF (F (1,35) = 0.61, p =
0.44; Schlaf/Wachheit), noch durch den Faktor STRESS (F (1,35) = 0.09, p = 0.77;
Corticosteron/Placebo). Darüber hinaus konnte keine signifikante Interaktion zwi-
50 Davon 7-mal in der Kombination Placebo und Wachheit, 9-mal in der Kombination Placebo und Schlaf, 10-mal in der Kombination Corticosteron und Schlaf und 13-mal in der Kombination Corti-costeron und Wachheit.
56
schen den vier Bedingungen (F (1,35) = 0.08, p = 0.78; SCHLAF x STRESS) fest-
gestellt werden. Der Einstichproben-t-Test belegte, dass alle Versuchstiere in den
unterschiedlichen Kombinationen besser als zufällig abschnitten (alle p > 0.01;
siehe auch Abbildung 7).
7.4.4.2 “object-place recognition” Test
Der “object-place recognition” Test wurde insgesamt 33-mal durchlaufen51. Genau
wie im “novel-object recognition” Test belegte die Auswertung des zweifaktoriellen
ANOVA-Tests weder eine signifikante Beeinflussung der Testperformanz durch den
Faktor SCHLAF (F (1,29) = 0.07, p = 0.80; Schlaf/Wachheit), noch durch den Faktor
STRESS (F (1,29) = 0.01, p = 0.92; Corticosteron/Placebo). Im Gegensatz zum Hip-
pocampus unabhängigen Test bestand hier jedoch eine signifikante Interaktion zwi-
schen den vier Bedingungen (F (1,29) = 11.09, p = 0.002; SCHLAF x STRESS).
Die Post-hoc-Analyse der Testergebnisse mittels des Student-Newman-Keuls-
Tests staffelte das Testabschneiden in den unterschiedlichen Konditionen wie folgt:
In den Schlaf-Bedingungen führte die Applikation von Corticosteron zu einer Ver-
schlechterung der Testergebnisse verglichen mit der Infusion von Placebo (t (15) =
2.32, p = 0.035; Schlaf + Corticosteron/Schlaf + Placebo). Dabei schien die Appli-
kation von Placebo während des Schlafs die Gedächtnisbildung insgesamt verbes-
sert zu haben (p = 0.007), während die mit Corticosteron während des Schlafs be-
handelten Ratten ein zufälliges Testabschneiden zeigten (p = 0.1). In den Wach-
Bedingungen lagen die Ergebnisse hingegen genau anders herum: Die Applikation
von Corticosteron schien das Testabschneiden verglichen mit der Infusion von Pla-
cebo insgesamt zu verbessern (t (14) = 2.39, p = 0.032; Wachheit + Corticoste-
ron/Wachheit + Placebo).
Dabei wiesen die mit Corticosteron behandelten Ratten eine deutlich verbesserte
Gedächtnisleistung (p = 0.001) auf, als die mit Placebo behandelten, deren Tester-
gebnisse sich nicht von zufälligen unterschieden (p = 0.018). Zusammenfassend
schnitten damit die Kombinationen Placebo und Schlaf sowie Corticosteron und
Wachheit deutlich besser im Hippocampus abhängigen “object-place recognition”
Test ab, als die Kombinationen Placebo und Wachheit und Corticosteron und Schlaf
(siehe auch Abbildung 7).
51 Davon 6-mal in der Kombination Placebo und Schlaf, 7-mal in der Kombination Placebo und Wach-heit, 9-mal in der Kombination Corticosteron und Wachheit und 11-mal in der Kombination Corti-costeron und Schlaf.
57
Histologische Untersuchung
Von den insgesamt 24 verwendeten Tieren wurden 20 einer histologischen Unter-
suchung unterzogen. Zu den ausgeschlossenen Ratten gehörte die Testratte für
das Operationsverfahren sowie drei weitere Tiere, die ihre Implantate vor Durchfüh-
rung des ersten Testlaufs verloren hatten. Bei den eingeschlossenen Ratten konnte
bei zwei Tieren gar keine Position der Kanülen bestimmt werden, bei zwei weiteren
Ratten war nur die Position auf einer der beiden Seiten zu detektieren. Die Positio-
nen der Implantate der übrigen 16 Versuchstiere verteilten sich wie folgt52: Die
Mehrheit der Implantate (14 = 41.2 %) befand sich im Stratum lacunosum und
moleculare, gefolgt vom Stratum oriens und Stratum radiatum (je 5 = 14.7 %), dem
Stratum pyramidale und Stratum moleculare des Gyrus dentatus (je 4 = 11.8 %) und
schließlich der CA1/CA3-Region des Hippocampus (2 = 5.9 %). Dabei entspricht
das Verteilungsmuster der Kanülen dem anatomischen Aufbau des Hippocampus
mit dem Stratum lacunosum und moleculare in der Mitte.
7.5 Diskussion
Vergleichen wir die in der Einleitung von mir aufgestellten Hypothesen mit den o.g.
