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Committee Chair signature: The role of serotonin in brain development and 3,4- methylenedioxymethamphetamine-induced cognitive deficits
A Dissertation submitted to the
Division of Research and Advanced Studies of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in the Graduate program in Molecular and Developmental Biology of the College of Medicine
May 2009
by
Tori Lynn Schaefer
B.S., College of Mount St. Joseph 2004
Committee Chair: Michael T. Williams, Ph.D.
Charles V. Vorhees, Ph.D. Kenneth Campbell, Ph.D. Gary A. Gudelsky, Ph.D. Steve Danzer, Ph.D.
ABSTRACT
Serotonin (5-hydroxytryptamine, 5-HT) is thought to be important during brain development and is one of the first neurotransmitters to appear. It appears to act as a neurotrophic factor supporting the growth and maturation of both serotonergic and non- serotonergic cells during the pre and early postnatal periods prior to its role as a neurotransmitter. Disruption of 5-HT functioning during human development is thought to be associated with autism and schizophrenia. Early developmental exposure to stress or drugs of abuse disrupt 5-HT development and produces altered cognitive ability. To better understand the relationship between the developing serotonergic system and long- term cognitive function we employed the use of a genetic model in which 5-HT levels are depleted ~80% throughout life and the use of a model of 3,4- methylenedioxymethamphetamine (MDMA) exposure in which administration from postnatal day (P)11-20 is used; a period analogous to the second half of human gestation.
Pet-1 is a transcription factor that is restricted in the brain to 5-HT neurons and has been shown to be important for their development and function. A loss of Pet-1 results in an
80% reduction of the number of 5-HT neurons as well as 5-HT tissue content. Pet-1 knockouts were tested as adults in both the Cincinnati and Morris water mazes that assess path integration and spatial learning, respectively. A reduction in cognitive ability was not observed in Pet-1 knockout mice, although they displayed decreased locomotor activity, increased marble burying, and increased startle reactivity. To better assess the role 5-HT plays during development for circuits involved in memory formation, we used a model of MDMA exposure that has previously been reported to produce protracted path integration and spatial learning deficits that last late into adulthood. In adults, MDMA
i produces an initial release of 5-HT followed by dramatic depletions, and these depletions can last for weeks. It was previously shown that neonatal MDMA administration on P11, the first day of the exposure period known to result in memory impairment (P11-20), also produced significant depletions in 5-HT. We further characterized the degree of 5-HT depletion that was experienced by rats exposed to P11 MDMA exposure and compared this to 5-HT reductions induced by other substituted amphetamines as well as determining the length and degree of depletion following P11-20 MDMA exposure.
Substantial 5-HT depletions existed during all ten days of MDMA exposure, however, these depletions were not observed on P30. Citalopram (CIT), a highly selective SSRI, was used in combination with P11-20 MDMA exposure to attenuate the 5-HT depletions.
Thereafter, cognitive ability was assessed in MDMA-treated animals with attenuated 5-
HT depletions. The combination of CIT and MDMA did not improve learning ability; however CIT treatment alone produced deficits in path integration learning. These data suggest that 5-HT alterations during vulnerable critical periods can affect later cognitive ability, and they suggest that depletions in 5-HT alone may not account for cognitive dysfunction in neonatal rats treated with MDMA. Examination of specific 5-HT receptors is likely the next step to understand the mechanism involved in 5-HT disruption during development and later cognitive ability.
ii
ACKNOWLEDGEMENTS
I would like to thank my thesis advisor, Dr. Michael Williams, for his patience, support, and guidance throughout my graduate career. I am grateful to have the opportunity to work with Michael and am thankful to have him as an advisor. I would also like to thank Dr. Charles Vorhees for his additional guidance throughout my graduate career. It has been an incredible experience to work with these excellent scientists who I can comfortably call friends as well as colleagues. I would also be remiss if not thanking current and former my lab mates: Matt Skelton, Devon Graham,
Curt Grace, Amanda Braun, and Mary Moran; I have been truly blessed to work with such wonderful and intelligent people.
I would also like to thank my dissertation committee members, Drs. Michael
Williams, Chip Vorhees, Gary Gudelsky, Steve Danzer, and Kenny Campbell for their support, guidance and interest in my research.
I am honored to be a graduate of the Molecular and Developmental Biology
Graduate Program. I would like to thank the staff of the Division of Developmental
Biology and the Division of Neurology, both past and present.
Finally, I would like to thank my family, especially my wonderful husband
Jonathan. His support, patience and encouragement have made this possible. I would like to thank my mother, Kim, for supporting whatever I decided to do with my life.
iv TABLE OF CONTENTS
List of figures vii
Chapter 1 Introduction 1 The serotonergic system 1 Serotonin depletion 5 Pet-1 7 MDMA 9 o History and prevalence 9 MDMA-induced 5-HT alterations 10 Other MDMA-induced alterations 12 o Behavior 14 o Learning and memory 14 Dissertation synopsis 19 References 20 Figures 40 Hypothesis and Specific Aims 41
Chapter 2: Mouse Pet-1 knock-out induced 5-HT disruption results in a lack of cognitive deficits and an anxiety phenotype complicated by hypoactivity and defensiveness
Title page 42 Abstract 43 Introduction 44 Material and Methods 46 Results 52 Discussion 56 References 63 Figures 71
Chapter 3: A comparison of monoamine and corticosterone levels 24 hours following (+)methamphetamine, (±)3,4-methylenedioxymethamphetamine, cocaine, (+)fenfluramine, or (±)methylphenidate administration in the neonatal rat
Title page 78 Abstract 89 Introduction 80 Materials and Methods 82 Results 86 Discussion 88 References 97 Figures 106
v Chapter 4: Short- and long-term effects of (+)-methamphetamine and (±)-3,4- methylenedioxymethamphetamine on monoamine and corticosterone levels in the neonatal rat following multiple days of treatment
Title page 113 Abstract 114 Introduction 115 Material and Methods 118 Results 122 Discussion 127 References 132 Figures 143
Chapter 5: Alterations to learning and memory following neonatal exposure to 5-HT altering drugs: individual and combined effects of MDMA and citalopram
Title page 153 Abstract 154 Introduction 156 Material and Methods 159 Results 168 Discussion 178 References 187 Figures 199
Chapter 6: Discussion
Conclusions 208 Discussion 208 Critical period of 5-HT development 210 Alternative 5-HT hypothesis 212 Future studies 214 References 219 Figures 227
vi LIST OF FIGURES
Chapter 1
Figure 1. Simplified schematic of serotonergic signaling 40
Chapter 2
Figure 1. Elevated zero maze 71
Figure 2. Locomotor activity 72
Figure 3. Marble burying 73
Figure 4. Light-Dark box exploration 73
Figure 5. MWM hidden platform trials 74
Figure 6. MWM shift phase initial heading error 75
Figure 7. Cincinnati water maze 76
Figure 8. Locomotor activity with MA challenge 77
Chapter 3
Table 1. Body weights 106
Figure 1. Corticosterone concentrations in plasma 107
Figure 2. Serotonergic markers in the neostriatum 108
Figure 3. Dopaminergic markers in the neostriatum 109
Figure 4. Serotonergic markers in the hippocampus 110
Figure 5. Comparison of MWM effects 111
Chapter 4
Figure 1. Body weights 143
vii Figure 2. Corticosterone concentrations in plasma 144
Figure 3. Serotonergic markers in the neostriatum after P11-15 exposure 145
Figure 4. Dopaminergic markers in the neostriatum after P11-15 146 exposure
Figure 5. Serotonergic markers in the neostriatum after P11-20 exposure 147
Figure 6. Dopaminergic markers in the neostriatum after P11-20 148 exposure
Figure 7. Serotonergic markers in the hippocampus after P11-15 149 exposure
Figure 8. Serotonergic markers in the hippocampus after P11-20 151 exposure
Figure 9. Time course of CORT and 5-HT changes 152
Chapter 5
Table 1. 5-HIAA, 5-HIAA/5-HT ratio, DA, DOPAC, DOPAC/DA ratio, 199 NGF, BDNF, and CORT on P12, 16, and 21 Table 2. Light-Dark test, Elevated zero maze, and Straight channel 201
Figure 1. Hippocampus 5-HT: P11-20 exposure 202
Figure 2. P12, P16, and P21 5-HT levels 203
Figure 3. Body weights 204
Figure 4. Locomotor activity 205
Figure 5. Cincinnati water maze 206
Figure 6. Morris water maze 207
viii Chapter 6
Table 1. Summary of hippocampal 5-HT, CORT, and CWM learning 227 Figure 1. Schematic of 5-HT neuron 228
ix
CHAPTER 1: INTRODUCTION
The monoamine 5-hydroxytryptamine (5-HT) was first isolated from serum and was named “serotonin” because of its vasoconstrictive effects (Rapport et al. 1948). Serotonin was later detected in the mammalian brain and had a wide distribution throughout the central nervous system. Many aspects of mammalian physiology are influenced by the effects of 5-HT, including cardiovascular regulation, respiration, gastrointestinal function, and more centrally controlled functions including circadian rhythm, appetite, aggression, sensorimotor activity, sexual behavior, mood, cognition, and learning and memory. Although many therapeutic drugs with serotonergic activity can alleviate some of the symptoms associated with psychological and cognitive dysfunction we still do not have a good understanding of the disease etiologies.
It has long been suggested that impaired development of the 5-HT system, due to genetic disruptions or environmental factors such as stress, malnutrition, sleep, hormones, or drug exposure, leads to altered synaptic plasticity, resulting in a variety of observable behavioral alterations. In the studies described herein, we focused on the cognitive deficits associated with developmental 5-HT disruption. Many illicit drugs, including 3,4- methylenedioxymethamphetamine (MDMA), are known to target the serotonergic system and can alter neuronal structure and produce spatial and path integration learning deficits following developmental exposure. Genetic manipulation of Pet-1, a transcription factor involved in the proliferation and function of the serotonergic system, or the exposure of immature rats to
MDMA provided a framework to investigate the effects produced from the developmental disturbance of the serotonergic system.
The serotonergic system
1
The serotonergic system is involved in many physiological and behavioral processes including
cognition, anxiety, and aggression (Whitaker-Azmitia et al. 1996a). Serotonin (5-
hydroxytryptamine, 5-HT) is one of the first neurotransmitters to appear during development and
is thought to aid in organizing the brain (Lauder and Bloom 1974;Lauder and Krebs
1978a;Lauder et al. 1981). Abnormal activity of this neurotransmitter system has been
implicated in a wide variety of disorders including autism, depression, schizophrenia, and
attention deficit disorder (Azmitia 1999). Both children and adults can be affected by these
disorders and successful treatments tend to target the serotonergic system. Interestingly, it has
been suggested that autism and schizophrenia have perinatal etiological roots and may involve 5-
HT and be the result of disruptions in the specification/organization of the developing brain
(Murray et al. 1992;Scott and Deneris 2005;Torrey et al. 1994;Whitaker-Azmitia 2005).
Early in embryonic development, before serotonin acts as a neurotransmitter, it appears to be a neurotrophic factor for the organization of the CNS. For example, 5-HT is released from sprouting axons even before synapses have been formed (Vitalis and Parnavelas 2003).
Serotonin not only regulates the growth and development of target regions, but it also regulates
its own development and is involved in cell division, cell differentiation, neuronal migration, and
synaptogenesis (Azmitia 2001;Lauder 1993;Vitalis and Parnavelas 2003;Whitaker-Azmitia et al.
1996b). Serotonin is first expressed in two domains (a caudal and rostral domain) in the hind
brain (Lauder et al. 1982) on E11 in the mouse (Pfaar et al. 2002) and E 13 in the rat (Lauder
1990). These 2 domains of 5-HT cells migrate and within 1 week the 9 major serotonergic
nuclei are visible (B1-9 groups (Dahlstrom and Fuxe 1964)) (Jacobs and Azmitia 1992). The
caudal domain sends projections to the spinal cord and the rostral domain innervates the
forebrain (Tork 1990). The B6 and B7 nuclei are considered collectively as the dorsal raphe
2
nucleus and send projections to the hippocampus and striatum and the B8 nucleus is referred to
as the median raphe nucleus and innervates the hippocampus, septum, and hypothalamus (Jacobs
and Azmitia 1992). Serotonergic projections are highly organized such that cell bodies found in
particular regions in the raphe nuclei project to a specific area in the forebrain. Initial projections
reach subcortical regions during gestation, however critical innervations of the hippocampus do
not begin until postnatal day 1 and continue through 3 months of age in the rat (Jacobs and
Azmitia 1992).
Serotonergic neuronal function is dependent on the ability to synthesize, package, export,
and regulate 5-HT (Fig. 1). Serotonergic neurons are confirmed by the presence of the rate
limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (TPH) (Goridis and Rohrer 2002).
The synthesis of 5-HT involves modification of amino acid L-tryptophan (TRP) by TPH and L-
aromatic amino acid decarboxylase (L-AADC), which is also necessary for dopamine synthesis
(Goridis and Rohrer 2002). Once synthesized, 5-HT is packaged by a vesicular monoamine transporter (Vmat2) and is then ready for neurotransmission (Gaspar et al. 2003). In addition to
TPH, serotonin levels are maintained largely by the high affinity serotonin transporter (SERT),
allowing neurons to bring 5-HT back into the cell for repackaging, (Lesch 1991) and by
monoamine oxidase (MAO) which degrades 5-HT (Borue et al. 2007). SERT expression is
highest during the first two postnatal weeks in the rodent (Narboux-Neme et al. 2008) and is expressed more broadly in the human embryonic brain compared to adult brain (Verney et al.
2002). During development, non-serotonergic neurons including glutamatergic neurons found in
the thalamus, limbic region, hypothalamus, and the retinal and superior olivary nuclei have been
shown to express SERT. These neurons can uptake 5-HT without the ability to synthesize it and
therefore can be influenced by fluctuations in developmental 5-HT levels (Beltz et al.
3
2001;Goridis and Rohrer 2002;Lebrand et al. 1996). The protracted development of the 5-HT
system likely produces multiple critical periods for neuronal development that can be affected by
alterations in 5-HT levels and subsequently result in cognitive deficits.
The serotonergic system has many receptors, encoded by 15 genes, which are categorized
by their second messenger coupling pathways. The 5-HT1 receptors (5-HT1A, 5-HT1B-C, and the
5-HT1D-F) are coupled to Ti, the 5-HT2 receptors (5-HT2A-C) are coupled to Gq, the 5-HT4, 5-HT6, and 5-HT7 receptors are coupled to Gs, and the 5-HT5 receptors (5-HT5Aand 5-HT5B) are
associated with an undetermined Gα subunit transduction cascade (Gaspar et al. 2003). In
addition to the 13 guanine nucleotide-binding protein coupled receptors (G protein), 5-HT3 and
5-HT3B are 5-HT-gated ion channel receptors (Chameau and van Hooft 2006). Alternative mRNA splicing and editing for these 15 receptor subtypes has also been described that create an additional twenty 5-HT receptor subtypes with different binding affinities and physiological functions (Raymond et al. 2001). The abundance of 5-HT receptors and the relatively few specific 5-HT receptor agonists and antagonists has made it difficult to fully understand the functions of each receptor, however 5-HT1A receptors have received the most attention and are thought to be the most influential for mediating the developmental effects of 5-HT (Patel and
Zhou 2005). During rat neonatal development, 5HT1A binding sites have been identified in the
brainstem, neurons and glia in the hippocampus, and cortex and are functional with regard to G
protein coupling (Daval et al. 1987;Kobilka 1992;Patel and Zhou 2005). Similarly in human
fetal brains in the hippocampus, 5-HT1A mRNA has been observed during midgestation (del et
al. 1998). Transient high expression during early rat postnatal life of 5-HT1A has also been
observed in the cerebellum even though mature structures do not retain this expression (Miquel
et al. 1994) further suggesting 5-HT1A mediates events specific to development. The 5-HT1A
4
receptor has been shown to control neurite extension via negative feedback (Brownstein et al.
1976;Jacobs and Azmitia 1992). This mechanism allows for neuron growth to occur until it
reaches an environment (i.e., target region) after which excess 5-HT is released. The surplus 5-
HT that leaks from the synapse interacts with presynaptic 5-HT1A receptors and results in cessation of that neuron’s growth (Jacobs and Azmitia 1992). Other 5-HT receptors are also thought to play a role in dendritic maturation and synaptic plasticity including the 5-HT1B and 5-
HT2 receptors (Gaspar et al. 2003). Therefore, disruption of either the number of receptors or
the amount of 5-HT that is released from vesicles during neurite outgrowth is likely to impede
proper synaptogenesis and prevent proper wiring of neuronal networks necessary for cognitive
functioning.
Serotonin depletion
Depletions in 5-HT or TPH activity have been linked to cognitive disturbances associated
with various human diseases including Huntington’s disease (Yohrling IV et al. 2002) and
Alzheimer’s disease (Schmitt et al. 2006). Interestingly, cognitive deficits associated with both
schizophrenia and autism are suspected to be related to perinatal changes to the 5-HT system
(Murray et al. 1992;Whitaker-Azmitia 2005). In addition, early developmental exposure to
various drugs of abuse, which are known to affect the 5-HT system, have been found to produce
long-term learning and memory deficits in rodents (Mazer et al. 1997;Morford et al.
2002;Williams et al. 2002;Williams et al. 2003).
Much of our understanding of the role 5-HT plays in behavior and many physiological processes is a result of experimentally depleting 5-HT via restriction of dietary tryptophan, administration of pharmacological agents that affect 5-HT biosynthesis, or administration of 5-
5
HT neurotoxins. 5-HT synthesis in the brain is affected by three factors: the total amount of free
plasma tryptophan, the amount of free tryptophan that crosses the blood brain barrier, and the
activity of the rate limiting enzyme TPH (Bell et al. 2001). To decrease tryptophan through
dietary manipulation it is necessary to not only limit tryptophan in the diet, but also to administer
a tryptophan-deficient amino acid load that includes the known amino acids that compete for
transport across the blood brain barrier (reviewed by (Reilly et al. 1997)). In adult rats,
nutritional decreases in tryptophan using this method do not affect spatial learning, but have been shown to produce impairments in object recognition tasks (Lieben et al. 2004). This deficit in recognition memory was only observed when animals were tested during the acute TPH depletion; once TPH activity resumed, deficits were no longer observed (Lieben et al. 2004) suggesting that long-lasting changes in cognition are not affected following acute TPH depletion in adults.
Administration of the irreversible tryptophan hydroxylase inhibitor, parachlorophenylalanine (PCPA) to pregnant rats, has been shown to delay the onset of differentiation of the raphe nuclei and presumptive serotonergic targets and alter postnatal expression of serotonergic receptors in the offspring (Lauder and Krebs 1978b;Lauder et al.
1985). PCPA administration on gestational day (GD) 14-17 significantly depleted 5-HT levels in
17-day-old rat embryos and, when tested as adults, resulted in spatial learning deficits in the
MWM, regardless of sex (Vataeva et al. 2007). PCPA-induced 5-HT depletions during the neonatal period has also been shown to result in decreased learning and memory function (Mazer
et al. 1997). This is in contrast to adult PCPA treatment in which spatial learning is not affected
(Richter-Levin and Segal 1989). The 5-HT neurotoxins parachloroamphetamine (PCA), and 5, 7
6
dihydroxytryptamine (5,7-DHT) produce decreases in 5-HT and dendritic spine density and
length in granule cells when administered to rats on P3 and P4 (Yan et al. 1997).
