ABSTRACT
Memory and Learning are diverse and complex entities attracting numerous studies with
different findings, some even contrasting each other. This study was conducted to make a
contribution towards unravelling this reality. Behavioural studies using the Morris Water
Maze task was elicited in Wistar rats which were grouped for weight and sex (nmale=18; n
female =12). Reference or latency time recordings for three (3) training at the start of the
experiment and after a 5week period of exposure to cigarette smoke (in case of exposed
Rats) or 5weeks training free period (in case of non-exposed Rats). Analysis of the time
recordings suggest that there is a significant decrease (p≤0.05)for the control male Wistar
rats unlike the insignificant(p≥0.05) change obtained for the control and experimental
female and experimental male. Similarly, assessment of the histological section of the
hippocampus of the Male Wistar rats from both tissue sections and hippocampal cell
suspension was done. Cell counting of normal and Pyknotic pyramidal cells was done using
Image J soft ware. Analysis of the cell count suggests a significantly higher (p≤0.05) count
in the Exposed Males only, for both the normal and pyknotic pyramidal cells compared to
the counts of the Control Male rats. In conclusion, male Wistar rats tend to retain Reference
memory of Morris Water Maze Task for a longer time (5 weeks in this case) compared to
female Wistar rats. Similarly, cigarette smoke maintained at 10-32ppm of Carbon Monoxide
for 4 days in a week for 5 weeks suffices in disrupting the memory capabilities of the Wistar
rats. With a corresponding level of higher rates of Pyknotic pyramidal cell.
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TABLE OF CONTENTS
Title page ………………………………………………………………………………………………………… i
Declaration ……………………………………………………………………………………………………… ii
Certification ……………………………………………………………………………………………………… iii
Dedication ………………………………………………………………………………………………………… iv
Acknowledgements ……………………………………………………………………………………………. v
Abstract…………………………………………………………………………………………………………….. vi
Table of contents ……………………………………………………………………………………………… vii
List of tables …………………………………………………………………………………………………….. xii
List of figures……………………………………………………………………………………………………… xiii
List of Plates ……………………………………………………………………………………………………… xiv
Abbreviations …………………………………………………………………………………………. ……….. xvi
CHAPTER ONE
1.0 Introduction …………………………………………………………………………………………………. 1
1.1 Background…………………………………………………………………………………1
1.2 Statement of Problem . …………………………………….………………………….. 4
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1.3 Study Hypothesis… ….………………………………………………………………… 4
1.4 Justification . ……………………………….…………………….…………………… 5
1.5 Aim ……………………………….………………………………………………… 6
1.6 Objectives …………………………………………………………………………… 6
CHAPTER TWO
2.0 Literature Review …………………………………………………………………… 7
2.1 Anatomy of Hippocampus ………………………….…………. …………………….. 7
2.2 Place Cells of The Hippocampus ………………………………………………….…11
2.3 Memory And Spatial Memory ….……………………………………………………12
2.4 Memory Systems And Models ……………………..…………………………………15
2.4.1 Atkinson- Shiffrin Model ……………………………………………………………15
2.4.2 Working Memory Model …………………………………………………………..17
2.5 Studies Relating Hippocampus to Memory ….……………………………………….19
2.6 Electrophysiology of Memory ………………………………………………………20
2.6.1 SRR And Hippocampal Memory Consolidation ……………………………………27
2.7 Memory Task For Rats …………………….…………………………………………28
2.7.1 Morris Water Maze Task ……………………………………………………………28
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2.8 Cigarette / Cigarette Smoke ………………………………………………………….31
2.8.1 Carbon Monoxide ……………………………………..……………………………32
2.8.1.1 CO And L.T.P …………………………………………………………………….35
2.8.2 Nicotine …………………………………..…………………………………………..37
2.8.2.1 Nicotine And Cognition …………………………………………………………38
2.8.2.2 Nicotine And Hippocampus ………………….………………………………….39
2.8.3 Hydrogen Sulphide …………………………….…………………………………40
2.8.4 Tar ………………………………………………………………………………….42
2.8.5 Polynuclear Aromatic Hydrocarbon ……………………………………………….42
2.8.6 Chlorinated Dioxins And Furans ……………………………………………………42
2.8.7 Cigarette Smoke In The Environment …………………………………………….43
2.8.7.1 Cigarette Smoke Exposure & Hippocampus ………………………………………49
CHAPTER THREE
3.0 MATERIALS AND METHOD ………………………………………………………52
3.1 Materials …………..…………………………………………………………………52
3.1.1 Rats ……………………………………………………………………………… 52
3.1.2 Carbon Monoxide Meter ……………….…………………………………………52
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3.1.3 Nicotine Screen Strip ……………………………………………………………..53
3.1.4 Commercial Available Cigarette (Aspen) .………………………………………..53
3.1.5 Morris Water Maze Training Tank ………………………………………………………..58
3.1.6 Fabricated chamber for static exposure …. …………………………………………58
3.1.7 Microscope ………………………………………………………………………58
3.1.8 Neuber Improved Bright Lines Counting Chamber…………………………………61
3.2 Method ……………………………………………………………………………………. 61
3.2.1 Morris Water Maze Task ………………………………………………………. 62
3.2.2 Tissue Preparation For Cell Suspension ………………………………………….65
3.2.2.1 Preparation of hippocampal cell suspension……………………………………..…68
3.2.3 Preparation of Histological Slides ..…………………………………………………71
3.2.4 Assessment of The Slides of the Hippocampus ……………………………………71