Testergebnissen, so finden wir all meine Annahmen durch die Experimente bestä-
tigt: Im Hippocampus unabhängigen “novel-object recognition” Test schnitten alle
Tiere unter allen Bedingungen gleich ab, es war weder eine Beeinflussung der Er-
gebnisse durch den Faktor SCHLAF, noch durch den Faktor STRESS, noch durch
deren Kombination zu beobachten. Im Gegensatz dazu bestand im Hippocampus
abhängigen “object-place recognition” Test eine von der Kombination der Bedingun-
gen abhängige Leistung: Während die Kombinationen aus Placebo und Wachheit
sowie Corticosteron und Schlaf jeweils Ergebnisse lieferten, die sich nicht von zu-
fälligen Testwerten unterschieden, verbesserte die Kombination aus Placebo und
Schlaf und Corticosteron und Wachheit die Testperformanz signifikant (s.o.).
Wenden wir uns der anfangs gestellten Frage, wo und wann Stresshormone mit
dem Konsolidierungsprozess Hippocampus abhängiger Gedächtnisinhalte interfe-
rieren, zu, können eingangs genannte Thesen ebenfalls bestätigt werden: Durch die
direkte Applikation und selektive Interaktion von Glukokortikoiden mit den dortigen
Mineralo- und Glukokortikoidrezeptoren (Joëls, 2008; McEwen et al., 1986; McE-
52 Siehe auch Abbildungen 8 und 9 und Anhang, S. VI und S. VII.
58
wen and Sapolsky, 1995; Reul and de Kloet, 1985) ist die Führungsrolle des Hippo-
campus in der Verarbeitung deklarativer Gedächtnisinhalte (Carr et al., 2011; Ei-
chenbaum, 1993) sowie die Existenz der Stresshormonbindungsstellen bewiesen.
Wie um das zu unterstreichen, hatte die Infusion von Placebo weder auf die Konso-
lidierung von Hippocampus abhängigem noch von Hippocampus unabhängigem
Gedächtnis einen Effekt (Lupien und Lepage, 2001).
Bezüglich der Vigilanz abhängigen Unterschiede in der Verarbeitung von Hippo-
campus abhängigen Gedächtnisinhalten belegen die Ergebnisse gleichsam die be-
reits in der Fachwelt geäußerte Annahme, dass die Reaktivierung von Informatio-
nen während eines aktiven Wachintervalls deren Speicherung im Langzeitgedächt-
nis stört, während die Reaktivierung im Schlaf sie fördert (Debiec et al., 2002;
Diekelmann et al., 2011; Gupta et al., 2010; Jezek et al., 2002; Nader and Hardt,
2009). Als Beispiel hierfür dient das unterschiedliche Abschneiden der mit Placebo
behandelten Ratten im Hippocampus abhängigen “oject-place recognition” Test:
Die Tiere, die zwischen “sample trial“ und “test trial” schlafen durften, schnitten deut-
lich besser ab, als die Schlaf deprivierten Tiere. Im Gegensatz dazu war im Hippo-
campus unabhängigen “novel-object recognition” Test kein Unterschied im Hinblick
auf die Vigilanz-Bedingung in der Performanz zu beobachten.
Verknüpfen wir nun den letzten Punkt – die Vigilanz abhängigen Unterschiede in
der Konsolidierung von Hippocampus abhängigen Gedächtnisinhalten – mit der Mo-
dulation durch Stresshormone, komme ich zur gleichen Schlussfolgerung, wie Plihal
und Born (1999), Plihal et al. (1999) und Wilhelm et al. (2011) in ihren Gedächtnis-
studien mit Menschen unter systemischer Applikation von Glukokortikoiden: Wäh-
rend die Applikation von Stresshormonen im Laufe eines Wachintervalls die Verar-
beitung von Hippocampus abhängigen Gedächtnisinhalten fördert, wird sie durch
die Infusion während des Schlafs verschlechtert. Hippocampus unabhängige In-
halte sind davon nicht betroffen.
Studien legen nahe, dass für oben genannten Effekt eine Verringerung in der Aus-
prägung bestimmter Hirnwellen durch hohe Stresshormonspiegel verantwortlich zu
machen ist, die die Reaktivierung von Hippocampus abhängigen Gedächtnisinhal-
ten begleiten und während Wachheit zu einer Stabilisierung, im Schlaf jedoch zu
einer Labilisierung der Informationen führt (Weiss et al., 2008; Wilhelm et al., 2011).
Eine andere mögliche Erklärung bietet die von Sazma et al. 2018 geäußerte Theorie
der „kontextuellen Bindung“. In ihrer Studie haben die Forscher festgestellt, dass
59
die Applikation von Stresshormonen nach der Enkodierung von Gedächtnisinhalten
die Gedächtnisleistung nur verschlechtert hat, wenn die Versuchstiere vorher an die
Versuchsumgebung gewöhnt worden waren. Bei Tieren, die nicht habituiert waren,
hatte die zusätzliche Gabe gar keinen oder sogar einen verbessernden Effekt.
Sazma et al. machen hierfür einen Wechsel des „mentalen“ Kontexts während des
Schlafes verantwortlich. Die zusätzlich applizierten Stresshormone sorgen hierbei
dafür, dass die Verbindung der gelernten Gedächtnisinhalte mit dem ursprünglichen
Kontext gekappt und stattdessen eine Verknüpfung mit dem neuen „mentalen“ Kon-
text hergestellt wird.