Pet-1
Pet-1 (plasmacytoma-expressed transcript 1) is an ETS domain transcription factor that is
restricted in the brain to 5-HT neurons and is suspected to play a vital role in the development
and differentiation of serotonergic progenitors as well as maintain the expression of TPH and
other genes required for serotonergic function in adulthood. Pet-1 and other winged helix-loop-
helix transcription factors are defined by a highly conserved 85 amino acid binding domain
(Donaldson et al. 1996) and they are reported to be involved in regulating cellular specification,
cell proliferation, migration, and apoptosis (Tootle and Rebay 2005). Several ETS factors have been found to regulate transcription in a cell type, specific manner within the hematopoetic
lineage (Wasylyk et al. 1993), and a few have been identified in the vertebrate and invertebrate
nervous systems suggesting that they may play a role in the development and maintenance of neural cell phenotypes.
Pet-1 was discovered while trying to identify trans-acting regulators of clustered neuronal nicotinic acetylcholine receptor (nAchR) genes in the PC12 (rat pheochromocytoma) neuroendocrine cell line (Fyodorov et al. 1998). Other cell lines, including rat CNS glioma, mouse neuroblastoma, rat fibroblast, rat normal liver, rat pancreatic tumor, HeLa, and human cervical carcinoma, and tissues, including intestine, liver, spleen, lung, heart, thymus, superior cervical ganglia (SCG), kidney, adrenal, brain, and eye, were also assayed for Pet-1 mRNA expression. Of these, Pet-1 was only expressed in PC12 cells and tissues from adrenal, small intestine, eye, and brain (Fyodorov et al. 1998). Interestingly, neural cells within the adrenal
7
gland and SCG arise from the same sympathoadrenal sublineage of post-migratory neural crest
cells (Anderson 1989) however, SCG cells do not express Pet-1 and the adrenal cells do. This
pattern of expression suggests that Pet-1 may be a regulator of terminal differentiation and helps
to stabilize cell phenotypes (Fyodorov et al. 1998).
In rodents, the appearance of Pet-1 mRNA precedes 5-HT immunoreactivity by 0.5 days
in both the rostral and caudal domains of the raphe nuclei and is restricted to 5-HT neurons in the
brain during development and in adults (Hendricks et al. 1999;Pfaar et al. 2002). The Ets
domain transcription factor Fev is the human homologue of Pet-1 and its mRNA has also been
shown to be restricted to the raphe nuclei of human tissue (Maurer et al. 2004). Pet-1 has been
found to bind upstream regions of the genes for SERT, TPH, and the 5-HT1A receptor; each of these is important for serotonergic neurotransmission (Hendricks et al. 1999). Pet-1 binding can activate transcription of the genes whose expression are characteristic of mature central 5-HT neurons in vitro and therefore PET-1 is thought to be a regulator of transcription in vivo
(Hendricks et al. 1999). These data suggest that Pet-1 is an important control element of the 5-
HT system and genetic manipulation of this factor will aid in our understanding of the importance of 5-HT during development and in adulthood.
Thus far, it has been shown using constitutive knock-out technology that Pet-1 is necessary for the development/differentiation of serotonergic neurons. In the absence of Pet-1, there is an 80% reduction of both the total number of serotonergic neurons and a 70-80% decrease in the level of 5-HT in target regions, such as, the hippocampus, striatum, and cortex
(Hendricks et al. 2003). Although the remaining serotonergic neurons do not appear to require
Pet-1 for differentiation, their observed TPH and SERT deficiencies support the idea that Pet-1 is a critical protein controlling transcription of these genes. Furthermore, the reductions in Pet-1
8
and subsequently 5-HT result in increased anxiety-like behavior and aggression, without
affecting motor behavior, in the Pet-1 nulls compared to wild-type mice (Hendricks et al. 2003).
No reports have been published describing the cognitive ability of Pet-1 null mice.
MDMA
History and Prevalence of MDMA
MDMA was originally patented in Germany in 1914 as a precursor to a therapeutically
active compound (Cohen 1998) and tested by the U.S. military for toxicity in the 1950s
(Hardman et al. 1973). Effects of its psychoactivity in humans were not reported until 1978 by
Shulgin and Nichols (Shulgin A.T. and Nichols D.E. 1978). In the 1980s, patient self-esteem
and therapeutic communication during psychotherapy were improved by 75-175 mg of MDMA
administered orally (Grinspoon and Bakalar 1986), although increased heart rate and blood
pressure and transient anxiety were reported. The U.S. Drug Enforcement Administration
classified MDMA as a Schedule 1 drug in 1985 because it has a high abuse potential, lack of
clinical application, lack of safety even under medical supervision, and a close chemical
relationship to MDA, a compound found to induce serotonergic nerve terminal degeneration in
rats (Lawn 1986). MDMA is also illegal in the United Kingdom under the Misuse of Drugs Act
(1971) and in Australia (1986) (Green et al. 2003a). Nevertheless, MDMA, in tablet or capsule form, containing between 80 and 150 mg, has become a popular recreational drug and commonly used at raves or all night dance parties (Green et al. 2003a).
The Monitoring the Future study, published by the National Institute on Drug Abuse, has included MDMA on its questionnaire since 1989. Secondary, college age, and young adults have been reported to abuse MDMA yearly anywhere from 0.7% in 1989 to 14% in 2001
9
(Johnston et al. 2004;Johnston 2008). Since then, abuse rates have declined, but remained steady
between 2005 and 2007 with 2.2% of college age students reporting use (Johnston 2008).
Among the highly abused substituted amphetamines, all of which affect monoaminergic
systems (Cadet et al. 2007), MDMA is thought to disrupt serotonergic terminal markers including increased release of 5-HT more than dopaminergic markers (De, I et al. 1997;Molliver et al. 1990). MDMA releases ten-fold more 5-HT and six times less DA than (+)- methamphetamine (Baumann et al. 2007). In humans, within 20-60 min after ingestion of
MDMA, a general euphoric state, emotional openness, reduction of negativity, and a decrease of inhibitions can be experienced and are attributed to the actions of MDMA on the serotonergic system (Green et al. 2003b). MDMA is thought to increase promiscuity (Buffum 1988) and is known to be abused throughout pregnancy (Ho et al. 2001). For example, in the United States during the 1990s and between 1998 and 2000 in Canada, over 100 pregnant women called a substance abuse helpline to inquire about the potential teratogenic effects of MDMA (Ho et al.
2001). Such abuse during pregnancy raises the likelihood that the developing fetus will experience disruptions in the serotonergic system. This section focuses on the MDMA-induced alterations observed during or following P11-20 administration because this appears to be a critical period for serotonergic development (relevant adult data are included for general knowledge and comparison).
MDMA-induced 5-HT alterations
The effects of MDMA on the serotonergic system are thought to be mediated through
SERT by MDMA or one of its metabolites (Berger et al. 1992;Crespi et al. 1997;Fitzgerald and
Reid 1993). MDMA has a high affinity for SERT (Rudnick and Wall 1992) and reverses the
10
direction of transport, thus facilitating 5-HT efflux to the synaptic cleft (Baumann et al. 2007)
and therefore allowing for increased postsynaptic receptor activation. MDMA was first shown to
cause release of radiolabeled 5-HT from adult hippocampal slices (Johnson et al. 1986) as well
as from neonatal neuronal cultures (Kramer et al. 1994). MDMA systemically administered to
adult animals has been shown to increase central extracellular 5-HT levels by in vivo
microdialysis (Gough et al. 1991a;Gudelsky and Nash 1996;Mechan et al. 2002;Sabol and
Seiden 1998a;Shankaran et al. 1999). In adult rats, a single day of MDMA administration (4 x
10 mg/kg) produces 48-82% reductions in 5-HT, depending on the brain region, and that persist
at least 8 weeks in the hypothalamus and at least 16 weeks in the hippocampus and striatum
(Scanzello et al. 1993). The same dose of MDMA administered on P11 was shown to reduce hippocampal 5-HT levels 1 h after the first dose and this continued through at least 72 h after the last dose, with a maximum 45% depletion observed 24 h after the last dose (Williams et al.
2005). Similarly, 5-HIAA in the hippocampus was reduced 24 and 30 h after the first dose
(Williams et al. 2005). P11-20 administration of MDMA to rats was shown to reduce hippocampal 5-HT by ~31% on P21 (Koprich et al. 2003). It was also demonstrated that P11-20
MDMA administration produced 5-HT depletions when examined in adulthood that range from
6-40% of control rats (Broening et al. 2001;Cohen et al. 2005a;Crawford et al. 2006). The range of effects for MDMA-induced 5-HT reductions observed between these studies may be the result of differential handling and/or testing of animals after the MDMA exposure. For example, monoamines were collected following behavioral testing in one experiment, a challenge dose of
SAL in another, and shipped to a different institution completely for analysis in the third
(Broening et al. 2001;Cohen et al. 2005a;Crawford et al. 2006).
11
In addition to the decreased 5-HT content, other markers of serotonergic function are
altered by MDMA treatment. In adult rats, SERT activity, as determined by paroxetine binding,
was reduced for 32 weeks in the striatum and cortex and for at least one year in the hippocampus
following MDMA administration (Battaglia and Napier 1998;Scanzello et al. 1993). However,
SERT activity was not altered by MDMA on P11only in the hippocampus (Williams et al. 2005) or after P10-13, P15-18, P20-23, or P25-28 in the cortex (Kelly et al. 2002). In adult rats, TPH activity was decreased as early as 15 min following MDMA treatment (Stone et al. 1987) and this persisted for two weeks (Schmidt 1987). MDMA has been shown to bind to 5-HT1A
receptors in frontal cortex sections of rat brain (Giannaccini et al. 2007), and 5-HT1A binding
sites were increased in the frontal cortex and decreased in the hippocampus and brainstem after 4 consecutive days of MDMA administration (Aguirre et al. 1997). Adult rats exposed to P11-20
MDMA, have increased GTPγS activation following exposure to either 5-HT or 8-OH-DPAT, a
5-HT1A/5-HT7 agonist. These data suggest that neonatal MDMA exposure increases 5-HT1A
receptor sensitivity in adulthood. Furthermore, taken together with the adult data, the developing
and mature brain are sensitive to MDMA-induced serotonergic changes, however with different
degrees of severity.
Other MDMA-induced alterations
In addition to 5-HT alterations, adult and neonatal MDMA administration also produces
changes in other biochemical systems. For example, MDMA is known to affect the
dopaminergic system in adult rats. MDMA stimulates dopamine (DA) release in the striatum
and decreases in vivo levels of its major metabolites, dihydroxyphenylacetic acid (DOPAC) and
homovanillic acid (HVA) (Yamamoto and Spanos 1988;Yamamoto et al. 1995;Nash and
12
Brodkin 1991;Nash and Yamamoto 1992;Gudelsky et al. 1994;Gough et al. 1991b;Koch and
Galloway 1997;Sabol and Seiden 1998b). DA levels in the striatum have also shown to be
depleted for up to 10 weeks (Commins et al. 1987;McGregor et al. 2003;Able et al.
2006a;Cohen et al. 2005b;Clemens et al. 2004). Following neonatal MDMA exposure, striatal
DOPAC was decreased by P11 MDMA administration 6, 24, and 30 h after the first of 4 MDMA doses (10 mg/kg) without alterations in DA at any time-point analyzed (1, 6, 24, 30, 78 h)
(Williams et al. 2005). Assessment of adult DA levels following P11-20 MDMA exposure demonstrated a significant 18.4% depletion in one study and a nonsignificant depletion of 8.3% in another (Broening et al. 2001;Crawford et al. 2006). The ratio of DOPAC/DA was also shown to be altered following neonatal MDMA exposure (Koprich et al. 2003;Williams et al.
2005) suggesting that even in the absence of changes to the parent compound, neonatal MDMA exposure may be altering the developing DA system and especially its metabolism.
Besides changes in monoamines, other changes have been noted as well. In adult rats, brain derived neurotrophic factor (BDNF) mRNA levels were increased in the frontal cortex and decreased in the hippocampus after acute MDMA exposure (Martinez-Turrillas et al. 2006). In neonates, BDNF was shown to be increased in the frontal cortex, striatum, hippocampus, and brainstem 24 h following P11-20 MDMA exposure (Koprich et al. 2003). BDNF has been shown to be influenced by a number of factors including stress (Smith et al. 1995). In this regard, activation of the hypothalamic pituitary adrenal (HPA) axis has been demonstrated in adult and neonatal rats following MDMA administration. In adults, corticosterone (CORT) and cortisol, the major glucocorticoid in rats and humans respectively, was elevated as early as 30 min and remained elevated for 6 h after MDMA (Connor et al. 1998;Pacifici et al. 2000).
Increases in CORT were also observed beginning 1 h after the first MDMA dose through 24 h
13
after the fourth MDMA dose when administered on P11 (Williams et al. 2005). Concurrent with changes in CORT, adult rats show hyperthermia induced by MDMA when the ambient room temperature is over 21°C (Green et al. 2004) and hyperthermia is also observed in human users
(Freedman et al. 2005). Developing animals seem to be resistant to the hyperthermic effects of
MDMA. For example, P10 administration of MDMA did not alter rectal temperatures (Broening et al. 1995).
Behavioral changes induced by MDMA
While changes in brain neurochemistry have been the major focus of the research on
MDMA, functional changes have also been reported. In rats neonatally exposed to MDMA hyperactivity is initially observed 30 and 60 min following a single MDMA dose on P10 (0.5, 1, or 10 mg/kg) (Winslow and Insel 1990). However, in adulthood, following P11-20 MDMA exposure, hypoactivity has been reported (Cohen et al. 2005a;Vorhees et al. 2007). P10 MDMA exposure has also been shown to diminish ultrasonic vocalizations as well as a negative geotaxic response (Winslow and Insel 1990).
Learning and memory after neonatal MDMA
In the first study to demonstrate learning and memory deficits following developmental
MDMA exposure, two time periods were assessed, P1-10 and P11-20 (Broening et al. 2001).
Interestingly, only the animals exposed during the later exposure period demonstrated learning and memory deficits. This period of brain development is analogous to the second half of human gestation (Bayer et al. 1993;Clancy et al. 2001;Clancy et al. 2007;Rice and Barone S Jr 2000).
Neurons in the dentate gyrus have the highest rate of proliferation during the second half of
14
pregnancy in humans and during the first 19 days in the neonatal rat (Bayer et al. 1993). During
this same time period, there are also twice as many synapses although there is a lack of
myelinated structures (Huttenlocher and de Court 1987). With regard to serotonergic development, there is an increased number of SERT and 5-HT1A receptors, a functional switch
between the 5-HT2A and 5-HT2C receptors in the rat hippocampus, adult-like levels of 5-HT are
achieved, and 5-HT innervation of the dentate gyrus is increasing (Hamon and Bourgoin
1979;Herlenius and Lagercrantz 2004;Ike et al. 1995;Jacobs and Azmitia 1992;Lidov and
Molliver 1982a;Lidov and Molliver 1982b).
In animals exposed to 20 mg/kg twice daily of MDMA from P11-20 and tested beginning
on P60, there were significant increases in both latency and errors committed in the Cincinnati
water maze (run under fluorescent room lighting), suggesting that MDMA produces alterations
in a combination of spatial and path integration learning. In a test of spatial learning, the Morris
water maze (MWM), MDMA-exposed rats performed consistently worse as demonstrated by
increases in latency, path length, and cumulative distance compared to SAL controls. Reference
memory during the probe trials in which the platform was removed was also disrupted in the
MDMA-treated pups as they crossed the platform position less frequently and were consistently
farther away from the platform than SAL controls. Following behavior, adult monoamine
content was analyzed and although only minor decreases in 5-HT in the frontal cortex and
hippocampus (< 15%) and a slight increase in hippocampal norepinepherine in MDMA exposed
rats were observed, these changes did not correlate with the learning and memory deficits. The
lack of cognitive deficits in the P1-10 MDMA-exposed rats and the observation by another group
that 10 mg/kg administered twice daily from P1-4 did not result in altered novel object
recognition learning (Piper and Meyer 2006) suggests that the maturational stage of the CNS at
15
the time of drug exposure likely plays a role in the cognitive dysfunction. Alternatively, since the 5-HT system is still developing, it is possible that higher doses of MDMA are required in order to produce an effect.
To further investigate the reliability of these cognitive deficits induced by P11-20
MDMA exposure (2x 20 mg/kg/day) various experiments were conducted. It was observed that
MDMA animals gained weight at a slower pace than SAL controls during dosing even though the body weights were similar in adulthood (Broening et al. 2001). Therefore, to control for and determine if undernutrition played a role in the cognitive deficits, a large litter design was employed to produce body weights similar to those animals exposed to MDMA. Litters of 16 pups treated with SAL and litters of 8 pups treated with MDMA were found to gain weight at a similar pace and were tested in the CWM (under fluorescent light) and MWM (Williams et al.
2003). This study demonstrated that undernutrition alone cannot account for the learning deficits observed following P11-20 MDMA exposure because SAL-treated animals that weighed similarly to MDMA-treated animals performed similarly to SAL-treated pups that grew at a faster rate (Williams et al. 2003). This study also included a handled only group to determine if multiple injections could produce a difference in learning ability, and this was not observed to produce a difference. SAL-injected (large and small litters) and handled animals (no injections) performed similarly on all learning measures except for probe trials in which the large litter
SAL-treated animals were further away from the platform than weighed only animals on one phase of the MWM (Williams et al. 2003). The findings suggest that maternal selective attention for control animals compared to MDMA-treated animals did not affect the cognitive outcome of
MDMA-treated pups as they were impaired regardless of whether a within (Broening et al. 2001) or between-litter (Williams et al. 2003) design was used. Overall, this study demonstrated that
16
factors such as growth retardation, multiple injections, and litter effects do not affect the
development of learning and memory impairments resulting from P11-20 MDMA exposure
since MDMA-treated pups performed worse in the CWM and MWM than any of the control groups (Williams et al. 2003).
To determine if spatial deficits following P11-20 MDMA exposure could be observed in
a non-swimming task, the Barnes maze was employed and run as an aversive task (Barnes 1979).
Litters were counterbalanced for treatment such that half of the animals were tested in the Barnes
maze prior to testing in the MWM (one phase using a platform 75% smaller than previously
reported methods) and vice versa; these were then followed by a working memory test (Vorhees et al. 2004). MDMA-treated animals showed increases in latency, path length, and cumulative distance during MWM testing at all doses administered (5, 10, or 20 mg/kg administered twice daily), however no effects were observed in the Barnes maze or the working memory tasks during this study (Vorhees et al. 2004). Animals that were tested in the Barnes maze first showed an amelioration of MWM deficits suggesting the possibility of transference of spatial
strategy from one spatial task to another (Vorhees et al. 2004). As in previous studies, no
deficits were observed in the visible platform phase of the MWM or in the ability of the animals
to swim a straight channel, thereby further solidifying the hypothesis that MDMA-induced
learning and memory deficits are not due to sensorimotor deficits (Vorhees et al. 2004).