3.2.5 Statistical Analysis ………………………………………………………………72
CHAPTER FOUR
4.0 Result ……………………………….……………………………………. 73
4.1 Physical Observation ……………………………………………………………73
4.2 Morris Water Maze ………………………………………………………………………..78
4.3 Cell Count of Hippocampal Cell Suspension … …………………………………… 81
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4.4 Cell Count of Slides …………………………….…………………………………..82
4.5 Histological Observations of M &G Stained Slides ………………………………………89
CHAPTER FIVE
5.0 Discussion ……………………………..…………………………………………92
5.1 Physical Characteristics …..…………………………………………………….…92
5.2 Morris Water Maze …………………………………………………………….…92
5.3 Cell Count ……………………………………………………………………..94
5.3.1 Cell Count for Hippocampal Suspension ………………………………………………………… 94
5.3.2 Cell Count of Slides ……………………………………………………………….94
5.3.3 Discussion on Cell Count ………………………………………………………..94
5.4 Summary and Conclusion ….……………………………………………………….. 95
5.4.1 Summary ……………………………………………………………………………..95
5.4.2 Conclusion …………………………………………………………………………..96
5.5 Recommendations …………………………………………………………………..97
References …………………………………………………………………………98
Appendix …………………………………………………………………………117
CHAPTER ONE
1.0 Introduction
1.1 Background
Memory is a complex, diverse and heterogeneous entity and in recent decades, it has
become one of the principal pillars of a branch of science called cognitive neuroscience, an
interdisciplinary link between cognitive psychology and neuroscience. In psychology,
memory is an organism’s ability to store, retain and recall information. Tulving and Craik
(2000) define memory as ‘the ability to recollect past events and to bring learned facts and
ideas back to mind’. An adequate definition of memory must incorporate other aspects of
this complex phenomenon including, both conscious and non-conscious aspects of memory.
Memory is ‘the demonstration that behaviour has been altered as a consequence of the
previous storage of information at some point in time ranging from a few seconds to several
decades’(Jonathan, 2002). For descriptive purpose, memory could be viewed from the
perspectives of duration, temporal and information type.
The duration perspective has the following entities: Sensory memory, which is the memory
that corresponds approximately to the initial 200 – 500 milliseconds after an item is
perceived. The ability to look at an item and remember what it looked like with just a second
of observation or memorization is an example of sensory memory (Sperling, 1960).
While, a short term memory is the memory that allows recall for a period of several seconds
to a minute without rehearsal. Long-term memory can store much larger quantities of
information for potentially unlimited duration sometimes a whole life span. The capacity can
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also approach infinity (unlimited). Memory could be viewed from temporal direction of the
information to be remembered, this addresses whether the content to be remembered is in
the past, “retrospective memory”, or whether the content to be remembered is in the future,
“prospective memory”. Retrospective memory includes semantic, episodic and
autobiographical memory. In contrast, prospective memory can be divided into event- and
time-based prospective remembering (Winograd, 1988). Finally is, the classification of
memory based on information type, which divides long-term memory into declarative
(explicit) and procedural (implicit) memories (Anderson, 1976). Procedural memory is
primarily employed in learning motor skills and should be considered a subset of implicit
memory since it is devoid of conscious recall of information. Procedural memory depends
on the cerebellum and basal ganglia. Where a declarative memory requires conscious recall,
it is sometimes called explicit memory, since it consists of information that is explicitly
stored and retrieved. It can be further sub-divided into: Semantic memory, which concerns
facts taken independent of context and Episodic memory, which concerns information
specific to a particular context, such as a time and place. Autobiographical memory and
spatial memory are subset of episodic memory (Conrad, 1964).
The hippocampus is hypothesized to be important in a memory system that participates in
encoding relationships among stimuli (Cohen and Eichenbaum, 1993). Nearly all
investigators agree that any event which significantly alters the function of the hippocampus,
either temporarily or permanently will cause amnesia in humans and rats.
Well-studied examples include drug administration (Toumane and Derkin, 1993),
electroconvulsive shock (Squire et al.,1981), brain trauma (Markowitsch et al., 1993), brain
ischemia (Zola-Morgan., 1986), hypoxia (De Renzi and Lecchelli, 1993), encephalitis
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(Yoneda et al., 1992), surgical excision of brain tissue (Corkin, 1984), and new learning
(Keppel,1984). There are many reports that damage to the hippocampus has a detrimental
effect on the recall of recent but not remote memories (Squire, 1992) amnesia. There are two
important properties of memories which determine their vulnerability to hippocampal
damage; Type and Age (Sutherland et al., 2001).