Wie bereits in der Einleitung beschrieben, scheint die Modulation durch Stresshor-
mone nicht nur durch deren generelle Verfügbarkeit, sondern auch deren absolute
Konzentration gekennzeichnet zu sein. Gemäß der bereits in der Einleitung zitierten
“Yerkes-Dodgsen-Regel” (Robert und John, 1908) bewirken niedrige bis normale
Glukokortikoid-Spiegel eine Förderung der Bildung von “long-term potentiation”,
während hohe bis sehr hohe Stresshormonspiegel zum Gegenteil führen (Andersen
et al., 2006; Beispielstudien: Okuda et al., 2004; Roozendaal, 2000; Roozendaal et
al., 2002; Sandi und Rose, 1994).
Übertragen auf das vorliegende Studiendesign und unter Berücksichtigung der An-
nahme, dass die Stresshormonspiegel zu Beginn der Ruhezeit des Tieres beson-
ders niedrig sind (Penalva et al., 2003) und durch Schlafdeprivation gesteigert wer-
den (Penalva et al., 2003), lässt sich folgende Staffelung nach Corticosteron-Spie-
geln vornehmen: Die niedrigste Konzentration findet sich in der schlafenden Pla-
cebo-Bedingung; mittlere Konzentrationen kommen in der schlafenden Corticoste-
ron-Bedingung und der wachen Placebo-Bedingung vor; die höchste Konzentration
findet sich in der wachen Corticosteron-Bedingung. Würde man diese Ergebnisse
graphisch auftragen, ergäbe sich eine U-förmige Kurve, ähnlich wie in der Studie
von Okuda et al. (2004). Okuda verwendete 2004 den gleichen “novel-object recog-
nition” Test, wie auch hier, mit dem Unterschied, dass die Versuchstiere drei unter-
schiedliche Konzentrationen von Corticosteron systemisch infundiert bekamen und
eine der beiden Gruppen vorher an den Experimentalraum gewöhnt wurde. Wäh-
rend die Ergebnisse der nicht an den Experimentalraum gewöhnten Tiere o.g. U-
förmige Kurve bezogen auf die Menge des applizierten Stresshormons ergaben,
fand sich kein Unterschied in der Performanz der an den Experimentalraum ge-
60
wöhnten Tiere. Die Experimentatoren schlossen daraus, dass der Effekt von Corti-
costeron an den durch die Versuchsobjekte hervorgerufenen Erregungszustand ge-
bunden ist.
Diese These belegen auch Beobachtungen von de Quervain et al., 2009, die erga-
ben, dass Stresshormone selektiv die Konsolidierung von emotional geladenen Ge-
dächtnisinhalten beeinflussen. Als mögliche Erklärung dient die Feststellung, dass
emotionale Erfahrungen in der Amygdala die Freisetzung von Norepinephrin bewir-
ken, das wiederum die Konsolidierung von Gedächtnisinhalten fördert (Galvez et
al., 1996; McIntyre et al., 2002; Quirarte et al., 1998). Zerstört man nun die
Amygdala oder blockiert deren Rezeptoren, führt dies zu einer Behinderung der
Überführung von Informationen in das Langzeitgedächtnis (Quirarte et al., 1997;
Roozendaal et al., 1999; Roozendaal et al., 2002).
Die Verbindung zwischen den Ergebnissen der Studie von Okuda und den hier be-
obachteten Vigilanz-abhängigen Effekten der Stresshormone liegt nun in der unter-
schiedlichen Konzentration von Norepinephrin. Analog zum körpereigenen Corti-
costeron-Spiegel ist der Norepinephrin-Spiegel ebenfalls während des Schlafs am
niedrigsten (Kalen et al., 1989; Tononi et al., 1994), während er durch aktive Schlaf-
deprivation künstlich gesteigert wird. Daraus folgt, dass der Effekt von Glukokor-
tikoiden in den schlafenden Bedingungen geringer ausfällt, als in den Schlaf depri-
vierten Bedingungen (analog dem Testergebnis von Okuda et al., 2004, das einen
fehlenden Effekt von Corticosteron auf die Performanz der an den Experimental-
raum gewöhnten Gruppe zeigt).
Einen ähnlichen Erklärungsansatz liefert das sogenannte „tagging“-Theorem, das
besagt, dass hohe Cortisol- und Norepinephrin-Spiegel während der Enkodierung
die Markierung („tagging“) von Gedächtnisinhalten fördern, die besonders ausge-
prägte Reaktionen im Hippocampus und der Amygdala hervorgerufen haben, indem
sie deren Verarbeitung auf neuronaler Ebene unterstützen. Somit dient Stress auch
als „Gedächtnisfilter“ (de Jesús and Fishbein, 2018; Payne and Kensinger, 2018;
Ritchey et al., 2017; Sazma et al., 2018).