The effect of dose distribution on P11-20 MDMA exposure was explored as well, since
there is evidence that differential release and subsequent depletion of monoamines in adults
administered MDMA and/or MA occurs (Vorhees et al. 2007). To this end, a total of 40 mg/kg
of MDMA was injected from P11-20 such that a pup from each litter received 1 dose of 40
mg/kg, 2 doses of 20 mg/kg, or 4 doses of 10 mg/kg while maintaining a total of 4 injections per
17
day (saline was administered at the other time points). It was observed that a single dose of 40
mg/kg produced the greatest deficits in the CWM (for this experiment the task was run under
dim red light conditions (Vorhees et al. 2007)). Rats could see well enough under this dim red
light that control performance was not impeded but only delayed compared to performance under
fluorescent lighting (Broening et al. 2001;Williams et al. 2003). Dose pattern had a different
effect in the MWM in which the 4x10 mg/kg regimen produced the longest latencies, path lengths, and cumulative distances in the acquisition phase compared to SAL controls suggesting that dose-pattern effects may depend on the type of learning or the difficulty of the task (Vorhees et al. 2007).
Exposure of animals to P11-20 MDMA and then re-exposing them as adults to an additional day of MDMA, i.e., a known monoamine depleting adult dose, was shown to have some additional effects on cognition compared to neonatal treatment alone (Cohen et al. 2005a).
It was shown that animals treated postnatally and in adulthood demonstrated increased latencies, cumulative distances, and angles of first bearing in the reversal phase of the MWM compared to those treated only neonatally (Cohen et al. 2005a). Deficits in MWM acquisition learning,
CWM, and novel object recognition were not exacerbated by an additional day of MDMA treatment in adulthood as these animals performed no worse than animals treated only during the neonatal period (Cohen et al. 2005a). This study also showed that adult MDMA-induced monoamine depletions were not altered by prior neonatal MDMA exposure. This experiment suggests that a complex monoaminergic interaction is involved in the physiology of early
MDMA-induced cognitive deficits (Cohen et al. 2005a).
Previous studies have shown the presence of P11-20 MDMA-induced cognitive deficits to be present when testing begins around P60, however, to further characterize the emergence
18
and stability of these cognitive deficits, behavioral testing was conducted beginning early on P30 or P40 or late on P180 or P360 (Skelton et al. 2006). Animals that were tested during the early period still demonstrated deficits in the CWM (under dim red light) and MWM (Skelton et al.
2006). Interestingly, animals tested later in adulthood only showed deficits in the MWM suggesting that spatial deficits are long lasting whereas deficits in path integration learning may recover over time (Skelton et al. 2006).
Neonatal MDMA-induced path integration deficits were further characterized by altering the lighting conditions in which the CWM task was run. P11-20 MDMA-exposed animals still demonstrated path integration deficits (increased latencies, errors, and start returns) when infrared lighting was used and all spatial cues were eliminated (Skelton et al. 2009). This requires the animals to rely on self movement as opposed to a combination of spatial and path integration strategies as occurs when some light is present in the room. This method increased the difficulty of the task compared to white or red visible light conditions because control animals require between 10 and 21 days in the dark (Herring et al. 2008;Skelton et al.
2009;Vorhees et al. 2009) to find the escape compared to controls that readily learn the task in 5 days (Able et al. 2006b;Broening et al. 2001;Skelton et al. 2006;Williams et al. 2003).
Dissertation synopsis
To further assess the relationship of cognitive ability and developmental serotonergic perturbations we used genetic manipulation of Pet-1 and a model of developmental MDMA exposure. In Chapter 2, we analyzed the cognitive ability of Pet-1 knockout mice that lack nearly 80% of the total number of 5-HT neurons and content throughout development and in adulthood. We used this genetic model of 5-HT depletion because many of the previously
19
mentioned pharmacological methods for depleting 5-HT also produce non-serotonergic
physiological changes. In Chapter 3, we employed several substituted amphetamines, some that
were known to decrease learning ability when administered during a period analogous to the
second half of human gestation. We sought to determine if the degree of 5-HT depletion following developmental exposure might reflect the severity of spatial learning deficits
previously reported. This further indicated that developmentally reducing 5-HT functioning resulted in a cognitive decline in adulthood. In chapter 4, we wanted to determine if 5-HT depletions were maintained following multiple days of MDMA exposure and if 5-HT levels returned to normal after MDMA treatment stopped. To determine if previously reported neonatal MDMA-induced learning deficits could be attributed to the 5-HT depletions reported in
Chapters 3 and 4, we used a pretreatment of citalopram to attenuate the 5-HT depletions. In
Chapter 5, we assessed the long-term cognitive outcomes in the Morris and Cincinnati water mazes following an attenuation of MDMA-induced 5-HT depletions. Because we were able to block or attenuate the 5-HT depletions induced by MDMA, we expected an improvement in animals administered citalopram in combination with MDMA compared to animals exposed to
MDMA alone.
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39
Figures
Figure 1. Simplified schematic of serotonergic signaling. Adapted from (Borue et al. 2007).
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Hypothesis and Specific Aims
Hypothesis
Abnormal serotonergic system maturation results in learning and memory deficits. Genetic
depletion of serotonin (5-HT) will result in altered spatial and path integration learning while
developmental methylenedioxymethamphetamine (MDMA)-induced learning deficits are the
result of 5-HT depletion during the exposure period and these deficits can be attenuated by
lessening the 5-HT depletions.
Specific Aims
1. To determine if an approximate 80% decrease in 5-HT via genetic deletion of Pet-1 (the
gene responsible for most serotonergic neurons) results in cognitive deficits.
2. To determine if drugs that differentially affect the serotonergic system in adults will
produce varying degrees of 5-HT depletions when administered to postnatal day (11) rat
pups.
3. To characterize the serotonergic changes in P11-20 MDMA- and MA-treated animals.
4. To determine if 5-HT depletions following P11-20 MDMA exposure play a role in spatial
and path integration learning deficits.
41
CHAPTER 2:
Mouse Pet-1 knock-out induced 5-HT disruption results in a lack of cognitive deficits and an
anxiety phenotype complicated by hypoactivity and defensiveness
Tori L. Schaefer, Charles V. Vorhees, Michael T. Williams
Cincinnati Children’s Research Foundation, Division of Neurology, Dept. of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229-3039, USA
As submitted (April 6, 2009)
42
ABSTRACT
Serotonin (5-HT) is involved in many developmental processes and influences behaviors including anxiety, aggression, and cognition. Disruption of the serotonergic system has been implicated in human disorders including autism, depression, schizophrenia, and ADHD.
Although pharmacological, neurotoxin, and dietary manipulation of 5-HT and tryptophan
hydroxylase has added to our understanding of the serotonergic system, the results are
complicated by multiple factors. A newly identified ETS domain transcription factor, Pet-1, has
direct control of major aspects of 5-HT neuronal development. Pet-1 is the only known factor
that is restricted in the brain to 5-HT neurons during development and adulthood and exerts
dominant control over 5-HT neuronal phenotype. Disruption of Pet-1 produces an ~80% loss of
5-HT neurons and content and results in increased aggression in male Pet-1-/- mice (Hendricks et
al., 2003). We hypothesized that Pet-1-/- mice would also exhibit changes in anxiety and
cognition. Pet-1-/- mice were hypoactive which may have affected the observed lack of anxious
behavior in the elevated zero maze and light-dark test. Pet-1-/- mice, however, were more
defensive during marble burying and showed acoustic startle hyper-reactivity. No deficits in
spatial, path integration, or novel object recognition learning were found in Pet-1-/- mice. These
findings were unexpected given that 5-HT depleting drugs given to adult or developing animals
result in learning deficits (Mazer et al., 1997;Morford et al., 2002;Vorhees et al., 2007). Lack of
differences may be the result of compensatory mechanisms in reaction to a constitutive knockout
of Pet-1.
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Introduction
The serotonergic system is involved in many physiological and behavioral processes
including cognition, anxiety, and aggression (Whitaker-Azmitia et al., 1996). Serotonin (5-
hydroxytryptamine, 5-HT) is one of the first neurotransmitters to appear during development and
aids in organizing the brain (Lauder and Bloom, 1974;Lauder and Krebs, 1978;Lauder et al.,
1981).
In humans, decreased cognitive ability in children and adults has been linked to decreases
in 5-HT. Cognitive deficits associated with schizophrenia and autism have been suggested to be
associated with perinatal insults to the 5-HT system (Murray et al., 1992;Scott and Deneris,
2005;Torrey et al., 1994;Whitaker-Azmitia, 2005). In animal studies, early developmental
exposure to drugs that decrease 5-HT also result in long-term learning and memory deficits
(Mazer et al., 1997;Morford et al., 2002;Williams et al., 2002;Williams et al., 2003b).
Most of the evidence for the role of 5-HT in brain development and function comes from
pharmacological agents that inhibit 5-HT biosynthesis, destroy 5-HT containing neurons, or
block 5-HT reuptake. For example, maternal administration (prenatal exposure) of the tryptophan hydroxylase inhibitor, p-chlorophenylalanine (PCPA), has been shown in the offspring to delay the onset of neuronal differentiation in the raphe nuclei and presumptive serotonergic targets, and alter postnatal expression of serotonergic receptors (Lauder and Krebs,
1978;Lauder et al., 1985). PCPA-induced 5-HT depletions during the neonatal period and in
older animals have been shown to result in decreased learning and memory ability (Matsukawa
et al., 1997;Mazer et al., 1997). Both p-chloroamphetamine (PCA) and 5, 7
dihydroxytryptamine (5,7-DHT), 5-HT neurotoxins, produce decreases in dendritic spine density
and length in granule cells when administered to developing rats (Yan et al., 1997). Although
44
pharmacological manipulations of 5-HT have been informative, drugs also produce peripheral
effects, secondary central effects, and variable responses even at their intended targets (Chang et
al., 1979;Choi et al., 2004;Knuth and Etgen, 2004;Stokes et al., 2000). Constitutive gene-
targeted deletions of several 5-HT receptors are also known to produce changes in learning ability, aggression, and anxiety, although these mutations are not cell-type specific (reviewed by
(Sodhi and Sanders-Bush, 2004)).
Pet-1 (plasmacytoma-expressed transcript 1) is an ETS domain transcription factor that is
restricted in the brain to 5-HT expressing neurons and is suspected to play a key role in the
development and differentiation of serotonergic progenitors, as well as maintain the expression
of tryptophan hydroxylase (TPH) and other genes required for serotonergic function in adulthood
(Cheng et al., 2003;Ding et al., 2003;Hendricks et al., 1999;Hendricks et al., 2003;Pfaar et al.,
2002). In the absence of Pet-1, there is an 80% reduction of both the total number of
serotonergic neurons in the brain and a 70-80% decrease in the level of 5-HT in target regions,
such as the hippocampus, striatum, and cortex (Hendricks et al., 2003). Although the remaining
serotonergic neurons do not appear to require Pet-1 for differentiation, observed TPH and SERT
deficiencies in Pet-1-/- mice supports the idea that it is a critical protein controlling transcription of these genes (Hendricks et al., 2003). Furthermore, the genetic deletion of Pet-1 and
subsequent reductions of 5-HT neuronal differentiation have been shown to increase anxiety-like
behavior and aggression in male Pet-1 nulls compared to male wild type mice without affecting
motor behavior (Hendricks et al., 2003). Our interest was primarily to test the role of 5-HT in
cognition. In order to more thoroughly phenotype these mice we also included tests of anxiety,
depression, locomotion, and sensorimotor gating. Because previous experiments have focused
45
on males (Hendricks et al., 2003), we evaluated both males and females to obtain a more comprehensive assessment.
Materials and Methods
Animals. Pet-1-/- and wild type (WT) males were mated to Pet-1+/- females in order to
obtain subjects and therefore both WT and null mutants were born and raised by females that
were heterozygous for the Pet-1 gene. It has been suggested that Pet-1-/- females are not as
nurturing and offspring have lower survival rates than control mice (Lerch-Haner et al., 2008).
All mice (129sv x C57BL/6J genetic background) were maintained on a 14 h light/10 h dark
cycle. Males and females, up to a maximum of 2 pups per litter of the same sex and genotype were used from a total of 31 litters. Ears were notched for identification and tails were clipped between P21-28 for genotyping. Mice were genotyped according to a previous protocol
(Hendricks et al., 2003). Mice were housed separately after weaning on postnatal day (P) 28 because of high-level aggression (both combative and sexual). Behavioral testing began between
P60-75 and was conducted in four separate cohorts in which both sex and genotype were equally represented in order to control for possible seasonal fluctuations in behavioral responses.
Breeding and testing was done at Cincinnati Children’s Research Foundation (CCRF) so that no shipping stress occurred. All procedures were approved by the institutional Laboratory Animal
Care and Use Committee. Behavior was assessed during the light portion of the light/dark cycle and food and water were available ad libitum. Tests intended to determine anxiety-related responses (elevated zero maze, spontaneous locomotor activity, marble burying, light-dark box exploration) were assessed before tests of learning and memory or acoustic startle (File et al.,
2004;Hogg, 1996).
46
Elevated zero maze (EZM). The first test administered was the elevated zero maze
(Shepherd et al., 1994) with minor modification (Williams et al., 2003a;Moseley et al., 2007).
Animals were brought to a room adjacent to the test room and allowed 1 h to acclimate before being tested. The test room was dimly lit based on preliminary experiments showing that this induced the proper conditions for mice to explore the test environment. The experimenter exited the room immediately after placing the mouse in one of the closed quadrants of the apparatus.
Sessions were video recorded and later scored for time in open, number of head dips, number of open arm entries, and latency to first enter an open quadrant (ODLog, Macropod Software).
Spontaneous locomotor activity. At least 1 h following completion of the elevated zero maze, locomotor activity was assessed in infrared photocell activity chambers (41 x 41 cm;
Accuscan Electronics, Columbus, OH) for 1 h (Moseley et al., 2007). Total horizontal, peripheral, and central distances were analyzed in 5 min intervals.
Marble Burying. Immediately after spontaneous locomotor activity assessment, animals were moved to an adjacent room and tested in a defensive marble burying task adapted from a previous procedure (Williams et al., 2007). Fifteen blue marbles (1.5 cm in diameter) were arranged in 5 rows of 3 using a template that spaced the marbles 4.5 cm apart, 4.5 cm from the long edge and 3.5 cm from the short edge of a 16 x 27 cm mouse cage containing 5 cm of wood chip bedding. Animals remained in these cages with a tall filter-top lid for 20 min. Latency to begin active digging and the number of marbles at least 2/3 buried at the end of the session were recorded. New cages and bedding were used for each animal and marbles were cleaned with
70% ethanol between animals.
Light-Dark test. In the light dark-chamber, assessment of boundary crossings and time spent in each compartment may be used to assess anxiolytic activity or screen for new 5-HT
47
reuptake inhibitors (Crawley and Goodwin, 1980). On the day after spontaneous locomotor assessment, the locomotor chambers were fitted with a dark enclosure (1/2 the size of the chamber) placed against one wall and mice were placed in the corner of the open (light) side facing the wall. Latency to enter the dark, number of transitions from light to dark, and the total time spent in each side of the chamber were recorded for 10 min. The room was illuminated with overhead fluorescent lights. The chambers were cleaned with a 70% ethanol solution.
Novel object recognition. Testing began 1-3 days following the light-dark test as described previously (Brunskill et al., 2005) with minor modification. Briefly, a gray circular
PVC arena (60 cm in diameter, 41 cm high) was used. Mice were habituated to the empty arena for 10 min/day for 4 days. On day 5, testing consisted of two phases. Phase-1 was familiarization which consisted of mice being placed in the test arena in the presence of two identical objects and the cumulative time exploring either object was recorded until a combined total of 30 s elapsed. Phase-2 was retention (1 h after completion of familiarization) with one object being an identical copy of the familiarization object and the other a never before seen novel object, and mice had to accumulate 30 s attending to either object. ODLog (Macropod
Software) was used to score this task by the experimenter in real-time. Any animal that failed to acquire 30 s of attending to the objects during either phase within 10 min was discontinued from the test.
Morris water maze (MWM). Spatial learning and memory were assessed in the MWM using procedures described previously (Vorhees and Williams, 2006). Testing began 1-4 days following novel object recognition and was performed in a 122 cm circular tank (Moseley et al.,
2007). Animals were first tested for proximal cue learning in the cued version of the task. This consisted of 6 trials on day-1 with the start position (west) and platform (east) in fixed positions.
48
This was done to train the animals to the task requirements (i.e., swimming, moving away from the perimeter, and climbing on the platform to escape). On the next 5 days, 2 trials per day were administered in which both the platform and start positions were randomly moved. On all cued trials, curtains were drawn around the maze to obscure distal cues. The platform (10 cm diameter) was submerged 1 cm below water level and had a spherical orange marker positioned
7 cm above the surface of the water on a brass rod. Latency to reach the cued platform was recorded using a stop watch and all trials had a maximum time limit of 1 min. Following the cued learning, animals were tested in 3 phases of the hidden platform version of the MWM: acquisition, reversal, and shift (Moseley et al., 2007). Briefly, animals received 4 trials per day
(maximum of 1 min) for 6 days followed by a 30 s probe trial on day 7 with the platform removed. For all platform trials (cued and hidden) mice either stayed on the platform or were placed on the platform for 15 s. Each phase used a platform of a different size (10, 7, and 5 cm diameter, respectively). During hidden platform trials, a video camera and tracking software were used to map the animals’ performance (AnyMaze, Stoelting Company,Wood Dale, IL). On platform trials, latency, cumulative distance, path length, and speed were analyzed. For probe trials, platform site crossovers, speed, and percent distance and time in the target quadrant were assessed.
Cincinnati water maze (CWM). The CWM test was used to assess path integration ability and began 1-3 days following the MWM. The maze, as described elsewhere (Vorhees,
1987), was originally designed and scaled for rats. The maze consists of a series of nine black Ts that branch from a central circuitous channel. For mice, the maze was scaled-down such that the width of the Ts and channel were ~50% narrower (8 cm) than for rats (15 cm), with walls 25 cm high filled half-way with water. The maze was fabricated of high density 0.6 cm black
49
polyethylene. Water was drained and refilled daily and allowed to equilibrate overnight to room
temperature (21 ± 1 °C). Testing was conducted in complete darkness using infrared light
emitters and a camera placed above the maze and connected to a closed circuit TV monitor
located in an adjacent room where the experiment watched and recorded performance. Testing
under infrared light was designed to eliminate extramaze cues and prevent the animal from using
distal cues for a spatial strategy to navigate the maze. The animals were started facing the wall
from the point originally described as position-B (Vorhees, 1987) and were allowed a maximum of 5 min per trial to find the escape platform with a minimum 15 min intertrial interval (ITI) if an animal failed to locate the goal. Animals were given two daily trials for 10 days. Errors, latency to escape, and number of start returns were recorded. An error was defined as a head and
shoulder entry into the dead-end portion of any T. Perseverations within a given T were counted
as individual errors. Occasionally an animal did not find the escape after completing all trials and sometimes stopped exploring. This lack of exploration left them with relatively few errors compared to animals that were escaping the maze in less than 5 min. To adjust for this, error scores for these animals are assigned a value of 1 more than the score of the worst performing animal in the experiment.