Regarding type of memory, spatial memory (Morris et al.,1990), conditioning to context
(Kim et al., 1993) are affected following hippocampal damage while learning tasks which
require navigation to a location marked by a single landmark, are spared (McDonald and
White, 1993). The second property of memories which determines the vulnerability of
memory to disruption by hippocampal damage is age. A commonly reported pattern of recall
in retrograde amnesia following hippocampal damage is that remote events are remembered
better than recent events. This pattern of recall has come to be known as temporally graded
retrograde amnesia. These suggest that memories become more resistant to disruption by
hippocampal damage or dysfunction as time passes after the learning episode. This
transformation of memories from a labile to a stable form is termed memory consolidation.
There are numerous reports of temporally graded retrograde amnesia lasting from a few
months to decades as a result of damage to the hippocampus in humans (Larry et al., 1996).
The well-studied patient Henry Molaison (H.M) exhibits temporally graded retrograde
amnesia. He is unable to recall events which occurred during several year intervals prior to
his surgery. More remote memories are apparently unaffected (Corkin, 1984).
In contrast, there are also many reports of retrograde amnesia which is not temporally
graded (Kapur et al., 1992). Patients with retrograde amnesia that is more or less equivalent
for all past events regardless of the time that the memory was encoded prior to hippocampal
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damage are said to have a “flat gradient” (Sutherland et al., 2001). The length of “recent”
memory loss in retrograde amnesia is reported to be days to many weeks in non-humans.
For example, memory seems to be fully consolidated after 5 days in the case of socially
transmitted food preference, since hippocampal damage after this point does not affect
performance (Winocur, 1990). However, hippocampal-dependent memory consolidation
takes 4 weeks in classical fear conditioning (Kim and Fanselow, 1992) and several months
in the case of place navigation tasks (Kubie et al., 1999).
1.2 Statement Of The Problem
The gap in knowledge stems from the absence of data relating the changes in spatial
memory tasks in mammals directly to anatomical changes in the hippocampus attributable
to cigarette smoke exposure maintained at 10-32 ppm of carbon monoxide. The findings in
this study will strengthen the scientific understanding of the toxicological effect of cigarette
smoke on the hippocampus and try to extend and correlate the findings to the spatial
memory function of the hippocampus.
1.3 Study Hypothesis
Hoa: The difference in the cell count of the cell suspension of the male hippocampus of the
study groups is statistically significant.
Hia: The difference in the cell count of the cell suspension of the hippocampus of the study
groups is statistically significant
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Hob: The pre and post exposure latency time in the Morris Water Maze task for the study
animals is not statistically significant.
Hib: The pre and post exposure latency time in the Morris Water Maze task for the study
animals is statistically significant.
Hoc: The difference in the cell count of the CA1 and CA2 regions of the hippocampus of the
study groups is not statistically significant
Hic: The difference in the cell count of the CA1 and CA2 regions of the hippocampus of the
study groups is statistically significant
1.4 Justification
In smokers and patients suffering from a variety of neuropsychiatric disorders, nicotinic
agonists act beneficially on several aspects of cognition, including working memory
attention, learning and memory (Levin et al., 2005). In contrast, in normal nonsmokers,
nicotine tends to have deleterious effects on cognitive performance (Newhouse et al.,
2004b). On the other hand, adolescent nicotine treatment evoked decreases in total cell
number as assessed by DNA measurements in cerebral cortex, midbrain and hippocampus
(Trauth et al., 2000). Likewise, Zoli et al. (1999) showed that stimulation of nicotinic
cholinergic receptors in mature cells can actually decrease the cell death elicited by injurious
treatments potentially by induction of neurotrophic factors. On the other hand, Olcay et al.,
(2007) showed an increase in apoptotic bodies in the hippocampus of rabbits that were
exposed to the cigarette smoke. There is a need for a study of 10-32 PPM (low
concentration) of carbon monoxide, with adequate counting of pyramidal cell in the
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hippocampus and relating the count to memory capacity of Wistar rats in Morris water maze
task.
1.5 Aim
To determine the structural and functional effects of exposure to cigarette smoke maintained
at 10-32 PPM of carbon monoxide on the hippocampus of Wistar rats.
1.6 Objectives
(1)To compare the memory ability of the rats using records of time from Morris
water maze task before and after exposure to cigarette smoke
(2)To compare the number of pyramidal cells in the sections of the hippocampus of
the male rats, between the normal and those exposed to cigarette smoke.
(3)To compare the number of pyramidal cells in the hippocampus cell suspension
of the male Wistar rats between the normal and those exposed to cigarette smoke
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