Einschränkungen
Der schwerwiegendste Kritikpunkt an der vorliegenden Studie ist sicherlich der vor-
zeitige Verlust an Ratten mit intakten Implantaten und die daraus resultierenden,
61
unterschiedlichen und teilweise sehr kleinen Gruppengrößen, die spezielle statisti-
sche Testverfahren (Student-Newman-Keuls-Test für unbalancierte Gruppen) zur
Vergleichbarkeit der Ergebnisse nötig machten. Zur Steigerung der Aussagekraft
der Studie wäre eine erneute Durchführung der Experimente mit – wie eingangs
gefordert – mindestens 24 Tieren von Nöten (Ennaceur et al., 1997).
Mögliche Verbesserungsvorschläge zur Verlängerung der Haltbarkeit der Implan-
tate betreffen das Material (leichtere Stoffe mit besserer Haftungsfläche), dessen
Fixierung am Kopf (andere/mehr Fixierungsschrauben, andere Punktionsstelle oder
Versiegelung zur Vermeidung von Liquorrhoe) sowie den Infusionsapparat (leich-
tere Schläuche, drehbare Fixierung der Schläuche, individuelle, leise, ferngesteu-
erte Pumpen für jede Ratte).
Abgesehen von den technischen Verbesserungen wären bei einer Wiederholung
der Studie auch Änderungen im Design zu diskutieren. Beispielsweise würde ein
fester Infusionszeitpunkt für die Substanzen in den Schlaf deprivierten Bedingungen
die Vergleichbarkeit untereinander verbessern und gleichzeitig der Beobachtung
Rechnung tragen, dass die Zeitdauer zwischen “sample trial” und Applikation von
Corticosteron oder Placebo die Abrufung von Informationen im “test trial” beein-
flusst53. Des Weiteren wäre eine simultane EEG- und EMG-Aufzeichnung wün-
schenswert, um den genauen Einschlafzeitpunkt und die Schlaftiefe (es galt schließ-
lich SWS, also das letzte Schlafstadium, zu erreichen) zu bestimmen. Neuere Stu-
dien (Sazma et al., 2018) legen zudem nahe, dass die Auswirkung der Stresshor-
mone auf die Konsolidierung von Gedächtnisinhalten entscheidend davon abhängt,
ob die Versuchstiere vor der Durchführung der Tests an die Lernumgebung gewöhnt
worden waren, oder nicht. Bei den Tieren, die nicht an die Umgebung habituiert
waren, bewirkte die Applikation von Stresshormonen nach dem Test nämlich teil-
weise sogar eine Verbesserung im Testabschneiden. Die Autoren führen dies auf
unterschiedliche Effekte von Cortisol und Norepinephrin in Abhängigkeit von deren
Konzentration zurück. Gerade weil auch ich einen Zusammenhang im Abschneiden
der einzelnen Testgruppen mit dem vorherrschenden Norepinephrin-Level vermute,
wäre es interessant zu sehen, wie die Ergebnisse in einer weiteren, nicht an die
Testumgebung gewöhnten Gruppe, ausfallen würden. Weiterhin wurde kürzlich
eine Studie mit einem etwas abgewandelten „object-place recognition“ Test, dem
53 Je mehr Zeit zwischen Enkodierung und Infusion der Glukokortikoide liegt, desto schlechter das spätere Testergebnis (de Quervain et al., 1998, 2009).
62
sogenannten „what-where-which“ Test vorgestellt (Oyanedel et al., 2018). In diesem
werden zwei verschiedene Objekte in zwei unterschiedlichen Testumgebungen prä-
sentiert. Dadurch wird statt der im „what-where-when“ Test abgefragten zeitlichen
Reihenfolge ein sogenannter „occasion setter“ etabliert, der laut Autoren im „test
trial“ schneller zum Tragen kommt („when“-Komponente erst nach 3 Minuten im
„test trial“ ausgeprägt). Möglicherweise ließen sich mit o.g. Test – auch im Hinblick
auf die begrenzte Versuchstieranzahl - noch bessere Ergebnisse erzielen. Sa-
wangjit et al. haben 2018 schließlich festgestellt, dass auch bis dato als Hippocam-
pus unabhängig geltende Gedächtnisinhalte hippocampaler Mechanismen zur Ver-
arbeitung bedürfen, weil alle Informationen mit ihrem zeitlichen und örtlichen Kon-
text abgespeichert werden. Somit erfüllen strenggenommen alle Gedächtnisformen
die Eigenschaften für „episodisches“ Gedächtnis und es wird eine neue Einteilung
– auch im Hinblick auf die für die Verarbeitung hauptverantwortlichen Gehirnregio-
nen - benötigt.
Abschließend ist noch die Zusammensetzung der Corticosteron-Lösung zu beden-
ken. Der Komplex54, der hier Verwendung fand, enthält als Lösungsmittel Cyclo-
dextrin, das möglicherweise eigene Effekte auf das Gehirn besitzt und nicht dem
Placebo hinzugegeben wurde. Obwohl vorangegangene Studien keine Auswirkun-
gen bei intracerebraler Anwendung auf die Gehirnaktivität oder das generelle Ver-
halten der Versuchstiere gezeigt haben, scheint Cyclodextrin jedoch die Verfügbar-
keit und den Abbau der in der Lösung enthaltenen Stoffe zu verändern (Yaksh et
al., 1991). Andererseits gibt es kaum andere Möglichkeiten, wasserunlösliches Cor-
ticosteron in Lösung zu bringen. Das von Yaksh et al. ebenfalls getestete Dimethyl-
sulfoxid bewirkt eine Abnahme von Susbtanz P und dem calcitonine-gene-related-
peptide. Des Weiteren beeinflusst es die Miktion und den Blutdruck und fällt im Bei-
sein von Natriumchlorid aus. Ethanol stört über die Stimulation von GABA-Rezep-
toren und die Inhibition von NMDA-Rezeptoren die synaptische Plastizität und be-
einflusst außerdem die Vigilanz. Öl kann wegen seiner hohen Adhäsionskräfte nicht
über 0.5 mm dünne Schläuche gegeben werden.