Acoustic startle/Prepulse Inhibition (PPI). Prepulse inhibition (PPI) of the acoustic startle response (ASR) tests sensorimotor gating and was assessed 1-7 days following completion of the CWM using a previously described procedure (Brunskill et al., 2005). Briefly, each mouse was placed inside a sound-attenuating test chamber (SR Lab apparatus, San Diego
Instruments, San Diego, CA) inside an inner cylindrical acrylic holder with sliding doors at each end and mounted on a platform. The platform had a piezoelectric force transducer mounted beneath it that was sensitive to the animal’s movements. Mice were placed in the cylindrical test
50
chamber for a 5 min acclimation period followed by a 4 x 4 Latin square sequence of trials that
were of four types. The trial types were: zero stimulus, startle stimulus alone, 70 dB prepulse + startle stimulus, or 76 dB prepulse + startle stimulus. The Latin square was repeated three times for a total of 48 trials. The ITI was 8 s and the interstimulus interval was 70 ms from prepulse onset to startle signal onset. The startle signal was 115 dB (SPL) and lasted for 0.2 s.
Tail Suspension. The tail suspension test is known to be affected by acute 5-HT reductions or synaptic release and reduced immobility is commonly used to evaluate the effects of antidepressants in rodents (Crowley et al., 2004;O'Leary et al., 2007). Animals were moved
to the testing room at least 1 h prior to testing. The tail suspension apparatus consisted of a clear
acrylic plate mounted 15 cm above the table on plastic legs. A 1.3 cm hole fitted with a rubber
grommet had an aperature large enough to accommodate the width of a mouse tail. Mice were
suspended with their tails pulled all the way through the hole to prevent grabbing of their tails.
Mice were disqualified if they used their claws to hold onto the grommet. Animals were scored
for 6 min for time spent immobile (absence of struggling).
Forced Swim. Animals were moved to the testing room at least 1 h prior to testing 4-8
days after the tail suspension test. Mice were placed in a clear plastic chamber 19 cm in
diameter and 50 cm tall. Water depth was 20 cm and immobility was scored for 6 min. Mice
were considered immobile if they were not actively moving.
Locomotor Activity with Methamphetamine Challenge. Activity levels following a
subcutaneous injection of the indirect dopamine agonist, (+)-methamphetamine (MA) (1 mg/kg),
were assessed 6-10 days following forced swim testing. Mice were placed in the chambers for
30 min to re-habituate them and were then briefly removed, injected s.c. with MA and returned to the chambers for an additional 120 min.
51
Statistical Analysis. Data were analyzed using mixed linear ANOVA models (SAS Proc
Mixed, SAS Institute 9.1, Cary, NC). The covariance matrix for each data set was checked using
best fit statistics. In most cases the best fit was to the autoregressive-1 (AR(1)) covariance
structure. Proc Mixed calculates Kenward-Rogers adjusted degrees of freedom that do not match
those obtained from general linear model ANOVAs and can be fractional. Measures taken
repetitively on the same animal, such as trial, day, or test interval, were repeated measure factors.
Significant interactions were analyzed using simple-effect slice ANOVAs at each level of the
repeated measure factor. Only genotype main effects and interaction F-ratios with genotype are
shown for clarity of data presentation. Some data are presented only by genotype when sex did
not influence the results. Significance was set at p ≤ 0.05 and trends at p< 0.10. Data are
presented as least square (LS) mean ± LS SEM.
Results
Elevated Zero maze
There was a main effect of genotype for time in open (F(1,45)=8.49, p < 0.006) (Fig. 1A)
and latency to enter an open quadrant (F(1,45)=7.97, p < 0.007) (Fig. 1 B). No main effect of
Sex or Genotype x Sex interaction was present therefore, data are averaged across sex. Pet-1-/- mice (n = 14 males, 12 females) spent more time in the open and took longer to enter the first open quadrant than WT mice (n = 9 males, 14 females). No significant effects were observed for the number of open quadrant entries (Fig. 1C).
Spontaneous Locomotor Activity
For distance, there was a main effect of Genotype (F(1,70.1) = 6.73, p < 0.02) and a trend
for a Genotype x Interval interaction (F(11,659) = 1.70, p < 0.07) (Fig. 2). For center and
peripheral distance there was also an effect of Genotype (F(1,70) = 12.73, p < 0.001 and
52
F(1,70.1) = 3.87, p < 0.05, respectively) and a Genotype x Interval interaction (F(11,770) = 2.73,
p < 0.002 and F(11,658) = 3.00, p < 0.001, respectively). Pet-1-/- mice (n = 18 males, 20
females) traveled less distance overall with comparable reductions in the center and periphery
(not shown) compared to WT mice (n = 18 males, 18 females).
Marble Burying
There was a trend (F(1,76)= 3.71, p < 0.06) for shorter latency to begin burying in Pet-1-/- compared to WT mice (n= 20/genotype/sex) (Fig. 3A). For number of marbles at least 2/3 buried there was a main effect of Genotype (F(1,76)=25.34, p < 0.0001) in which Pet-1-/- buried more marbles than WT mice (Fig. 3B).
Light-Dark Test
There was an effect of Genotype (F(1,55) = 4.36, p < 0.04) for latency to enter the dark in
which Pet-1-/- mice (n = 13 males, 17 females) took longer to move from the light side to the dark side compared to WT mice (n = 17 males, 15 females; Fig. 4A). There were no differences in the number of light-side entries or time spent in the dark (Fig. 4B-C).
Novel Object Recognition
For time spent with the novel object there were no differences between Pet-1-/- (20.31 ±
0.5 s; n = 16 males, 20 females) and WT mice (20.29 ± 0.62 s; n = 16 males, 14 females) (not
shown).
Morris Water maze
During cued platform trials, latency to find the platform was not affected by Genotype or
Sex (not shown; n = 20 male Pet-1-/-, 20 male wild type, 20 female Pet-1-/-, and 19 female WT
mice).
53
During hidden platform trials, for the acquisition phase, there was a Genotype x Sex x
Day interaction for path length (F(5,287) = 2.31, p < 0.04) and cumulative distance (F(5,283) =
2.20, p < 0.05). There were no differences between males (Fig. 5A), but in females, path length
(Fig. 5B) and cumulative distance for Pet-1-/- mice were increased on day-1 and decreased on day-2 compared to female WT mice. Path lengths are plotted for each phase since this measure has been suggested to be less subject to performance effects than latency. Initial heading error showed a significant genotype x day interaction (F(5,290) = 2.97, p < 0.01). Pet-1-/- mice had a larger initial heading error on days 3 and 6 of testing (not shown). There was a trend for
Genotype to affect latency (F(1,70.1) = 3.87, p < 0.05) such that Pet-1-/- mice had decreased latency to find the hidden platform compared to WT mice, even though speed did not differ between Pet-1-/- and WT mice (not shown).
On the probe trial, average distance to the target, crossovers, percent time and distance in the target quadrant, heading error, and speed were not affected by Genotype or Sex (not shown).
During reversal with the 7 cm platform, there was a main effect of Genotype for latency
(F(1,76.3) = 5.01, p < 0.03). Pet-1-/- mice had shorter latencies compared to WT mice (Fig. 5D).
Path length (Fig. 5C), cumulative distance, initial heading error, and swim speed showed no significant effects (not shown).
On the reversal probe trial, there was a main effect of Genotype on crossovers (F(1,75) =
6.19, p < 0.02) and speed (F(1,75) = 4.26, p < 0.04). Pet-1-/- mice had more crossovers and swam faster than WT mice (not shown). No significant effects were seen on average distance or percent time or distance in the target quadrant.
During shift with the 5 cm platform, there was a main effect of Genotype for latency
(F(1,75) = 4.95, p < 0.03) (Fig. 5F) and a Genotype x Day interaction (F(5,375) = 3.08, p <
54
0.01); the latter also being seen for path length (F(5,375) = 2.93, p < 0.01) (Fig. 5E). On days 4,
5, and 6, Pet-1-/- mice had decreased latencies compared to WT mice and conversely on day-1
Pet-1-/- mice had increased path lengths compared to WT mice. There was a main effect of
Genotype (F(1,75.7) = 6.23, p < 0.01) and a Genotype x Sex x Day interaction (F(5,295) = 2.23,
p < 0.05) for initial heading error. On day-2 male Pet-1-/- mice had an increased initial heading error (Fig. 6A) and on days 1, 2, and 5, female Pet-1-/- mice had an increased initial heading error
(Fig. 6B) compared to WT same-sex controls. Speed was not affected.
During the shift probe trial, there was a significant effect of genotype for speed (F(1,75)
= 5.53, p < 0.02). Pet-1-/- mice swam faster than WT mice. Crossover, average distance, and
percent time and distance in the target quadrant were not significantly affected by Genotype or
Sex (not shown).
Cincinnati water maze
For latency (F(1,77.1) = 5.5, p < 0.02) and errors (F(1,77.1) = 3.97 p < 0.05) there was a
Genotype x Sex interaction (n = 20 male Pet-1-/-, 20 male wild type, 20 female Pet-1-/-, and 19
female wild type mice). Pet-1-/- males had had shorter latencies (Fig. 7A) and committed fewer
errors (Fig. 7C) compared to WT male mice. No differences were observed in females for
latency (Fig. 7B) or errors (Fig. 7D). For start returns, there was a main effect of Genotype
(F(1,77) = 13.54, p < 0.0004) and a Genotype x Day interaction (F(9,571) = 2.68, p < 0.005). On days 2 through 5, Pet-1-/- mice had more start returns than WT controls (Fig 7E).
Acoustic startle/PPI
There was a Genotype x Prepulse interaction on startle amplitude (Vmax, F(2,150) = 5.99,
p < 0.003). Slice effect analysis showed there was only a significant effect when no prepulse
55
was presented. Pet-1-/- mice (251.14 ± 21.15 mV) (n = 20 male, 20 female) had increased startle
compared to WT mice (144.73 ± 21.15 mV) (n = 20 male, 19 female).
Tail Suspension
No immobility time differences were found (s); Pet-1-/- male (n=20, 183.3 ± 15.233) and
female (n=20, 178.2 ± 14.804); WT male (n=20, 196.68 ± 14.044) and female (n=19, 174.83 ±
15.7015). Many mice grabbed their hind legs during testing despite our attempt to prevent this
behavior. In order to determine if this affected the results, a separate analysis was performed in
which time spent engaged in limb holding was subtracted from immobility time. No significant
effects of genotype were obtained.
Forced Swim test
No effects of genotype were found. For Pet-1-/- mice, immobility times (s) were: male
(94.9 ± 18.2) (n = 12) and female (99.5 ± 18.2) (n = 12) and for WT they were male (145.0 ±
22.3) (n = 8) and female (98.0 ± 18.2) (n = 12).
Methamphetamine challenge
Following methamphetamine challenge, there was a significant effect of Genotype
(F(1,71.6) = 4.22, p < 0.04), Genotype x Interval (F(23, 1478) = 1.7, p < 0.02), and Genotype x
Sex x Interval (F(23,1478) = 1.74, p < 0.02) by analysis of covariance with the last 10 min of the pre-challenge re-habituation period used as the covariate to ensure that post-challenge effects were not contaminated by baseline activity levels. All groups showed a significant
methamphetamine-induced hyperactivity response, however, Pet-1-/- male mice (n = 18) showed
an exaggerated increase and traveled more distance than WT male mice (n = 18) during intervals
3-16 (Fig. 8A). Female mice (n = 20 Pet-1-/-, 18 WT) did not differ from each other (Fig. 8B).
Discussion
56
Pharmacological, lesion, and neurotoxin experiments suggest that 5-HT is involved in
cognition. Accordingly, we postulated that Pet-1-/- mice, with 80% fewer 5-HT-expressing
neurons and 70-80% reductions in 5-HT levels that originate from early in development onward,
would exhibit cognitive deficiencies. The results, however, reveal that the expected cognitive
deficits were not observed in the 5-HT depleted, Pet-1-/- mice. The lack of 5-HT throughout development did not negatively impact spatial learning in the MWM, path integration learning in the CWM, or recognition memory in a novel object recognition task. Emotional behaviors
including anxiety in the elevated zero maze and light-dark box exploration task and depression-
related behaviors in the forced swim and tail suspension tests were not increased in this genetically-induced model of decreased 5-HT functioning, although the behavior of Pet-1-/- mice in the marble burying task suggested increased defensive anxiety and/or aggression. This is not to say that the alterations of the serotonergic system were without effect. For example, Pet-1-/-
mice did exhibit hypoactivity in spontaneous locomotor activity, increased acoustic startle
amplitude, and an increased sensitivity in locomotor responsiveness to a methamphetamine
challenge. It may be that the lack of cognitive phenotypic changes in these mice was attributable to compensatory mechanisms that prevented the emergence of a ‘5-HT cognitive syndrome.’
Alternatively, perhaps 5-HT is not as vital to cognitive and emotional functioning as reported, however, this interpretation is difficult to reconcile with the extensive accumulated evidence that acute reductions of 5-HT affect these functions or that increasing 5-HT in organisms with endogenous reductions tend to normalize these functions. It may be that the acuity of the 5-HT change is critical to the induction of cognitive effects. If so, the Pet-1 mouse is not a suitable model in which to test this notion.
57
Much of the work assessing the developmental role of 5-HT on adult cognition involves
disrupting 5-HT function during critical periods. In these experiments, initially the 5-HT system
develops normally until pharmacological or neurotoxic manipulation inhibits or ablates some or most serotonergic cellular function. In disease states, such as schizophrenia, autism, depression,
and anxiety disorders, there is evidence that endogenous 5-HT is dysregulated, however while the dysregulation may be chronic it is not as severe or as early in development as that induced by the Pet-1 deletion (Murray et al., 1992;Scott and Deneris, 2005;Torrey et al., 1994;Whitaker-
Azmitia, 2005). We have shown that P11-20 MDMA exposure in rats produces approximately a
50% decrease in 5-HT content in the hippocampus and neostriatum during dosing (P11 through
P21) (Schaefer et al., 2006;Schaefer et al., 2008;Williams et al., 2005). This results in adult learning deficits in the MWM and CWM that are long lasting (Skelton et al., 2006;Vorhees et al.,
2004;Vorhees et al., 2007;Williams et al., 2003b). Fenfluramine produces more dramatic decreases in 5-HT content when administered neonatally and produces more impaired learning in
the MWM than developmental MDMA exposure (Morford et al., 2002;Schaefer et al., 2006).
Others have shown that P10-20 exposure to the tryptophan hydroxylase inhibitor p-
chlorophenylalanine produces spatial learning deficits in the eight-arm radial maze and that these
animals fail to extinguish learned behaviors as adults (Mazer et al., 1997). Given such findings,
it is surprising that Pet-1-/- mice do not exhibit learning deficits since they are lacking 70-80% of
the normal level of 5-HT throughout development and in adulthood. While there is no direct
evidence to support the idea of developmental compensation to explain the absence of cognitive
deficits in Pet-1-/- animals, it is difficult to explain these data by other means. Alternatively, it
may be that previously mentioned learning changes following developmental pharmacologic
58
manipulations of 5-HT may have been dependent on changes induced by these drugs to other
neurotransmitter systems, hypothalamic-pituitary-adrenal axis, or other factors.
In terms of assessing spatial learning in the MWM, path length has been suggested to be
a more accurate index of spatial ability than latency because it is less affected by swimming
ability or speed. It is noteworthy, therefore, that Pet-1-/- mice performed similarly in the cued
phase of MWM suggesting no differences in swimming ability or motivation to escape from
water. There were minor differences in path lengths between Pet-1-/- mice and WT controls (i.e.,
females in acquisition on day-1 and -2; males and females on shift day-1) although these
differences occurred early in the phases in which they were seen and were not maintained on
later test days. These data demonstrate that no profound differences in spatial learning ability
exist in Pet-1-/- compared to WT mice. There were some differences during the most spatially
demanding, shift, phase of MWM testing. Pet-1-/- mice exhibited increased path lengths on day 1
and larger initial heading errors in females on days 1, 2, and 5 and in Pet-1-/- males on day 2.
These changes suggest that Pet-1-/- mice may have a deficit in spatial orientation that is not
reflected in the principal indices of spatial learning. We surmise that the Pet-1-/- mice may leave
the tank wall more off-course than WT controls, but correct their initial heading error quickly
enough to not affect their performance for the trial as a whole.
The CWM is a task that assesses path integration ability which requires egocentric
learning and reliance on self-movement cues of the animal to determine their position within an
environment (Etienne and Jeffery, 2004). This is one of the first experiments to show that mice
will perform this task and successfully use path-finding ability in the absence of distal cues. This
task may be useful for assessing genetic manipulations in mice in which disruptions of path
integration substrates are evaluated, including presubiculum head-direction cells, entorhinal
59
cortex grid and border cells (Solstad et al., 2008), and subsets of hippocampal place cell (Fuhs and Touretzky, 2006;McNaughton et al., 2006;Rondi-Reig et al., 2006;Sargolini et al.,
2006;Whishaw et al., 1997;Witter and Moser, 2006) that together constitute egocentric circuitry.
We showed that male Pet-1-/- mice learn this task more rapidly (shorter latencies and fewer
errors) than male WT controls whereas female Pet-1-/- mice perform the task similarly to WT
females. It is conceivable that Pet-1-/- mice swam faster than controls which may result in more
frequent chance encounters with the escape platform and therefore decreased latencies on later
trials, however, the decreases in errors argue against this interpretation.
The serotonergic system is the target for numerous pharmacological agents including
those for depression, anxiety, and affective disorders. This suggests that the 5-HT system is
involved in the etiology of these behaviors, however, we do not show Pet-1-/- mice to have
depressive-like symptoms or exhibit an anxiety phenotype. The elevated zero maze and light-
dark box exploration tasks assess conflict between the drive to explore a novel environment
versus neophobia. Pet-1-/- mice of both sexes, remained in the closed areas of the elevated zero
maze longer than WT mice, which suggests increased anxiety, but this may also be the result of
decreased activity levels since once the Pet-1-/- mice emerged from a closed quadrant they spent
more time in the open arm than WT mice without a difference in the number of zone crossings
suggesting, contrary to the longer latencies to leave the enclosed areas, that these mice were less
anxious. The light-dark box exploration results also suggest that they may be less anxious since
Pet-1-/- mice remained in the open side longer than controls. Here again, whether these results reflect hypoactivity or reduced anxiety is unclear based on the present data.
It was previously reported that Pet-1-/- males do not spend time in the open arms of the
elevated plus maze suggesting that they may be more anxious (Hendricks et al., 2003). The
60
apparent discrepancy between these data and the current results may be the product of testing
differences. Firstly, the mice previously tested in the elevated plus maze experienced a variety of tests prior to anxiety assessment including the resident-intruder assay in which Pet-1-/- mice were more aggressive. The stress of this test may have played a role in their subsequent response to the elevated plus maze. Secondly, when tested in the elevated plus maze, standard lighting was used previously whereas in the present experiment animals were tested in the elevated zero maze under low-level lighting. Thirdly, like the elevated zero maze, the elevated plus maze results may have been influenced by the hypoactivity of the Pet-1-/- mice. Accordingly, a
number of factors may explain the differences between this and the previous experiment in
regard to anxiety differences.