Abgesehen von den Zusatzstoffen des HBC-Komplexes gab es auch Probleme bei
der Zubereitung der Corticosteron-Lösung. Weil die Zuliefererfirma Sigma-Aldrich
keine kleinen Mengen des HBC-Komplexes verschickt, musste jeden Tag 5 mg des
54 C174 SIGMA – Corticosterone: HBC complex, Sigma Aldrich, St. Louis, MO, USA; http://www.sig-maaldrich.com/catalog/product/sigma/C174?lang=de®ion=DE, 27.10.2016.
63
sehr feuchtigkeitsanfälligen Stoffes mit einer groben Feinwaage abgewogen und
mittels Vortex mit 16.6 ml 0.9% iger Kochsalzlösung in Lösung gebracht werden. In
diesem Zusammenhang ist nicht ganz auszuschließen, dass durch Ungenauigkei-
ten beim Herstellungsprozess Unterschiede in der Konzentration der Glukokor-
tikoid-Lösung zwischen den einzelnen Gruppen aufgetreten sind. Wäre eine
Stammlösung für alle Experimentaltage erstellt oder abgewogene Mengen von der
Firma verwendet worden, wären diese sicherlich kleiner ausgefallen.
Schließlich wurde während des Review-Prozesses kritisiert, dass keine Kontrollin-
fusionen in anderen Gehirnregionen vorgenommen wurden und damit nicht ausge-
schlossen werden konnte, dass die beobachteten Effekte auch durch Interaktion der
Stresshormone mit Glukokortikoid-Rezeptoren in anderen Hirnarealen zustande ge-
kommen wären. In eine ähnliche Richtung ging der Vorwurf, dass keine Messungen
der lokalen oder systemischen Corticosteron-Konzentration unternommen wurden
und damit auch nicht bewiesen werden konnte, dass der Stoff nur im Hippocampus
akkumulierte.
Ausblick
Wie bereits unter den Einschränkungen erwähnt, wäre eine gleichzeitige EEG- und
EMG-Aufzeichnung nicht nur zur quantitativen sondern auch zur qualitativen Aus-
wertung der einzelnen Schlafstadien der Versuchstiere wünschenswert. In der Wis-
senschaft bestehen zur Zeit im Wesentlichen zwei unterschiedliche Hypothesen be-
züglich der selektiven Beeinflussung von unterschiedlichen Gedächtnisformen
durch verschiedene Schlafphasen: Die “dual-process hypothesis” von Maquet aus
dem Jahre 2001, die besagt, dass SWS-reicher Schlaf nur die Bildung Hippocam-
pus abhängigen Gedächtnisses fördert, während REM-reicher Schlaf die Bildung
Hippocampus unabhängigen Gedächtnisses unterstützt. Dem gegenüber steht die
“sequential hypothesis” von Gais et al. (2000) und Giuditta et al. (1995), in der die
Wissenschaftler behaupten, dass die Konsolidierung von Gedächtnisinhalten jed-
weder Form allein von der Abfolge von REM-Schlaf auf SWS abhängt. Letztere
These wird auch durch neuere Studien (Lewis et al., 2018) belegt, in denen die
Autoren nur in der Abfolge der beiden Schlafformen aufeinander die Grundlage für
die Bildung komplexer Gedächtnisstrukturen und damit kreativem Denken sehen.
Es wäre interessant herauszufinden, inwiefern Glukokortikoide und andere Neuro-
transmitter in diese Prozesse eingreifen und möglicherweise den einen oder ande-
ren Prozess fördern.
64
7.6 Zusammenfassung
Während in der Wissenschaftswelt mittlerweile viele Belege dafür existieren, dass
Stresshormone die Aufnahme (Enkodierung) von Gedächtnisinhalten jedweder Art
fördern55, gibt es bei den Auswirkungen auf die Abspeicherung (Konsolidierung)
konträre Ergebnisse. Als mögliche Erklärung dienen Vigilanz-abhängige Unter-
schiede in der Verarbeitung von Glukokortikoiden56.
In Weiterentwicklung der Studien von Born und Wagner (2004), Plihal und Born
(1999) und Plihal et al. (1999) fanden Wilhelm et al. 2011 heraus, dass die Applika-
tion von Cortison während eines Wachintervalls die Gedächtnisbildung fördert, wäh-
rend sie sie bei Applikation im Schlaf stört. Weil es sich bei o.g. Studie um ein Hu-
manexperiment mit systemischer Verabreichung von Cortison handelte, ist der ge-
naue Zeitpunkt und der genaue Wirkort der Stresshormone in der Gedächtnisbil-
dung nicht genau zu bestimmen.
Um dem Abhilfe zu verschaffen, wurde hier ein Tiermodell entwickelt, in dem o.g.
Bedingungen genau festgelegt werden konnten. Dabei wurden 24 Ratten jeweils
zwei Kanülen in den dorsalen Hippocampus implantiert57. Anschließend durchliefen
die Tiere zwei unterschiedliche Gedächtnistests (Hippocampus unabhängiger “no-
vel-object recognition” Test und Hippocampus abhängiger “object-place recogni-
tion” Test) unter vier verschiedenen Bedingungen (Injektion von Corticosteron oder
Placebo während eines 80-minütigen Behaltensintervalls, in dem die Tiere entwe-
der schlafen durften oder wach gehalten wurden).
Wie bereits vermutet, bestanden im Hippocampus unabhängigen “novel-object
recognition” Test weder signifikante Unterschiede in der Testperformanz, wenn man
den Faktor SCHLAF oder den Faktor STRESS allein betrachtete (F (1,35) = 0.61, p
= 0.44 bzw. F (1,35) = 0.09, p = 0.77), noch in deren Kombination (F (1,35) = 0.08,
p = 0.78). Demgegenüber hatte die Interaktion der Faktoren SCHLAF und STRESS
auf die Konsolidierung Hippocampus abhängiger Gedächtnisinhalte (F (1,29) =
11.09, p = 0.002) folgende Auswirkungen: Während die Applikation von Corticoste-
ron im Zuge eines Wachintervalls die Testperformanz der Ratten im Vergleich zur
55 de Quervain et al., 1998; de Quervain et al., 2009; Kirschbaum et al., 1996; Maheu et al., 2004; Rimmele et al., 2012; Wolf, 2009. 56 de Kloet et al., 1999; de Quervain et al., 2009; McIntyre et al., 2012; Schwabe et al., 2012. 57 Studien belegen, dass dort eine besonders hohe Dichte an Mineralo- und Glukokortikoid-Rezep-toren herrscht (Joëls, 2008) und Stresshormone besonders starke Auswirkungen auf Hippocampus abhängige Gedächtnisinhalte haben (Lupien et Lepage, 2001).
65
Infusion von Placebo verbesserte (t (15) = 2.32, p = 0.035), führte die Infusion wäh-
rend des Schlafs zu einer Verschlechterung der Gedächtnisleistung im Vergleich
zur Placebo-Bedingung (t (14) = 2.39, p = 0.032).
Daraus folgt, dass die Konsolidierung Hippocampus abhängiger Gedächtnisinhalte
während des Schlafs und bei Wachheit auf unterschiedliche Weise stattfinden muss
und Corticosteron diese Vigilanz abhängig beeinflusst.
66
8 Own contributions to the doctoral thesis
step persons involved
idea Marion Inostroza
study design Marion Inostroza
technical realization Marie Gessert, Marion Inostroza
practical application Marie Gessert, Kimberley Petersen, Ma-
rion Inostroza, Sonja Binder
surgeries Marion Inostroza, Marie Gessert, Kim-
berley Petersen
sacrifice and perfusion of animals Sonja Binder, Marie Gessert
histological examination Marion Inostroza
scoring of videos Marie Gessert, Marion Inostroza
statistical analysis Marie Gessert, Carlos Oyanedel, Ma-
rion Inostroza
67
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basal forebrain during sleep deprivation. Brain research 1399, 40–48.
9.2 Books
Abdi, H., Williams, L.J., 2010. Newman-Keuls Test and Tukey Test. In: Salkind, N.J., 2010. Encyclopedia of Research Design. SAGE.
Andersen, P., Morris, R., Amaral, D., Bliss, T., O’Keefe, J., 2006. The hippocampus
book. Oxford University Press, USA.
76
Eichenbaum, H., 1993. Memory, amnesia, and the hippocampal system. MIT press.
Köhler, W., Schachtel, G., Voleske, P., 2012. Biostatistik. Springer DE.
Paxinos, G., Watson, C., 2006. The Rat Brain in Stereotaxic Coordinates: Hard
Cover Edition. Academic Press.
Schacter, D.L., Tulving, E., 1994. Memory systems 1994. Mit Press.
Squire, L.R., Cohen, N.J., Lynch, G., McGaugh, J., Weinberger, N.M., 1984.
Neurobiology of learning and memory. Guilford Press New York, NY.
Tulving, E., n.d. Elements of episodic memory. 1983. Clarendon Press, Oxford.
Tulving, E., Terrace, H., Metcalfe, J., 2005. Episodic memory and autonoesis:
Uniquely human. New York: Oxford University Press.
9.3 Web pages
(listing according to appearance in the text)
1. http://tayloredge.com/reference/Science/BiologySlides/LimbicSystem.gif, 27.10.2016.
2. http://www.human-memory.net/images/memory_types.jpg, 27.10.2016.
3. http://media.wiley.com/mrw_images/cp/cpns/articles/ns0901/image_n/ nns090102.gif, 27.10.2016
4. http://kaylab.uchicago.edu/images/rat_brain.png, 27.10.2016.