We also included a different measure of anxiety, marble burying, in which the Pet-1-/- mice buried more marbles than the WT mice. While the marble burying task is regarded as a test of anxiety or depressive-like behavior (Kobayashi et al., 2008;Li et al., 2006), the previous observation that Pet-1-/- males are highly aggressive in the resident-intruder assay (short attack
latencies;(Hendricks et al., 2003)) suggests that the increase in marbles buried and the tendency
towards decreased latencies to begin to bury may be related to this aggressive phenotype rather
than to anxiety (Sluyter et al., 1996).
Male Pet-1-/- mice appear to be more affected by the MA challenge than female Pet-1-/- mice even though their activity levels during the spontaneous locomotor assessment and immediately after methamphetamine injection (at the 5 min interval) are similar (Fig 7). 5-HT is thought to support the development of DA neurons in the substantia nigra (Lauder and Krebs,
1978;Lauder, 1993) and the 5-HT depletions during development in Pet-1-/- animals may play a
61
role in the observed hyper-responsiveness to the indirect DA agonist methamphetamine. Why this pattern did not emerge in females is unclear.
Other reports of genetic manipulations of 5-HT content (constitutive mutants) have hinted at a lack of 5-HT involvement in emotional behavior. Tryptophan hydroxylase (TpH) 1 and 2 double knockouts do not exhibit any overt phenotypes even with nearly complete 5-HT depletion. For instance, no differences were observed in locomotor activity, acoustic startle, or tail suspension (cognitive function was not assessed) in TpH1/2 double knockouts (Savelieva et al., 2008). This group did show TpH1/2 double knockouts to bury more marbles in the defensive marble burying task just as we observed in Pet-1-/- mice, so on this assay good agreement was obtained. However, whereas the TpH double knockouts showed no change in acoustic startle,
the Pet-1-/- mice showed augmented startle reactivity. This indicates a significant difference between complete and partial 5-HT ablation. It may be that partial ablation induces a response akin to neurotoxin-induced receptor supersensitivity to acoustic stimulation. Further experiments will be needed to test this hypothesis.
In conclusion, it has been shown that the incipient 5-HT neurons in Pet-1-/- mice do not
undergo apoptosis, but rather their development is arrested (Krueger and Deneris, 2008). Other
neurotransmitter systems may respond by aiding in compensatory development and
reorganization of the brain under these circumstances. It is possible that a conditional mutant in
which Pet-1 and or 5-HT depletion can be deleted at different times during development may
give better insight into the involvement of neonatal 5-HT disruption and subsequent impact on
emotional and cognitive behaviors.
62
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Figures
Elevated Zero Maze A BC 120 * 60 * 12 100 50 10
80 40 8
60 30 6
40 20 entries Open 4 Timein open (s)
20 10 2 Latency to enter the open (s) open the enter to Latency
0 0 0 WT Pet-1-/- WT Pet-1-/- WT Pet-1-/-
Figure 1. Elevated Zero Maze: A) Time in open (s), B) Latency to enter the open (s), C) Open quadrant entries during a 5 min test session. LS mean ± LS-SEM with males and females combined. *p < 0.05 vs. WT.
71
1200
-/- 1000 Pet-1 WT
800
600
400
Total Distance (cm) * 200
0 5 1015202530354045505560
Min
Figure 2. Locomotor Activity: Mice were tested for 1 h during the light phase of the light/dark cycle. Total distance (cm) as a function of genotype in 5 min intervals. Data are LS mean ± LS-
SEM with males and females combined. *p < 0.05, vs. WT.
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Marble Burying A B 600 10 * 500 8 † 400 6
300
4 200 Latency to bury (s) to bury Latency Marbles 2/3 burried 2 100
0 0 WT Pet-1-/- WT Pet-1-/-
Figure 3. Marble Burying: A) Latency to start burying (s); B) Marbles at least 2/3rd buried.
Mice were tested for 20 min. Data are LS mean ± LS-SEM with males and females combined.
*p < 0.05, †p < 0.10 vs. WT.
Light/Dark A B C 160 * 20 500 140 400 120 15
100 300
80 10
60 200 Time in dark (s) dark in Time Entries into light 40 5
Latencyto enter dark(s) 100 20
0 0 0 WT Pet-1-/- WT Pet-1-/- WT Pet-1-/-
Figure 4. Light-Dark Box Exploration: A) Latency to enter the dark (s); B) Number of entries into the light; C) Time in dark (s). Animals were tested for 10 min. Data are LS mean ± LS-
SEM with males and females combined. *p < 0.05 vs. WT.
73
MWM Acquisition A B 12 12
10 10 Male WT Female WT -/- Male Pet-1 Female Pet-1-/- 8 8 * 6 6
4 4 Path length (m) length Path (m) length Path
2 2 *
0 0 0123456 0123456
Day Day
MWM D C Reversal 12 50 10 40 8
30 6
Latency (s) Latency 20 * 4 Path length (m) length Path WT WT -/- 2 Pet-1 10 Pet-1-/-
0 0 0123456 0123456 Day Day
F MWM E MWM Shift 14 Shift 50
12 * 40 * * WT * 10 -/- Pet-1 30
8 20 WT 6 (s) Latency -/-
Path length (m)Path length Pet-1
10 4
2 0 0123456 0123456 Day Day
Figure 5. Morris water maze (MWM) hidden platform trials: A) acquisition path length males;
B) acquisition path length females; C) reversal path length ; D) reversal latency; E) shift path
length; F) shift latency. Data are LS mean ± LS-SEM (path length was averaged across 4
trials/day). *p < 0.05 vs. same sex WT controls.
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MWM Shift AB 80 80 * * * *
60 60
40 40
WT females 20 WT males 20 Pet-1-/- females Pet-1-/- males Initial heading error (degrees) (degrees) error heading Initial
0 0 0123456 0123456 Day Day
Figure 6. Initial heading error (degrees) during the Shift phase of the Morris water maze
(MWM) hidden platform trials: A) males B) females. Data are LS mean ± LS-SEM (initial heading error was averaged across 4 trials/day). *p < 0.05 vs. same sex WT controls.
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A B 300 300 WT males WT female -/- Pet-1 males -/- 250 Pet-1 female 250
200 200
150
150 Latency (s) Latency 100 * (s) Latency 100 50
0 50 0246810 0246810
Day Day
C 60 60 D
WT males WT females 50 50 -/- Pet-1-/- males Pet-1 females
40 40
30 30 Errors Errors 20 * 20 10 10
0 0 0246810 0246810 Day Day
E 3.5 * WT 3.0 Pet-1-/- 2.5 * 2.0 * * 1.5 Start returns Start 1.0
0.5
0.0 0246810 Day
Figure 7. Cincinnati water maze (CWM): A) Latency (s) males; B) Latency (s) females; C)
Errors males; D) Errors females; E) Start returns males and females combined. Data are LS mean ± LS-SEM averaged across trials (2 trials/day). *p < 0.05 vs. same sex WT for panels A,
B, C, D; *p < 0.05 vs. WT with sexes combined.
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A * * *
2000 * * * * * * * * * * 1800 1600 * 1400 1200 1000 800 600 WT 400 Pet-1-/- 200 Male 0 B 2000
Total Distance (cm) Total Distance 1800 1600 1400 1200 1000 800 600 400 200 Female 0 10 20 30 40 50 60 70 80 90 100 110 120
min.
Figure 8. Locomotor activity with methamphetamine challenge: Total distance (cm) for: A) males; B) females. Methamphetamine dose was 1 mg/kg given s.c. Data are LS mean ± LS-
SEM shown in 5 min intervals. *p < 0.05 vs. same sex WT controls.
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CHAPTER 3:
A comparison of monoamine and corticosterone levels 24 hours following (+)methamphetamine,
(±)3,4-methylenedioxymethamphetamine, cocaine, (+)fenfluramine, or (±)methylphenidate
administration in the neonatal rat
Tori L. Schaefer1, Lisa A. Ehrman2, Gary A. Gudelsky3, Charles V. Vorhees1, Michael T.
Williams1
Divisions of 1Neurology and 2Developmental Biology, Cincinnati Children’s Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH 45229-3039, USA 3 College of Pharmacy, University of Cincinnati, OH 45267-0004, USA
As published Neurochem. 98, 1369-1378.
78
ABSTRACT
We have previously shown that neonatal administration of (±)3,4- methylenedioxymethamphetamine and (+)fenfluramine produce deficits in spatial and path integration learning, while (+)methamphetamine causes deficits in spatial learning. Conversely, cocaine and (±)methylphenidate have no effect on either form of learning following neonatal administration. The purpose of the present study was to determine if corticosterone and/or monoamine levels were changed following subcutaneous administration of 10 mg/kg of
(+)methamphetamine, (±)3,4-methylenedioxymethamphetamine, (+)fenfluramine,
(±)methylphenidate, or cocaine every 2 hours (4 total injections) on postnatal day 11. Twenty- four hours after the first dose, plasma, striatum, and hippocampus were collected. Corticosterone levels were increased in methamphetamine-, fenfluramine-, methylenedioxymethamphetamine-, and methylphenidate-treated rats relative to saline-treated rats while cocaine-treated rats were unaffected. In the striatum and hippocampus, serotonin and 5-hydroxyindolacetic acid were reduced in animals treated with methylenedioxymethamphetamine or fenfluramine, compared to saline. Dopamine levels were not changed by any of the drugs although 3,4- dihydroxyphenylacetic acid was decreased following methylenedioxymethamphetamine or methamphetamine. Minimal effects were seen in neurotransmitter levels following cocaine or methylphenidate. These data suggest that drugs that affect corticosterone and hippocampal serotonin are associated with both spatial learning and path integration deficits and those that affect corticosterone and 3,4-dihydroxyphenylacetic acid are associated only with spatial learning deficits.
Keywords: corticosterone, ecstasy, methamphetamine, cocaine, methylphenidate, fenfluramine
Running title: Developmental effects of psychostimulants
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Introduction
The abuse of substituted amphetamines has been reported in women of childbearing age
(EMCDDA 2004;Johnston et al. 2004). (±)3,4-Methylenedioxymethamphetamine (MDMA),
(+)methamphetamine (MA) (±)methylphenidate (MPH), and cocaine (COC) can sometimes increase the likelihood of sexual activity (Buffum 1988) and are often abused in social gatherings. However, the anorectic properties of these drugs and others such as (+)fenfluramine
[FEN, even though it has been associated with valvulopathy (Khan et al. 1998)] may be
appealing since it has been suggested that some women who become pregnant still strive to be
thin (Franko and Walton 1993). These factors increase the likelihood of fetal exposure and it is
known that MDMA (Ho et al. 2001), MA (Smith et al. 2003;Chomchai et al. 2004), MPH
(Debooy et al. 1993), COC (Richardson et al. 1999), and FEN (Jones et al. 2002)) have been
used during pregnancy, including the third trimester. Despite this, the ramifications of late fetal
exposure are largely unknown.
Developmental psychostimulant exposure in humans is suspected to alter normal brain
function that can last into adulthood (Billing et al. 1985;Cernerud et al. 1996;Chang et al.
2004;Plessinger 1998;Noland et al. 2005), but the hormonal and biochemical changes that occur
following exposure to these drugs remain to be elucidated. In rats, we have shown that
developmental exposure to MA (Vorhees et al. 2000a;Williams et al. 2002;Williams et al.
2003d;Williams et al. 2003b), MDMA (Broening et al. 2001;Vorhees et al. 2004;Williams et al.
2003c), or FEN (Morford et al. 2002) produce deficits in latency, path length, and cumulative
distance as well as probe trial performance in the Morris water maze, a hippocampally mediated
spatial learning task (Morris et al. 1982). Neonatal administration of MDMA (Broening et al.
2001;Williams et al. 2003c) and FEN (Morford et al. 2002) also produce an increased number of
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errors of commission and latency to locate an escape platform in the Cincinnati water maze which assesses path integration learning. Conversely, COC (Vorhees et al. 2000b) or MPH
(Vorhees, et al., unpublished results) administration does not affect learning assessed by the
Morris or Cincinnati water mazes (only COC-treated animals were tested in the Cincinnati water maze). Few studies have detailed the immediate changes induced by neonatal drug exposure during the sensitive period when long-term learning and memory changes originate, i.e., from postnatal day (P)11-20 (Broening et al. 1994;Koprich et al. 2003;Williams et al. 2000;Williams et al. 2005). The limited and existing studies suggest there are changes in adrenal hormone production as well as monoamine levels in the brain following exposure to some of these drugs.
We previously showed that administration of MDMA or MA to postnatal day (P)11 rats caused an increase in corticosterone (CORT) levels that remained significantly elevated for 24 h following the first dose of MDMA and at least 1 h after MA exposure (Williams et al.
2005;Williams et al. 2000). We and others have shown that administration of a divided daily dose of 40 mg/kg of MDMA on P10 or P11 or P10-20 also produces decreases in serotonin (5-
HT) in the hippocampus and striatum the day after the last administration (Williams et al.
2005;Broening et al. 1995;Koprich et al. 2003).
It has been proposed that alterations in glucocorticoids and monoamine levels during early developmental stages may disrupt normal brain development and alter later learning and memory. For example, fluctuations in CORT interfere with the proliferation of neurons during development, especially in the hippocampus (Gould et al. 1991;Liu et al. 2003;Woolley et al.
1990). The hippocampus is known to be involved in learning and memory functions and is still going through neurogenesis in the late gestational period in humans and comparably in early postnatal weeks in the rat (Bayer et al. 1993). 5-HT is abundant in the brain during neurogenesis
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and is known to be important in neuronal development. 5-HT is important for the differentiation of neurons and receptive target cells, and is involved in organizing the developing brain (Lauder
1993;Lauder and Krebs 1978). Furthermore, disruptions to 5-HT in rats during early development produce learning and memory deficits when the animals are tested as adults (Mazer et al. 1997).
The main purpose of this study was to compare the effect of the drugs noted above following a single day of administration on plasmatic CORT and on monoamine content in the striatum and hippocampus. This is the first study to directly compare the changes that occur following administration of MDMA, MA, MPH, COC, or FEN on P11, the first day of the critical period for the induction of later cognitive deficits following substituted amphetamine exposure.
Materials and Methods
Animals and housing
Nulliparous female Sprague-Dawley CD (International Genetic Strain) rats (Charles
River Laboratories, Raleigh, NC) were mated with males of the same strain and supplier. Rats were housed in a 22 ± 1°C environment at 50% humidity with a 14/10 h light/dark cycle (lights on at 0600 h). Prior to mating, animals were allowed at least 2 weeks to acclimate to the vivarium. Each female spent two weeks housed with one male in hanging wire cages before being housed singly in a polycarbonate cage (45.7 x 23.8 x 20.3 cm) containing wood chip bedding and ad libitum food and water. Pups from the matings were used as subjects. The
Cincinnati Children’s Research Foundation’s Institutional Animal Care and Use Committee
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approved all procedures and the vivarium was fully accredited by the Association for the
Assessment and Accreditation of Laboratory Animal Care (AAALAC).
The day of birth was considered P0 and on P1, litters were culled to 8 pups with 6 males and 2 females. Only males were used for this study since we have previously shown no or minor effects of sex and neonatal drug treatment on the spatial learning abilities of these animals in adulthood Further, no significant differences between males and females in adrenal-pituitary hormone levels were noted following P11 methamphetamine (Williams et al. 2000) or MDMA administration (Williams et al. 2005). On P7, pups were weighed and individually identified by ear-punch.
Dosing procedures
(+)Methamphetamine HCl (NIH, NIDA), (±)3,4-methylenedioxymethamphetamine HCl
(NIH, NIDA), (+)-fenfluramine HCl (Sigma, St. Louis, MO), cocaine HCl (Sigma, St. Louis,
MO ), and (±)-methylphenidate (Mallinckrodt, St. Louis, MO) were dissolved in isotonic saline at a concentration of 10 mg/3 ml (each expressed as the freebase; purity >95%). All drugs were administered at a dose of 10 mg/kg. This was a within litter design such that the 6 treatments were represented within each of the 8 litters, n= 8 and therefore treatment was considered a repeated factor (Kirk R.E. 1995;Winer 1978). Therefore, one male from each litter was randomly assigned to one of the following treatment groups: 1) MA; 2) MDMA; 3) FEN; 4)
MPH; 5) COC; or 6) saline (SAL). Each dose was administered through a subcutaneous injection in the dorsum. On P11, each animal received 1 injection every 2 h for a total of 4 injections. Each animal’s weight was recorded just prior to drug administration and injection sites were varied to prevent irritation to the dermis.
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Blood and Tissue Collection
Each animal was decapitated 24 h after the first injection (i.e., 18 h following the last
injection) since, in a previous study, we demonstrated that the greatest reduction in neurotransmitter content was induced with MDMA at this time point (Williams et al. 2005). All
animals were decapitated between 0900 and 1100 h in order to control for ultradian and circadian
rhythm. Trunk blood was collected in tubes containing 2% EDTA (0.05 ml) and then
centrifuged (1399 RCF) for 25 min at 4 °C. Plasma was collected and stored at –80 °C until
assayed for corticosterone.
The brain was removed and a chilled brain block (Zivic-Miller, Pittsburgh, PA) was used
to aid in dissection of the neostriatum and hippocampus. For the neostriatum, a coronal cut was
made at the optic chiasm and then another cut made 2 mm rostral to the first cut. The
neostriatum was dissected from this 2 mm slab. Hippocampi were removed from the remaining
tissue. All dissections were done on a chilled dissection plate and tissues frozen immediately on
dry ice. Tissue weights were determined by placing the tissues in pre-weighed tubes and then re-
weighing the tubes. All tissues were stored at –80 °C until monoamines were quantified by high-
pressure liquid chromatography with electrochemical detection.
Plasmatic Corticosterone assessment
Plasma was diluted 3:1 for CORT in a kit specific assay buffer prior to the determination
of hormone. CORT was assayed in duplicate using a commercially available EIA (ALPCO
Diagnostics, Windham, NH). The CORT EIA has little cross-reactivity with other hormones or
precursors, < 0.05 %.
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Monoamine determination
The tissue concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) in the neostriatum and 5-HT and 5-
HIAA in the hippocampus were quantified using high-pressure liquid chromatography with electrochemical detection. Tissues were homogenized in 50 volumes of 0.2 M perchloric acid and centrifuged for 6 min at 10,000 x g. Aliquots of 20 μl were injected onto a C18-column
(MD-150, 3x150mm; ESA, Chelmsford, MA) connected to either a LC-4B amperometric detector (Bioanalytical Systems, West Lafayette, IN) or a Coulochem (25 A, Chemsford, MA) detector and an integrator recorded the peak heights that followed each injection. The potential for the LC-4B was 0.6 V and the potentials of E1 and E2 on the analytical cell (model 5014B) of the Coulochem were –150 and 160 mV, respectively. The mobile phases consisted of 35 mM citric acid, 54 mM sodium acetate, 50 mg/l of disodium ethylenedeamine tetraacetate, 70 mg/l of octanesulfonic acid sodium salt, 6% (v/v) methanol, 6% (v/v) acetonitrile, pH 4.0, and pumped at a flow rate of 0.4 ml/min. Quantities of the analytes were calculated on the basis of known standards. Retention times for DOPAC, DA, 5-HIAA, and 5-HT were approximately 6, 8, 11, and 17 minutes respectively.