5. https://en.wikipedia.org/w/index.php?title=File:ElevatedPlusMaze.svg&page=1, 27.10.2016.
6. http://www.sigmaaldrich.com/catalog/product/sigma/C174?lang=de®ion=DE. 27.10.2016.
7. https://de.wikipedia.org/wiki/Post-hoc-Test#Student-Newman-Keuls-Test, 27.10.2016.
8. https://de.wikipedia.org/wiki/Einstichproben-t-Test, 27.10.2016.
9. http://www.sigmaaldrich.com/Graphics/COfAInfo/SigmaSAPQM/COFA/C1/ C174/C174-BULK_________041M4606V_.pdf, 27.10.2016.
I
10 Appendix
10.1 List of tables
Table 1. Timeline of the experiment.
............................................................................................................................. 10
10.2 List of figures
Figure 1. Anatomy of the limbic system. ................................................................. 2
Figure 2. Distinct neural circuits. CA1 = cornu ammonis 1; dSub = dorsal subiculum;
EC5 = medial entorhinal cortex 5; MB = mammillary body. .................................... 3
Figure 3. Memory types, steps of memory formation and role of the hippocampus.
............................................................................................................................... 4
Figure 4. The cannulas’ position in a stereotactic and anatomic model. .............. 13
Figure 5. Setting and proportions of the elevated plus maze test. ........................ 15
Figure 6. Experimental setup. ............................................................................... 17
Figure 7. Results of the test trials. The numbers associated with the bars indicate
the number of animals, which underwent the test. The star (*) marks the groups,
that were significantly different from the other groups by Newman-Keuls posthoc
test and t-test (each with p < 0.05). The (+) indicates that the test performances
were above chance level (p < 0.05). The error bars indicate standard deviations.
Sleep/Vehicle = infusion of vehicle during sleep; Wake/Depri = infusion of vehicle
during sleep-deprivation; Sleep/Cort = infusion of corticosterone during sleep;
Wake/Cort = infusion of corticosterone during sleep-deprivation. ........................ 26
Figure 8. Results of the histological examination. ................................................ 27
Figure 9. Example photomicrograph illustrating the placement of the needle tips in
one rat. The large arrows point to the cannula tips, the small ones to the infusion
needle tips. ........................................................................................................... 28
Figure 10. Summary of the results. ...................................................................... 46
II
10.3 Exemplary protocols
Handling
Hippocampus, glucocorticoids and sleep
Handling
ID:
Code of experiment: 42/g/11
Experimenter: Dr. Marion Inostroza Parodi
M.D. students: Marie Gessert, Kimberley Petersen
Experiment: Hippocampus, glucocorticoids and sleep
Date of arrival: 24.08.2011
Weight:
date activity oberservations
III
Postoperative control
Hippocampus, glucocorticoids and sleep
Postoperative control
ID:
Code of experiment: 42/g/11
Experimenter: Dr. Marion Inostroza Parodi
M.D. students: Marie Gessert, Kimberley Petersen
Experiment: Hippocampus, glucocorticoids and sleep
Date of arrival: 24.08.2011
day after surgery weight wound
IV
Habituation
Hippocampus, glucocorticoids and sleep
Habituation
ID:
Code of experiment: 42/g/11
Experimenter: Dr. Marion Inostroza Parodi
M.D. students: Marie Gessert, Kimberley Petersen
Experiment: Hippocampus, glucocorticoids and sleep
Date of arrival: 24.08.2011
Weight:
date session activity time (begin-
ning)
time (end) observations
V
Memory tasks
Hippocampus, glucocorticoids and sleep
Memory tasks
date … object one … object two … memory task conditions group …
sample
trial
test trial
ID … 2-5 min 3 min
5 min 80 min
time
start of sample
trial
end of sample
trial infusion
start of test trial
end of test trial
observations
VI
10.4 Histological results
animal’s
number
position of the right cannula position of the left cannula
1 Rad - radiatum layer of the hippocampus Py - pyramidal cell layer of the hippocam-
pus
2 - -
3 LMol - lacunosum moleculare LMol - lacunosum moleculare
4 LMol - lacunosum moleculare CA1/CA3 - field CA1 and CA3 of the hip-
pocampus
5 LMol - lacunosum moleculare Or - oriens layer of the hippocampus
6 LMol - lacunosum moleculare LMol - lacunosum moleculare
7 Py - pyramidal cell layer of the hippo-
campus
Rad - radiatum layer of the hippocampus
8 Py - pyramidal cell layer of the hippo-
campus
Py - pyramidal cell layer of the hippocam-
pus
9 - -
10 ? ?
11 - -
12 Or - oriens layer of the hippocampus MoDG - molecular layer of the dentate gy-
rus
13 - -
14 Or - oriens layer of the hippocampus? -
15 LMol - lacunosum moleculare Rad - radiatum layer of the hippocampus
16 Rad - radiatum layer of the hippocampus Rad - radiatum layer of the hippocampus
17 CA1/CA3 - field CA1 and CA3 of the hip-
pocampus
LMol - lacunosum moleculare
18 LMol - lacunosum moleculare LMol - lacunosum moleculare
19 - Or - oriens layer of the hippocampus?