Statistics
Data were analyzed with repeated measures ANOVA utilizing the general linear modeling procedure (Proc GLM, SAS, Cary, NC). The within-subject factors were treatment and for body weights, time of day. Litter membership was treated as a matching factor, therefore, treatment was handled in the ANOVA as a repeated measures factor as per both Kirk
(1995) and Winer (1978). The experimental unit was therefore the litter (n= 8) (Holson and
Pearce 1992). The Greenhouse-Geisser correction was used in instances in which symmetry of
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the variance-covariance matrices were significantly non-spherical. Significant main effects were
analyzed further to determine differences using the step-down F-test procedure (Kirk R.E. 1995).
Simple effect F-tests were used to analyze significant interactions followed by step-down F-tests for individual group comparisons. In a few cases, where previous data from this lab on the same dependent variable was known, preplanned comparisons between specific groups were made where indicated. Significance was set at p ≤ 0.05 and trends at p ≤ 0.10.
Results
Body weight
No main effect of Time or Treatment was observed for the analysis of body weight during P11 dosing, but there was a Time x Treatment interaction, F(5,35) = 4.88, p < 0.04.
Simple effect analyses revealed no significant differences among treatments across time points.
The interaction was apparently the result of a reduction in body weight gain during dosing in the
FEN- and MDMA-treated animals that caused the growth curves to be non-parallel even though these groups did not differ from any of the other groups at any single time point (Table 1).
Corticosterone
CORT samples obtained 24 h after the first dose demonstrated a significant Treatment effect (F(5,35) = 6.43 p< 0.01). Step-down analysis demonstrated increases in CORT in the animals treated with MA, FEN, and MPH when compared to SAL-treated animals. Because we previously demonstrated an increase in CORT following MDMA on P11 (Williams et al. 2005), a planned comparison was used and the CORT increase was significant compared to SAL-treated animals, p< 0.05 (Fig. 1). COC-treated animals did not differ from SAL-treated animals. MA treatment produced the greatest increase and was significantly different from all other treatments except FEN. The second largest increase was found in the FEN group, while the MDMA and
86
MPH increases were the smallest and were similar to one another. Other than the MA group,
none of the other drug treated groups differed among themselves.
Monoamines
Striatum
5-HT in the striatum showed a significant effect of Treatment (F(5,35) = 7.86 p< 0.004).
Post hoc analysis demonstrated FEN, MDMA, and MPH administration significantly decreased
5-HT compared to SAL (Fig. 2A), whereas no differences were noted following MA or COC administration. FEN produced the greatest 5-HT decrease. The rank order for magnitude of effect on 5-HT was FEN ≥ MDMA ≥ MPH ≥ COC = MA = SAL.
As with 5-HT, 5-HIAA in the striatum was altered by Treatment (F(5,35) = 11.86 p<
0.0004). Post-hoc analysis showed that only FEN and MDMA administration produced
significant decreases in 5-HIAA compared to SAL administration (Fig 2B). For 5-HIAA the
rank order of drug effect was MDMA ≥ FEN > MA = MPH = COC = SAL.
There was a significant Treatment effect on striatal turnover, i.e., the 5-HIAA/5-HT ratio
(F(5,35) = 3.89 p<0.02). Only FEN-treated animals demonstrated a significant increase in the 5-
HIAA/5-HT ratio compared to SAL-treated animals (Fig. 2C). The turnover of FEN-treated
animals was also significantly different from MA-treated animals, which produced the lowest
turnover ratio. The decrease in the 5-HIAA/5-HT ratio observed in MA-treated animals was
significantly lower than that of MPH- and COC-treated animals although it did not differ from
SAL-treated animals.
No significant Treatment effects were observed on DA levels in the striatum (Fig. 3A).
Treatment effects were exhibited for DOPAC, F(5,35) = 13.45 p< 0.0001 (Fig. 3B). Post hoc
analysis revealed that animals treated with MDMA or MA had decreased levels of DOPAC
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compared to SAL-treated animals and there was a trend for an increase in the MPH-treated animals relative to SAL.
Although there were no differences observed in DA concentrations in the striatum, the
DOPAC/DA ratio demonstrated a significant Treatment effect (F(5,35) = 27.81, p<0.0001).
MDMA and MA treatment produced a decrease in the DOPAC/DA ratio, while MPH treatment significantly increased and COC tended to increase the DOPAC/DA ratio compared to SAL treatment (Fig. 3C). The rank order for magnitude of decreased DA turnover was MA ≥ MDMA
> FEN > SAL; rank order for increased turnover was MPH > COC > SAL.
Hippocampus
In the hippocampus, both 5-HT (F(5,35) = 18.60 p< 0.0001) and 5-HIAA (F(5,35) =
21.53 p< 0.0001) were significantly affected by Treatment (Fig. 4A & B, respectively). FEN- and MDMA- treated animals showed significantly decreased 5-HT and 5-HIAA levels compared to SAL-treated animals. There was also a trend for 5-HIAA to be decreased in the MA-treated animals and increased in the MPH-treated animals relative to SAL-treated animals. The rank order effect of the drugs at reducing 5-HT was FEN > MDMA > MA = MPH = COC = SAL.
For 5-HIAA the rank order effect was similar and was FEN = MDMA > MA > MPH = COC =
SAL.
The 5-HIAA/5-HT ratio in the hippocampus showed a significant Treatment effect on 5-
HT turnover (F(5,35) = 11.81 p<0.0008). FEN-treated animals had a higher ratio compared to all other treatments (Fig. 4C). No other drug-treated animals differed from SAL-treated animals.
MA and COC treatments were significantly different from one another.
Discussion
88
This study is the first to directly compare the developmental effects of several
psychostimulants and fenfluramine on early hormonal and monoamine changes 24 h following
administration on P11. There were several major findings. Firstly, MA and FEN and to a lesser
extent MDMA and MPH administration produced prolonged adrenal activation as demonstrated
by increases in plasmatic CORT long after the last treatment. The extent of this increase varied
with MA producing a greater than 3-fold increase, FEN a greater than 2-fold increase and
MDMA and MPH about a 50% increase in CORT. Secondly, FEN- and MDMA-treatment produced dramatic decreases in 5-HT and 5-HIAA in both the striatum and hippocampus, whereas MA administration produced only a trend toward decreased 5-HIAA in the hippocampus. Thirdly, unlike adult administration of several of these drugs (especially MA),
DA levels were not affected. Since it has previously been established that treatment for 10 days with FEN (20-60 mg/kg/day)(Morford et al. 2002), MA (20-60 mg/kg/day) (Williams et al.
2003d), or MDMA (10-40 mg/kg/day) (Broening et al. 2001;Williams et al. 2003c) beginning on
P11 results in long-term spatial learning and memory deficits, whereas treatment with MPH (20-
120 mg/kg/day) (Vorhees et al., unpublished observation) or COC (60 mg/kg/day) (Vorhees et al. 2000b) does not, and the substituted amphetamines when administered for 1 day dramatically affect 5-HT and/or CORT, but little or no effect of MPH or COC is observed, it appears that these early drug-induced perturbations may be antecedent to and possibly associated with later cognitive outcomes. However, it must remain clear that behavioral deficits were seen after a 10- day dosing regimen of FEN, MA, or MDMA (40 mg/kg/day for each drug) and the monoamine and CORT data gathered from this study were observed following only a single day of drug administration, albeit, the fact that there is a pattern of associations with later learning and memory deficits raises the possibility that some or all may be causally linked.
89
In previous studies, we have examined the effect of neonatal drug exposure on learning
and memory ability in the offspring as adults. We have found that administration of FEN
(Morford et al. 2002), MDMA (Broening et al. 2001;Vorhees et al. 2004;Williams et al. 2003c),
and MA (Vorhees et al. 2000a;Williams et al. 2002;Williams et al. 2003d) at similar doses when
given on P11-20 all cause deficits in Morris water maze performance. Moreover, FEN (Morford
et al. 2002) and MDMA (Broening et al. 2001;Williams et al. 2003c;Cohen et al. 2005) also
produce path integration deficits in the Cincinnati water maze. Neither COC (Vorhees et al.
2000b) nor MPH (Vorhees et al., unpublished observations) administered on these same days,
even at higher doses, produce similar deficits. The efficacious drugs are all similar in that they
all are substituted amphetamines whereas the ineffective drugs are not. It is likely that the long-
term effects of the substituted amphetamines originate from changes induced by these drugs
during the period of exposure. For example, the behavioral differences we observe might
involve changes in CORT and/or monoamine levels in the neonate that lead to altered stress
responsiveness or miswiring of critical learning circuits. This is supported by data that increases
in CORT or depletions of 5-HT during the neonatal period produce cognitive deficits (Mazer et
al. 1997;Olton et al. 1975).
In adult animals, MDMA, MA, COC, MPH, and FEN are known to affect monoaminergic systems, but reports describing the ramifications of fetal or neonatal exposure
are virtually non-existent, with the exception of COC. In adult subjects these drugs produce
effects on serotonergic and/or dopaminergic neurons. For example, FEN primarily affects 5-HT
(Rothman et al. 2001;Rowland and Carlton 1986) and MDMA has also been reported to
preferentially affect the serotonergic system, but to some extent it also affects the dopaminergic
system (Lyles and Cadet 2003). In addition to decreasing monoamine levels, MDMA, MA, and
90
FEN have been shown to cause short-term release of various neurotransmitters from axon
terminals in adults (Rothman et al. 2001). However, we felt that the protracted changes in
neurotransmitter content would be more likely to affect brain development and possibly alter function later in life than the immediate release of neurotransmitters. Similar to adult effects, in developing animals we found decreases in striatal and hippocampal 5-HT levels that were reduced by 72% and 66%, respectively, following FEN administration and 50% and 49%, respectively, following MDMA administration. Adult MA exposure produces depletions in both dopaminergic and serotonergic neurons (Frost and Cadet 2000;Cappon et al. 1997), however in this study, 10 mg/kg of MA x 4 administrations did not produce any reductions in either 5-HT or
DA at the 24 h time point. Therefore, there appears to be a different action for MA on monoaminergic pathways during brain development relative to that seen in the mature brain.
This is evident because MA has been shown to produce a 40-60% decrease in DA and 5-HT even three days after drug administration in adult animals (Cappon et al. 2000;O'Callaghan and
Miller 2002;Wallace et al. 1999), whereas no effect was observed in the current study after developmental exposure. Cocaine, like MPH primarily blocks the dopamine transporter (Kollins et al. 2001;Muller et al. 2003), but does not affect levels of brain DA. While we found no effects of COC in this study, MPH produced a slight decrease of 5-HT in the striatum, although without changes in 5-HIAA, and trends for increased DOPAC in the striatum and 5-HIAA in the hippocampus. The dissimilarities between COC and MPH are difficult to reconcile, but these small monoamine changes might imply that MPH may produce modest alterations in brain development that the Morris water maze, as used previously, could not detect.
In the developing brain, 5-HT is not only functioning as a neurotransmitter, but is also thought to influence neuronal differentiation and synaptogenesis (Whitaker-Azmitia et al. 1996).
91
Depletion of 5-HT is thought to cause disruptions in brain development (i.e., reductions in
neuronal number and disruption of neuronal differentiation and synaptogenesis) that may
manifest later in life as cognitive deficits. For example, transient depletion of 5-HT using p-
chloroamphetamine or permanent reduction by 5,7-dihydroxytryptamine has been shown to produce fewer spines on the dendrites of neurons in the dentate gyrus without affecting dendritic length (Yan et al. 1997). We have found a similar effect in spine densities following MA
administration from P11-20 (Williams et al. 2004). Although we did not see a decrease in 5-HT
levels at the 24 h time point following only one day of MA administration, it is possible that
multiple days of treatment may affect brain 5-HT concentrations. It has also been found that the
depletion of 5-HT from P10-20 using the tryptophan hydroxylase inhibitor p-
chlorophenylalanine causes spatial learning and memory deficits (Mazer et al. 1997). Taken
together, it is evident that 5-HT is important in the development of circuits involved in cognitive
ability and that FEN and MDMA can alter this developmental pattern and lead to later cognitive
dysfunction.
Not only is 5-HT important in development, but adrenal hormones also play a role in
neuronal differentiation and survival. Rodents exhibit an endogenously generated decreased
responsiveness to stressors during the neonatal period that is hypothesized to ensure optimal
glucocorticoid levels for normal brain development (Sapolsky and Meaney 1986). This period is
termed the stress hyporesponsive period (SHRP) and lasts for approximately the first two weeks
of life. The decreased HPA axis responsiveness during the SHRP is thought to prevent stress-
induced increases in glucocorticoids from interfering with neuronal proliferation , especially in
regions that express abundant glucocorticoid receptors such as the dentate gyrus (Sapolsky and
Meaney 1986;Vazquez 1998). During the sensitive period of drug administration for substituted
92
amphetamine-induced cognitive deficits that begins on P11, the hippocampus is growing and differentiating rapidly (Liu et al. 2003;Bayer et al. 1993) and is an essential mediator involved in spatial learning and memory (Morris et al. 1982). In the current study, we observed that just one day of FEN, MDMA, or MA treatment produces increases in CORT that remain elevated for up to 18 h following the last dose (24 h following the first dose), much longer than we previously documented using MA (e.g., 60 min after the last dose) (Williams et al. 2000). Moreover, MA induced a >3-fold increase in CORT that persisted over this time span during the SHRP. This effect has an early onset and can be seen by 30 min following the 1st dose of MA (Williams et al., unpublished data). This is unlike any other chemical or stressor effect reported in the literature during this age while pups are with their mother, although it is known that maternal separation is a potent stressor during the SHRP. Maternal separations ≤ 2 h can induce small, but significant, increases in CORT (Pihoker et al. 1993;Huot et al. 2002); however, it takes at least 8 h of separation before a substantial increase in CORT is achieved (Levine et al. 1991).
MPH produced a small increase in CORT, but not of the magnitude observed following MA or
FEN administration and COC had no effect. Consistent with the CORT levels found for MPH and COC, no spatial learning deficits were found after exposure to these drugs. Hence, MA treatment appears unique among the psychostimulant/monoamine releasers tested in terms of the magnitude of adrenal output it induces at this age.
The central aim of this study was to identify neurochemical changes important in neurodevelopment such as CORT and 5-HT that are perturbed by psychostimulants. In this study, we examined a single time point, 24 h after the first, which was 18 h after the last, drug exposure. This time point was selected based on previous time-course data following MDMA, since this was when the largest decrease in 5-HT occurred (Williams et al. 2005). Nevertheless,
93
we do not know if the other drugs administered in this study have a temporal profile of effects similar to those of MDMA, therefore, there are limitations to the present results. For example, there might be transient changes in monoamines following administration of the other drugs studied here that were not detected 24 h after the first dose. However, we were most interested in the lasting, rather than transient changes in this study. The age chosen (P11) was selected because this is the first day of the critical period of induction of later cognitive deficits after exposure to substituted amphetamines. As described previously, we found that FEN, MA, and
MDMA cause cognitive deficits following drug administration from P11-20 although evidence suggests shorter exposure periods are likely to have these same effects. For example, MA administration from P11-15, but not P16-20, is sufficient to cause the deficits in spatial learning
(Williams et al. 2003b). In addition, a single exposure of MA on P14 in gerbils has been shown to cause alterations in behavior, neuroanatomical structure, and neurotransmitter content
(Blaesing et al. 2001;Busche et al. 2002;Dawirs et al. 1996;Neddens et al. 2002), suggesting that even a single day of exposure is enough to perturb some aspects of neurodevelopment.
If we compare studies in which MA, MDMA, FEN, MPH, or COC were administered to rats from P11–20 and tested during adulthood in the Morris water maze acquisition phase and express the data as a percent of control, there is a clear rank ordering of the behavioral effects. It should be noted that each of the behavioral studies presented also used a within litter design. In future studies it would be valuable to compare the behavioral effects of the drugs within the same study. As illustrated in Fig. 5, FEN administration (40 mg/kg/day) produced the greatest deficits in the Morris water maze (Morford et al. 2002) followed by MDMA (40 mg/kg/day) (Williams et al. 2003c) which was followed by MA (40 mg/kg/day) (Williams et al. 2002), while MPH (40 mg/kg/day) (Vorhees, et al., unpublished results) and COC (60 mg/kg/day) (Vorhees et al.
94
2000b) had no effects. In the current study, we have shown that FEN produces the second
largest increase in CORT and the most dramatic decrease in striatal and hippocampal 5-HT
levels. MDMA produced a decrease in 5-HT in both the striatum and hippocampus and a modest
increase in CORT. Therefore, both FEN and MDMA produce extended changes in early
signaling molecules that are important for normal brain development and are likely to be related
to later learning ability. MA administration caused significant deficits in the Morris water maze
(Williams et al. 2002), but they were not as severe as after FEN or MDMA administration.
However, MA produced the largest increases in CORT, but no changes in 5-HT, at least when
measured at 24 h. This suggests that the CORT increases produce damage that is less severe
than the combination of increased CORT and reduced 5-HT. COC (Vorhees et al. 2000b) or
MPH treatment (Vorhees, et al.,. unpublished results) produced no learning or memory deficits after P11-20 exposure (Fig. 5) nor did COC affect CORT or 5-HT. MPH, on the other hand, produced small changes in CORT and 5-HT levels in the striatum but not enough to affect
cognition. Although we did not see learning deficits, MPH during this sensitive period does
cause developmental problems such as growth retardation and may produce other yet
uncharacterized behavioral effects (Gauron and Rowley 1974). It should be noted that in the
behavioral studies reported, drug was administered for 10 days instead of a single day as in the
present study. In addition, there is a possibility that some of the drugs produce different pharmacological profiles than MDMA on early neurochemical changes and the prospect of these differences needs to be considered when evaluating the neurochemical/behavioral comparison.
While we used similar mg/kg doses in this study, it should be noted that these doses were not meant to be equivalent in relative potency but that they represented doses of the substituted
amphetamines that were known to produce long-term learning and memory differences when
95
administered for 10 days. Nonetheless, higher levels of MPH (up to 120 mg/kg/day) or COC (60
mg/kg/day) did not produce learning deficits. Therefore, we used similar dosing concentrations
of each drug for this comparison. The data from the present study considered together with the degree of impairment observed following FEN, MDMA, MA, MPH, or COC in the Morris water
maze suggest that developmental disruptions in CORT and 5-HT are important in the etiology of
substituted amphetamine-induced learning and memory deficits. Therefore, closer examination
of these effects on the long-term learning and memory deficits warrants further investigation.
Acknowledgments
Portions of these data were presented at the 12th annual meeting of the International
Behavioral Neuroscience Society meeting in San Juan, Puerto Rico, 2003. Supported by
National Institutes of Health grants DA014269 (MTW) and DA006733 (CVV), DA007427
(GAG) and training grant ES007051 (TLS & LAE).
96
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Table 1: Body weights taken during the first and last dose
Treatment N Weight Dose 1 (g) Weight Dose 4 (g)
SAL 8 25.59 ± 0.95 25.75 ± 1.02
FEN 8 26.14 ± 0.91 25.64 ± 1.01
MDMA 8 26.89 ± 1.26 26.10 ± 1.35
MA 8 26.89 ± 0.94 25.96 ± 0.92
MPH 8 26.15 ± 1.09 26.25 ± 1.10
COC 8 26.81 ± 1.04 26.73 ± 1.14
106
* c,e,f 30
25
* 20 CORT (ng/ml) 15 * * d d d
10
5 SAL FEN MDMA MA MPH COC Treatment
Figure 1. Corticosterone concentrations in plasma 24 h after the 1st of 4 doses (every 2 h)
of FEN, MDMA, MA, MPH, and COC (10 mg/kg) that began on P11 (mean ±
SEM). MA produced the greatest increase in CORT and was significantly
different from SAL, MDMA, MPH and COC. FEN also produced an increase in
these P12 animals. MDMA and MPH produced similar increases compared to
SAL. *p < 0.05 from SAL control; c= different from MDMA; d= different from
MA; e= different from MPH; f= different from COC.