20 MoDG - molecular layer of the dentate
gyrus
LMol - lacunosum moleculare
21 LMol - lacunosum moleculare LMol - lacunosum moleculare
22 - -
23 Or - oriens layer of the hippocampus LMol - lacunosum moleculare
24 MoDG - molecular layer of the dentate
gyrus
MoDG - molecular layer of the dentate gy-
rus
Histological results. For their arrangement see “10.5 Stereotaxic coordinates of the rat brain” in the appendix, p. VII.
VII
10.5 Stereotaxic coordinates of the rat brain
Stereotaxic coordinates of the rat brain – adapted from Paxinos and Watson: The rat brain in stereotaxic coordinates, 6th edition. Elsevier (2007).
VIII
10.6 Photos of the experimental room
Photo 1. Retention boxes and electric pump for the syringes.
IX
Photo 2. Tubing connecting the syringes to the cannulas. Because their weight influenced the head’s movement and the implants’ stability of the rats, the metal coat was removed during the experiment.
X
Photo 3. Rotatable attachment of the tubing to a clothesline stretched in the middle above the reten-tion boxes.
XI
10.7 Poster for the „6. Lübecker Doktorandentag 2013“
XII
10.8 Further attachments
- “Antrag auf Genehmigung eines Tierversuchsvorhabens”
- “Genehmigung zur Durchführung von Versuchen an Wirbeltieren”
Please find these documents attached to the thesis.
XIII
11 Acknowledgment
First and most of all, I have to thank Dr. Marion Inostroza for quickly and
straightforwardly recruiting me for her project, when I was still a greenhorn in her
second clinical semester, for providing me with papers and private lessons, for
assisting me in every step of the experiment and letting me take part in her
publications. Although I will probably not end up in research, I am grateful for having
got practical insight in the scientific mode of operation and hope that I did not confirm
all the prejudices, which are held against medical students.
Then, I am deeply indebted to Kimberley Petersen, a fellow student, work-mate, and
sufferer, with whom I lived through all ups and downs of a doctoral candidate and
who almost never lost her sunny temper.
Furthermore, my sincere thanks go to Dr. Walter Häuser, who naturally advised me
on the application of water insoluble drugs; to Sonja Binder, who assisted us with
sacrificing the animals and employed Kim and me as student assistants in her
projects; to the cardiosurgical lab, whose premises and instruments we were
allowed to use; to their research assistants, who refreshed our skills of the
biochemical course and endured the smell of twenty-four rat cages in the summer;
to Carlos Oyanedel, whom I have never personally met, but who backed up the
statistical analysis; to Prof. Dr. Lisa Marshall, who spontaneously overtook the
position as first reporter and did not only give critical advise to the contents of my
thesis but also to my style.
Last but not least, I am owing to my husband Felix Gessert, who not only solved
every computer problem I confronted him with but also never grew tired of trying to
teach me the basics of statistical analysis and built me up every time I wanted to
quit.
I dedicate this work to my parents and hope that I will not remain the only one to
complete her doctorate.
XIV
12 Curriculum vitae
Personal data:
Name: Marie Karoline Gessert,
née Bahrendt
Date of birth: 07.02.1989
University career:
- student of human medicine at the University of Lübeck from 2008-2014
- pre-clinical elective subject: advanced anatomy (“Anatomische Präparation für
Fortgeschrittene”)
- degree in the preliminary medical examination (summer 2010): 2,5
- clinical elective subjects: neuroradiology, neurology, emergency medicine
- degree in final examination (December 2014): 1,8
- current position: assistant doctor for psychiatry at the “Asklepios Klinik Ochsen-
zoll” in Hamburg (since January 2019), before: assistant doctor for neurology at
“St. Georg`s Hospital” in Hamburg (2015-2019)
Timetable of the doctoral thesis:
- June 2011: „Antrag auf Genehmigung eines Tierversuchsvorhabens“
- 26.04. – 23.08.2011: planning of the experiments
- 07.07.2011: approval of an English dissertation
- 31.08.2011: application for the approval of a dissertation
- 24.08. – 12.10.2011: execution of the experiments
- 21.10. – 01.11.2011: scoring of the sample and the test trails
- 12.09. – 20.09.2012: scoring of the sleep videos
- 19.06.2013: poster presentation at the “13. Lübecker Doktorandentag”
- 17.03.2014: publication of the paper in the journal Hippocampus
- April 2018: submission of the doctoral thesis
- August 2019: oral examination
XV
13 List of own publications
Kelemen, E., Bahrendt, M., Born, J., & Inostroza, M. (2014). Hippocampal corti-
costerone impairs memory consolidation during sleep but improves consolidation in
the wake state. Hippocampus, 24(5), 510-515.
XVI
14 Honesty declaration
I hereby declare that the work submitted is my own and that all passages and ideas
that are not mine have been fully and properly acknowledged. I did not use other
personnel, technical or material resources than claimed. Furthermore, I did not ap-
ply for approval or submit my dissertation elsewhere during this process nor did I
take part in other dissertational proceedings.
I do neither contradict the use of an anti-plagiarism software on my thesis nor a
public oral examination.
(date, signature)