107
0.12 b,c * * b b,c 0.08 * d,f d,e,f 0.04
Striatal 5-HT (ng/mg tissue) (ng/mg 5-HT Striatal 0.00
0.25
0.20 b,c b,c,d b,c,f 0.15 * d,e,f * 0.10 d,e,f
0.05
0.00 Striatal 5-HIAA (ng/mg tissue) (ng/mg 5-HIAA Striatal
* 4 d
3 d d b,e,f 2
1 Striatal 5HIAA / 5-HT / 5-HT 5HIAA Striatal
0 SAL FEN MDMA MA MPH COC Treatment
Figure 2. Serotoninergic markers in striatum (mean ± SEM; n = 8/group). (A)
Concentrations of 5-HT (top), (B) 5-HIAA, and (C) 5-HIAA/5-HT ratio (bottom)
in the striatum of animals exposed to 4 doses of FEN, MDMA, MA, MPH, or
COC on P11 and examined on P12. Decreases in 5-HT were observed in FEN,
MDMA, and MPH-treated animals and FEN and MDMA also decreased 5HIAA.
For the ratio, FEN produced a significant increase compared to the SAL group. *
p < 0.05 from SAL control; b= different from FEN; c= different from MDMA; d=
different from MA; e= different from MPH; f= different from COC
108
3
2
1
0 Striatal DA (ng/mg tissue)
† b,c,d 0.5 b,c,d 0.4 d,e,f * e,f 0.3 b,e,f* 0.2
0.1
0.0 Striatal DOPAC (ng/mg tissue) * b,c,d,f † 0.20 † b,c,d,e 0.15 c,d,e,f b,e,f* 0.10 b,e,f*
0.05 Striatal DOPAC / DA / DOPAC Striatal 0.00 SAL FEN MDMA MA MPH COC Treatment †
Figure 3. Dopaminergic markers in striatum (mean ± SEM; N = 8/group). (A)
concentrations of DA, (B) DOPAC, and (C) and the DOPCA/DA ratio in the
striatum on P12 following administration of FEN, MDMA, MA, MPH, or COC
on P11. No changes were observed in DA. MA and MDMA produced a
significant decrease in DOPAC. The ratio of DOPAC/DA was significantly
decreased following MDMA and MA treatments and increased in the MPH group.
There was also a trend for the DOPAC/DA ratio to be decreased following FEN
and increased following COC (bottom). *p < 0.05 from SAL control; b= different
from FEN; c= different from MDMA; d= different from MA; e= different from
MPH; f= different from COC; †= p<0.10.
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0.20 b,c b,c b,c 0.15 * 0.10 * b,d,e,f c,d,e,f 0.05
0.00 Hippocampal 5-HT (ng/mg tissue) † b,c,d b,c,d
0.3 † b,c,e,f
* 0.2 * d,e,f d,e,f
0.1
0.0 Hippocampal 5-HIAA (ng/mg tissue) * c,d,e,f
4
3 b b,d b 2 b,f
1 Hippocampal 5HIAA / 5-HT 0 SAL FEN MDMA MA MPH COC Treatment
Figure 4. Serotonergic markers in the hippocampus (mean ± SEM; N = 8/group). (A)
concentrations of 5-HT, (B) 5-HIAA, and (C) the 5-HIAA/5-HT ratio in the
hippocampus of animals on P12 after 4 doses of FEN, MDMA, MA, MPH, or
COC on P11. Decreases in 5-HT were observed in FEN and MDMA-treated
animals. FEN and MDMA treatment also produced a significant decrease in
5HIAA. There was a trend for a decrease in 5-HIAA in the MA group and an
increase following MPH. For the ratio, only FEN produced a significant increase
compared to the SAL group. *p < 0.05 from SAL control; b= different from
FEN; c= different from MDMA; d= different from MA; e= different from MPH;
f= different from COC; †= p<0.10.
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160 * * 140 * 120
100 SAL
80
60
LATENCY (% control) 40
20
0 FEN MDMA MA COC MPH TREATMENT
Figure 5. Comparison of FEN, MDMA, MA, COC, and MPH effects on Morris water
maze spatial learning from previous experiments. Each of the drugs was
administered at a dose of 40 mg/kg/day from P11-20 with the exception of COC
that was administered at 60 mg/kg/day. There is a clear rank order of effects
(expressed as percent of SAL from each study) in the acquisition phase of the
Morris Water maze. FEN produced the longest latencies to find the hidden
platform (Morford et al. 2002). In the current study, FEN-treated animals showed
the most dramatic decreases in both striatal (Fig. 2 top) and hippocampal 5-HT
(Fig. 4 top) and significant increase in CORT (Fig.1). MDMA produced the next
greatest deficit in the Morris maze (Williams et al. 2003c) and next largest
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decreases in striatal (Fig. 2A) and hippocampal (Fig. 4A) 5-HT levels. MA produced significant increases in latency (Williams et al. 2002), however not to the degree of FEN or MDMA and MA did not produce any changes in 5-HT (Fig.
2 and 4 top), but produced the largest increase in CORT (Fig. 1). Neither MPH
(Vorhees, et al.,. unpublished results) nor COC (Vorhees et al. 2000b) produced any changes in latency and only MPH slightly affected 5-HT in the striatum (Fig.
2 top). *p < 0.05 relative to saline controls (100% reference line)
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CHAPTER 4:
Short- and long-term effects of (+)-methamphetamine and (±)-3,4-
methylenedioxymethamphetamine on monoamine and corticosterone levels in the
neonatal rat following multiple days of treatment
Tori L. Schaefer1, Matthew R. Skelton1, Nicole R. Herring1, Gary A. Gudelsky2, Charles V. Vorhees1, Michael T. Williams1
Division of 1Neurology, Dept. of Pediatrics, Cincinnati Children’s Research Foundation and University of Cincinnati College of Medicine, Cincinnati, OH 45229-3039, USA 2James L. Winkle College of Pharmacy, University of Cincinnati, OH 45267-0004, USA
As published J. Neurochem. 104, 1674-1685.
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ABSTRACT
Rats treated with (±)-3,4-methylenedioxymethamphetamine (MDMA) or (+)-
methamphetamine (MA) neonatally exhibit long-lasting learning impairments (i.e., after
treatment on postnatal days (P)11-15 or P11-20). Although both drugs are substituted
amphetamines, they each produce a unique profile of cognitive deficits (i.e., spatial vs.
path integration learning and severity of deficits) which may be the result of differential
early neurochemical changes. We previously showed that MA and MDMA increase
corticosterone and MDMA reduces levels of 5-HT 24 h after treatment on P11, however,
learning deficits are seen after 5 or 10 days of drug treatment, not just one day.
Accordingly, in the present experiment rats were treated with MA or MDMA starting on
P11 for 5 or 10 days (P11-15 or P11-20) and tissues collected on P16, P21, or P30. Five-
day MA administration dramatically increased corticosterone on P16, whereas MDMA did not. Both drugs decreased hippocampal 5-HT on P16 and P21, although MDMA
produced larger reductions. Ten day treatment with either drug increased dopamine
utilization in the neostriatum on P21, whereas 5 day treatment had no effect. No corticosterone or brain 5-HT or dopamine changes were found with either drug on P30.
Although the monoamine changes are transient, they may alter developing neural circuits
sufficiently to permanently disrupt later learning and memory abilities.
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Introduction
Substituted amphetamines, including 3,4-methylenedioxymethamphetamine
(MDMA) and methamphetamine (MA), are being abused by women of childbearing age
(EMCDDA 2006;Johnston 2007). MDMA is known for its popularity at raves, or all
night dance parties, although its use is increasingly being documented in suburbs, rural
areas, and college campuses (Wish et al. 2006). The abuse of MA is a worldwide
problem due to its ease of production, low cost, stimulatory effects, and addictive
properties (Rawson et al. 2002). Both MDMA and MA are thought to increase high-risk
sexual behavior that may lead to pregnancy and potentially fetal exposure (Buffum
1988;Degenhardt 2005;McElrath 2005). Pregnant women have been reported to abuse
MDMA or MA into the third trimester and up to the point of delivery (Chomchai et al.
2004;Ho et al. 2001;Smith et al. 2003). It is suspected that these exposures to MDMA or
MA during fetal development have lasting effects that alter neural development and subsequent cognitive function, but this remains to be proven.
To investigate long-term consequences of MA or MDMA, we have used the neonatal rat as a model of late second through third trimester human brain development, specifically hippocampal and cortical development (Bayer et al. 1993;Clancy et al.
2007;Herlenius and Lagercrantz 2004). Although MA and MDMA are both amphetamine analogues, there are differences between the two in the behavioral outcomes of animals exposed to either drug neonatally. For example, we find that both
MDMA or MA, administered from postnatal day (P) 11-20, produce spatial learning deficits in the Morris water maze (MWM), however neonates exposed to MDMA exhibited more severe deficits (Broening et al. 2001;Schaefer et al. 2006;Skelton et al.
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2006;Vorhees et al. 2000a;Vorhees et al. 2004;Williams et al. 2002;Williams et al.
2003c;Williams et al. 2003b) than those exposed to MA. We also find that MDMA, but not MA, produces deficits in the Cincinnati water maze (CWM), a task solved using a combination of spatial and path integration learning when tested under fluorescent or low-level visible red light conditions (Broening et al. 2001;Skelton et al. 2006;Williams et al. 2003c;Williams et al. 2003b). Furthermore, we find that the majority of the spatial learning effects after MA are attributable to the first 5 days (P11-15) of MA treatment
(Williams et al. 2003a); shorter treatment periods have not yet been examined with
MDMA. In addition to long-term cognitive changes, MDMA and MA exposure produces hypoactivity following neonatal exposure (Vorhees et al. 1994;Vorhees et al. 2007).
Neonatal mice have also been shown to be affected in spatial learning and novel location recognition following neonatal MA administration (Acevedo et al. 2007). These differences in behavior following MA or MDMA treatment are likely the result of differential effects on neural development.
Brain development is influenced by changes in hormonal and monoamine levels including corticosterone (CORT) and serotonin (5-HT). Disruptions of the processes these signaling molecules control during development can cause short- and long-term changes in brain physiology and cognitive function. Although it is recognized that some level of CORT is essential for neuronal development, excess CORT has been shown to produce decreases in hippocampal cell proliferation (Gould et al. 1991a;Liu et al.
2003;Woolley et al. 1990). The hippocampus is important for several types of learning
(i.e., spatial learning) and undergoes neurogenesis and neuronal pruning during the first few weeks of life in the rat (Bayer et al. 1993;Gould et al. 1991a). In addition, 5-HT is
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one of the first monoamines to appear in the developing brain and is thought to be
required for neurogenesis, the development of serotonergic target fields, and organization
of some areas of the brain (Lauder and Krebs 1978;Lauder 1990;Whitaker-Azmitia et al.
1996). p-Chlorophenylalanine-induced inhibition of 5-HT synthesis, after essentially the
same days of exposure (P10-20) that we find to be sensitive for MDMA and MA (i.e.,
P11-20), altered synaptogenesis and produced deficits in learning (Mazer et al. 1997).
Taken together, these data suggest that factors influencing CORT and/or 5-HT may disrupt cognitive development.
We previously showed that one day of MDMA treatment produces significant increases in CORT and decreases in 5-HT and MA treatment produces only increases in
CORT levels 24 h later with MA producing the largest increase in CORT and MDMA producing the largest decrease in 5-HT (Schaefer et al. 2006). In addition, we showed that P11 exposure to MDMA produces CORT increases and 5-HT decreases within 1 h after exposure and that 5-HT decreases are still apparent 78 h following 1 day of treatment (Williams et al. 2005). In other experiments, we showed that MA produces increases in CORT 30 min following single or multiple days of exposure (Williams et al.
2006;Williams et al. 2000). These experiments represent the effects of these drugs at the beginning of the exposure intervals that lead to later learning and memory impairments, but the effects of multiple day treatment (P11-15 or P11-20) remain to be elucidated.
Therefore, the purpose of this experiment was to investigate the effects of MDMA and
MA on CORT and monoamine levels 24 h after the first treatment on the last day of exposure (P16 or P20) and on P30 following either 5 days (P11-15) or 10 days (P11-20) of exposure.
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Materials and Methods
Animals and housing
Nulliparous female Sprague-Dawley CD, International Genetic Strain, rats
(Charles River Laboratories, Raleigh, NC) were mated with males of the same strain and
supplier. Rats were housed in a 22 ± 1 °C environment at 50 ± 10% humidity with a
14/10 h light/dark cycle (lights on at 600 h). Prior to mating, a period of at least 1 week ensued to allow the animals to acclimate to the vivarium. Each female spent two weeks housed with one male in hanging wire cages before being housed singly in a polycarbonate cage (46 x 24 x 20 cm) containing wood chip bedding and ad libitum food and water. Pups from the resulting litters were used as subjects. The Cincinnati
Children’s Research Foundation’s Institutional Animal Care and Use Committee approved all protocols and the vivarium was fully accredited by the Association for the
Assessment and Accreditation of Laboratory Animal Care (AAALAC).
The day of birth was considered P0, and on P1 litters were culled to 8 pups with 6 males and 2 females. On P7, pups were weighed and individually identified by ear- punch. Only males were used for this study for several reasons. Firstly, we have previously shown only subtle if any drug x sex interactions on learning and memory following neonatal MA and MDMA administration in rats (Morford et al. 2002;Vorhees et al. 2000a;Williams et al. 2003c;Williams et al. 2003b;Vorhees et al. 2000b;Broening et al. 1994;Broening et al. 2001;Williams et al. 2003a). Secondly, our model produces no significant differences between males and females in adrenal-pituitary hormone levels during neonatal MA exposure (Williams et al. 2000) with only slight differences following exposure from P11-20 (Skelton et al. 2007). In regard to MDMA, no sex
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differences in CORT or monoamine levels have been observed.(Williams et al. 2005). It should be noted that others have suggested sex dependent changes exist following MA exposure in neonatal rats and mice. For example, some have found sex differences in DA levels in rats (Gomes-da-Silva et al. 2004) and behavioral differences in mice following neonatal MA (Acevedo et al. 2007). Nonetheless, taken together with the fact we wanted to maintain similar litter sizes between the current study and previous experiments, and the minor and sparsely observed sex differences during or after the drug dosing, only males were used.
Dosing procedures
(+)-Methamphetamine HCl and (±)-3,4-methylenedioxymethamphetamine HCl
(National Institute on Drug Abuse), were dissolved in isotonic saline (SAL) at a dose of
10 mg/kg (each expressed as the freebase; purity >95%) in a volume of 3 ml/kg. On each
day of drug administration one animal from each litter received 1 injection every 2 h for a
total of 4 injections of MA, MDMA, or SAL from P11-15 or P11-20. Each animal’s
weight was recorded prior to each drug treatment, however only the first (am dosing
time) and last (pm dosing time) weights taken each day were used for body weight
analysis and are described as dosing time am or pm. Injection sites were varied to prevent irritation to the dermis.
As noted, we investigated two dosing periods (P11-15 and P11-20). These were chosen based on prior behavioral experiments that demonstrated long-term effects using these exposure periods (Broening et al. 2001;Skelton et al. 2006;Vorhees et al.
2000a;Vorhees et al. 2004;Williams et al. 2002;Williams et al. 2003a;Williams et al.
2003c;Williams et al. 2003b). Two different time points were analyzed for each dosing
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regimen: 24 h after the first dose on the last day of drug treatment (P16 following P11-15 dosing or P21 following P11-20 dosing) or on P30. Therefore, the treatment groups were as follows: (1) treated from P11-15 and killed 24 h later on P16 (n = 13 litters); (2) treated from P11-15 and killed on P30 (n = 6 litters); (3) treated from P11-20 and killed
24 h later on P21 (n = 16 litters); and (4) treated from P11-20 and killed on P30 (n = 8 litters). Within each litter, 3 animals (one each treated with MA, MDMA, or SAL) were dosed and sacrificed at the earlier age (P16 or P21) and the other 3 animals in the litter
(one each treated with MA, MDMA, or SAL) were sacrificed on P30. In this design, treatment group was balanced for litter membership.
Blood and Tissue Collection
Each animal was decapitated at the appropriate time point and blood was collected in tubes containing 2% EDTA (0.05 ml) and then centrifuged (1399 RCF) for
25 min at 4 °C. Plasma was collected and stored at –80 °C until assayed for CORT.
The brain was simultaneously removed and placed in a chilled brain block (Zivic-
Miller, Pittsburgh, PA) to aid in dissection of the neostriatum and hippocampus as defined previously (Williams et al. 2007). Briefly, for the neostriatum, a coronal cut was made at the optic chiasm and then another cut made 2 mm rostral to the first. The neostriatum (caudate-putamen) was dissected from this 2 mm slab. Hippocampi were removed from the remaining tissue. Dissections were done on a chilled dissection plate and tissues were immediately frozen on dry ice. Tissue weights were determined by placing the tissues in pre-weighed tubes and then re-weighing the tubes. All tissues were stored at –80 °C until monoamines were quantified by high-pressure liquid chromatography with electrochemical detection.
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Corticosterone assessment in plasma
Plasma was diluted 3:1 for CORT in a kit specific assay buffer prior to the determination of hormone. CORT was assayed in duplicate using a commercially available EIA (Immunodiagnostic Systems Inc., Fountain Hills, AZ) and measured on a
SpectraMax Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA). The
CORT EIA has little cross-reactivity with other hormones or precursors (< 1.4%) with the minor exceptions of 11-dehydrocorticosterone and 11-deoxycorticosterone (< 6.7%).
Monoamine determinations
The tissue concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid
(DOPAC), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) in the neostriatum and 5-HT and 5-HIAA in the hippocampus were quantified using high-pressure liquid chromatography with electrochemical detection (Schaefer et al. 2006). Briefly, tissues were homogenized in 50 volumes of 0.2 N perchloric acid and centrifuged for 6 min at
10,000 x g. Aliquots of 20 μl were injected onto a C18-column (MD-150, 3x150mm;
ESA, Chelmsford, MA) connected to either a LC-4B amperometric detector
(Bioanalytical Systems, West Lafayette, IN) or a Coulochem detector (25A, Chemsford,
MA) and an integrator recorded the peak heights for each injection. The potential for the
LC-4B was 0.6 V and the potentials of E1 and E2 on the analytical cell (model 5014B) of the Coulochem were –150 and 160 mV, respectively. The mobile phase consisted of 35 mM citric acid, 54 mM sodium acetate, 50 mg/l of disodium ethylenediamine tetraacetate, 70 mg/l of octanesulfonic acid sodium salt, 6% (v/v) methanol, 6% (v/v) acetonitrile, pH 4.0, and pumped at a flow rate of 0.4 ml/min. Quantification of the
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analytes was calculated on the basis of known standards. Retention times for DOPAC,
DA, 5-HIAA, and 5-HT were approximately 6, 8, 11, and 17 min, respectively.
Statistics
Data were analyzed with repeated measures ANOVA utilizing the general linear
modeling procedure (Proc GLM, SAS Institute, Cary, NC). Body weights during dosing
were subjected to a separate ANOVA for the P11-15 and for the P11-20 treatment
regimens. Separate ANOVAs were performed on neurochemical and corticosterone data
for each of the 2 sacrifice days within the 2 exposure periods (5 day exposure, P16; 5 day
exposure, P30; 10 day exposure, P21; 10 day exposure, P30). Litter membership was
treated as a matching factor, therefore, drug treatment was handled in the ANOVA as a
repeated measures factor as per (Kirk 1995) and (Winer 1978). The experimental unit
was the litter at each time-point (Holson and Pearce 1992). Significant main effects were
analyzed further to localize differences using the step-down F-test procedure (Kirk 1995).
Simple effect F-tests were used to analyze significant interactions followed by step-down
F-tests for individual group comparisons. Only drug treatment main effects and
interaction F-ratios are shown for clarity of data presentation. Significance was set at p ≤
0.05.
Results
Body Weight
Animals treated from P11-15 showed significant main effects of Treatment (F2,38
= 19.97, p < 0.0001), Day (p < 0.0001), and Dosing time, i.e., am or pm (p < 0.0001), and the highest order significant interaction was Treatment x Day x Dosing time (F8,152 =
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2.96, p < 0.004). All animals showed increased weight gain from P11-15, however
analysis of the interaction showed that MDMA-treated animals weighed less than SAL-
treated animals beginning on P12 and through the remainder of drug treatment. MA-
treated animals weighed the least compared to SAL- and MDMA-treated animals
beginning on P13 and throughout the rest of the treatment period (Fig. 1A).
For animals treated from P11-20 there were significant effects of Treatment (F2,40
= 33.18, p < 0.0001), Day (p < 0.0001) , Dosing time (p < 0.0001), and the interaction of
Treatment x Day x Dosing time (F18,360 = 2.02, p < 0.009). Examination of the
interaction revealed that MDMA and MA treatment prevented animals from gaining
weight similarly to SAL-treated animals beginning on P13 and these differences
continued through the remainder of the treatment period. MA treatment produced the
greatest decrease in weight gain since it was different from MDMA treatment beginning
on P14 and lasted through the remainder of dosing (Fig. 1B). This pattern in body
weights continued through P28 (data not shown).
Corticosterone
Following drug treatment from P11-15, CORT in plasma on P16 was significantly
affected by Treatment (F2,24 = 6.77 p< 0.005). Step-down analysis demonstrated
significant CORT increases in the animals treated with MA compared to those treated with either SAL or MDMA (Fig. 2A). No effect of MDMA was observed at this time point. For animals examined on P30, no differences in CORT were observed in either drug group (Fig. 2A).
Following P11-20 treatment, no differences in CORT were observed on either
P21 or on P30 (Fig. 2B).
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Monoamines
Neostriatum
5-HT levels in the neostriatum following P11-15 dosing were significantly
affected by Treatment on P16 (F2,24 = 8.01 p< 0.003). Post hoc analysis demonstrated a significant decrease in neostriatal 5-HT only in MDMA-treated animals compared to
SAL- treated animals. No differences were detected among the treatments on P30 (Fig.
3A).
Neostriatal 5-HIAA was also affected by Treatment on P16 (F2,24 = 46.8 p<
0.0001). Post-hoc analysis showed significantly decreased 5-HIAA levels in MA- and
MDMA-treated animals compared to SAL-treated animals. Animals exposed to MDMA
had the largest decreases in 5-HIAA. No differences were observed on P30 among
treatment groups (Fig. 3B).
In the neostriatum no changes were observed on either P16 or on P30 in the ratio of 5-HIAA/5-HT or DA, DOPAC, or the ratio of DOPAC/DA (Fig. 3C, 4A-C,
respectively).
Following 10 days of drug administration from P11-20, 5-HT in the neostriatum
was significantly affected by Treatment on P21 (F2,30 = 11.27 p< 0.0002) but not on P30
(Fig. 5A). Post hoc analysis revealed that both MA and MDMA treatment significantly
decreased neostriatal 5-HT on P21.
Neostriatal 5-HIAA was also affected by Treatment on P21 following P11-20
dosing (F2,30 = 27.87 p< 0.0001). Post-hoc analysis showed significantly decreased 5-
HIAA levels in MA- and MDMA-treated animals compared to SAL-treated animals on
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P21. No differences were demonstrated among treatment groups on P30 (Fig. 5B).
Similarly, no changes in the 5-HIAA/5-HT ratio were observed on P21 or P30 (Fig. 5C).
In the neostriatum, DA levels were unaffected on P21 or P30 (Fig. 6A).
However, DOPAC levels were affected by Treatment (F2,30 = 14.70 p< 0.0001) on P21.
Post hoc analysis revealed neostriatal DOPAC was significantly increased on P21 in both
the MA- and MDMA-treated groups compared to SAL-treated animals. No treatment
group differences were observed on P30 (Fig. 6B).
The DOPAC/DA ratio in the neostriatum showed a significant Treatment effect
(F2,28 = 14.75, p<0.0001) on P21, but not on P30. As with DOPAC, both MA and
MDMA treatments produced a significant increase in the DOPAC/DA ratio compared to
SAL treatment (Fig. 6C).
Hippocampus
Analysis of the hippocampus following P11-15 dosing revealed that both 5-HT
(F2,24 = 73.09 p< 0.0001) and 5-HIAA (F2,24 = 30.47 p< 0.0001) were affected on P16 by
Treatment. Post hoc analysis revealed significant decreases in 5-HT and 5-HIAA in the hippocampus of MA- and MDMA-treated animals when compared to SAL-treated animals (Fig. 7A, 7B, respectively). 5-HT and 5-HIAA levels in the MA and MDMA groups were also different from each other with MDMA producing the larger decrease.
No changes in 5-HT in the hippocampus were noted on P30 (Fig. 7A), however for 5-
HIAA there was a significant Treatment effect. Both MA and MDMA treatment
produced a decrease in 5-HIAA compared to SAL controls (Fig 7B).
The 5-HIAA/5-HT ratio in the hippocampus was unaffected by MDMA or MA
treatment on P16. On P30 the 5-HIAA/5-HT ratio showed a significant Treatment effect
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(F2,10 = 12.42 p<0.002). Post hoc analysis revealed decreases in the 5-HIAA/5-HT ratio in MA- and MDMA-treated animals compared to SAL-treated animals (Fig. 7C).
Following P11-20 drug treatment, analysis of the hippocampus revealed that both
5-HT (F2,28 = 64.52 p< 0.0001) and 5-HIAA (F2,28 = 14.64 p< 0.0001) were affected on
P21 (Fig. 8A, 8B, respectively), and only 5-HIAA/5-HT ratio was altered on P30 (Fig.
8B), but not on P21 (Fig. 8A). Post hoc analysis for P21 revealed significant decreases in
5-HT and 5-HIAA in the hippocampus of MA- and MDMA-treated animals when compared to SAL-treated animals. MDMA treatment produced the largest decrease in 5-
HT since levels were significantly lower than MA (Fig. 8A). On P30, 5-HIAA levels in animals treated with MA were decreased compared to SAL-treated animals (Fig. 8B);
MDMA had no significant effect at this time point.
The 5-HIAA/5-HT ratio in the hippocampus was not affected on P21. However, the 5-HIAA/5-HT ratio in the hippocampus on P30 showed a significant Treatment effect
(F2,14 = 3.91 p<0.05). Post hoc analysis revealed decreases in 5-HIAA/5-HT ratio in MA- treated animals compared to SAL-treated animals (Fig. 8C), while MDMA-treated animals showed no effect.
Time-course of CORT and hippocampal 5-HT changes
We also examined the CORT and 5-HT changes induced by drug treatment as a percent of control at three different time periods. The time periods selected were all 18 h from the last drug administration which was 24 h from the first administration on the last day of dosing. The days selected were P12, 16, and 21. The P12 data were from a previous study (Schaefer et al. 2006). An ANOVA performed on the data, expressed as percent control, showed that MA produced a significant increase in CORT on P12 and
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P16, but not on P21. MDMA produced an initial increase in CORT on P12, but significant increases were not detected on P16 or P21. In the hippocampus, MA produced moderate decreases in 5-HT on P16 and P21 and MDMA administration lead to a 5-HT decrease at all three ages (Fig 9).
Discussion
We have shown differential CORT and 5-HT alterations 24 h following 5 or 10 days of MA or MDMA treatment during a period of development when cognitive deficits are induced by these drugs at these doses. We showed that MA treatment increased levels of CORT in plasma by approximately three-fold compared to SAL and two-fold compared to MDMA following 5 days of exposure when examined 24 h later (cf. Fig 2 &
9). However, no differences in CORT among treatments were observed on P21
following 10 days of exposure. In the hippocampus we identified significant 5-HT decreases of approximately 50% after 5 and 10 days of MDMA exposure, while MA administration produced a decrease following 5 days of exposure (~40%) which was attenuated on P21 (~30%; Fig. 7,8,9). 5-HT decreases in the neostriatum were similar to changes in the hippocampus for MDMA-exposed animals; however we only observed significant neostriatal 5-HT decreases following 10 days of MA injections (Fig. 3,5), not after 5 days of MA treatment. These findings suggest that MA and MDMA affect CORT
and 5-HT differentially and that these differences may contribute to the unique pattern of
long-term behavioral changes following administration of these drugs.
The increases in CORT following neonatal MA administration are likely to affect
cell proliferation and we have shown that neonatal MA affects dendritic morphology in the hippocampus (Williams et al. 2004); an area important for spatial learning and
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memory as assessed using the MWM (Morris et al. 1982). The greatest number of proliferating dentate granular cells, the dominant neuronal type in the hippocampus, occurs during the neonatal period at a time when environmental stress-induced CORT increases are normally blunted (Sapolsky and Meaney 1986;Schlessinger et al. 1975;Liu et al. 2003). Humans are also thought to experience a period of blunted cortisol (the major glucocorticoid in humans) induction following stress during prenatal development; in humans rapid cell proliferation in the dentate gyrus occurs from second through third trimester (Bayer et al. 1993;Brosnan 2001;Clancy et al. 2007;Rice and Barone S Jr
2000). During the period when CORT should be blunted, large increases in CORT or exposure to stress can prevent the proliferation of dentate granular cells in such a way that the number of proliferating cells is inversely proportional to the concentration of
CORT and this alteration in development may influence long-term cognitive function
(Sapolsky and Meaney 1986;Aisa et al. 2007). We previously showed the most sensitive period for MA induced spatial learning deficits occurs following P11-15 exposure
(Williams et al. 2003a) which coincides with the observed elevated CORT levels at the
24 h time point in the current study. These data along with previously reported results suggest that MA-treated animals experience CORT increases beginning just after the first dose on P11 and repeatedly through at least P16 at the 24 h time point following 5 days of dosing (Fig. 9) (Williams et al. 2006;Williams et al. 2000;Schaefer et al. 2006).
Although following MDMA treatment we do not find any significant CORT changes following 5 or 10 days of exposure at the 24 h time-point we have previously shown
CORT increases on P11 1 h after a single dose (~2.5x higher), 1 h following 3 doses (~5x higher), and 24 h following 4 doses on P11 (< 2x higher, Fig. 9) compared to SAL
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controls (Schaefer et al. 2006;Williams et al. 2005). Although we suspect that increased
CORT following these drugs is affecting later spatial learning performance at this time
direct assessment of spatial learning ability following neonatal CORT treatment during
these exposure periods and at the drug induced magnitude is not currently available. One
study that exposed pups to lower CORT increases than the observed elevations in the
current study via dam’s drinking water (P13-17) did not show detrimental effects in
spatial learning and even appeared to be beneficial (McCormick et al. 2001). Others
have demonstrated long-term learning and memory deficits in novel object recognition
and spatial memory following neonatal maternal separation, a stressor that triggers CORT
release (Aisa et al. 2007). It appears that the magnitude of CORT increase may influence any changes in spatial learning ability.
In addition to glucocorticoids, the developing brain requires critical concentrations of monoamines for proper development including long-term cognitive ability. For example, temporal lobe morphological changes are induced by depletion of
5-HT, and spatial learning deficits have been observed following P10-20 administration
of p-chlorophenylalanine, a tryptophan hydroxylase inhibitor (Mazer et al. 1997;Yan et
al. 1997). The decreases in 5-HT following both MA and MDMA may hinder
proliferation, migration, and synaptogenesis and this could contribute to the impaired
cognitive ability we see given that 5-HT levels are altered throughout a known critical
period (i.e., hippocampal neurogenesis) (Herlenius and Lagercrantz 2004;Bayer et al.
1993). During the 24 h time points investigated in this study, MDMA had larger and
more protracted effects on the 5-HT system in the hippocampus than MA since there is
an earlier-onset of 5-HT decreases observed following MDMA (Fig 3A, 7A, 8A, 9). The
129
5-HT depletions resulting from MA or MDMA treatment may influence cognitive
development since there is a rough correlation between the magnitude of the 5-HT
reductions and the magnitude of the later spatial learning impairment (i.e., MDMA >
MA). It will be important to test this hypothesis, perhaps by interfering with 5-HT reductions by pretreatment with another drug to determine the role of 5-HT in mediating later spatial learning effects. Others have also reported a decrease in 5-HT on P21 following P11-20 MDMA treatment (Koprich et al. 2003) and the present study shows similar reductions on P21 and shows that even greater depletions are seen on P16. Taken together with the CORT data, the learning and memory deficits in MDMA-treated animals may be more dependent on 5-HT depletions, whereas the large CORT increases in MA-treated animals may account for the learning and memory deficits in those animals. Disentangling these two effects on learning will require additional studies.
Interestingly, neonatal MA and MDMA treatment affect the dopaminergic system differently than in adult animals. We have observed no changes in DA following 5 or 10 days of drug exposure 24 h later, even though a single day of MA treatment, at the same dose as used in this study, to an adult rat produces a 40-60% DA decrease that lasts for days and even weeks or months (Cappon et al. 2000;O'Callaghan and Miller
2002;Wallace et al. 1999;Cass 2000;Cass and Manning 1999). Although we do not show changes in DA or 5-HT on P30 it is evident that lasting alterations to the 5-HT system remain because 5-HIAA and the 5-HIAA/5-HT ratio are decreased following both drugs on P30. Interestingly, we have previously demonstrated changes in 5-HT and DA in adults following similar neonatal drug exposure including decreases in striatal DA following MA treatment (Crawford et al. 2003) and hippocampal 5-HT and striatal DA
130
following MDMA (Broening et al. 2001;Crawford et al. 2006). This may be attributed to changes in monoamine innervations including differentiation, migration, and synapse formation or alterations in the rate of monoamine synthesis and/or degradation. It may be that developmental mechanisms are disrupted in such a way that the full effect of the drugs does not become apparent until later in life. Similar age-dependent changes in
[3H]paroxetine binding to serotonin transporters following P1-4 MDMA administration
have been reported in which no differences were observed on P25, however a decrease in
binding was observed on P60 (Meyer et al. 2004). Hence, early changes produced by
MA or MDMA treatment may alter the wiring of developing neural circuits such that absolute levels of neurotransmitters at P30 are not as indicative of the damage as are cytoarchitectural changes (Williams et al. 2004). This notion will require further experimental investigation.
In conclusion, MA produces increases in CORT and changes in 5-HT during the neonatal period that are associated with spatial learning deficits in the MWM whereas
MDMA produces a smaller initial increase in CORT and larger decreases in 5-HT than
MA that result in augmented long-term decreases in 5-HT, larger MWM deficits, and impaired learning in other tasks, such as the CWM. The mechanism by which MA or
MDMA prevent proper spatial learning ability is unknown however, we have shown protracted changes in two neurochemicals that are known to affect the hippocampus
(Gould et al. 1991a;Gould et al. 1991b;Gould and Tanapat 1999). It is likely that two different but perhaps overlapping mechanisms lead to the resulting spatial learning disruptions following neonatal MA or MDMA treatment.
131
Acknowledgments
Portions of these data were presented at the 13th annual meeting of the
International Behavioral Neuroscience Society meeting in Key West, Florida, 2004.
Supported by National Institutes of Health grants DA014269 (MTW) and DA006733
(CVV), DA007427 (GAG) and training grant ES007051 (TLS and MRS).
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Figures
60 A
SAL 50 MA MDMA
# 40 # # #
Weight (grams)Weight # * * * * 30 * * * * * * *** *
20 0 11 12 13 14 15 Day
B 60
# # SAL 50 MA # # MDMA # # # # * * 40 # * # * * # # * * # # * Weight (grams) Weight * * * * * * * 30 * * * ** * * * * * * * * * ** * 20 0 11 12 13 14 15 16 17 18 19 20 Day
Figure 1. Body weights taken during the first and last dose (mean ± SEM). (A) P11-15
and (B) P11-20 administration of MA or MDMA (10 mg/kg) 4 times each day
with an inter dose interval of 2 h caused a decrease in body weight gain compared
to SAL controls beginning on approximately the third day of dosing. MA
produced the least amount of body weight gain compared to MDMA and SAL
which was evident beginning on approximately P14 in both groups. *p < 0.05
from SAL control; # = MDMA and MA are different.
143
# * A 80
60
40 CORT (ng/ml)
20
0
P16 P30
SAL MA B MDMA 80
60
40 CORT (ng/ml)
20
0 P21 P30
Figure 2. Corticosterone concentrations in plasma (mean ± SEM). (A) CORT levels
following P11-15 dosing of MDMA or MA (10 mg/kg) 24 h after the 1st of 4
doses on P15 (sacrificed on P16) and CORT levels on P30 following P11-15
dosing. (B) CORT levels on P21 following P11-20 dosing and CORT levels on
P30 following P11-20 dosing. On P16 CORT levels were significantly increased
following MA treatment compared to both SAL and MDMA treatments. There
were no changes observed in CORT levels on P21 or on P30 following either drug
or dosing regimen. *p < 0.05 from SAL control; # = MDMA and MA are
different.
144
A 0.5
0.4
0.3
0.2
5-HT (ng/mg tissue)5-HT (ng/mg 0.1 *
0.0
P16 P30
B 0.7 Sal 0.6 MA MDMA 0.5 # 0.4 * 0.3 * 0.2 5-HIAA (ng/mg tissue) 5-HIAA 0.1
0.0
P16 P30
C 3.5
3.0
2.5
2.0
1.5 5-HIAA/5-HT 1.0
0.5
0.0 P16 P30
Figure 3. Serotoninergic markers in neostriatum following P11-15 drug administration
on P16 (n = 13/group) and P30 (n = 6/group; mean ± SEM). (A) P16
concentrations of 5-HT and P30 5-HT, (B) P16 5-HIAA and P30 5-HIAA, (C)
P16 5-HIAA/5-HT ratio and P30 5-HIAA/5-HT ratio in the neostriatum of
animals exposed to 5 days of MDMA or MA. Decreases in 5-HT were observed
in the MDMA-treated animals on P16. 5-HIAA levels were decreased following
MDMA and MA on P16. There were no differences in the 5-HIAA/5-HT ratio on
P16 or any of the neostriatal serotonergic markers on P30. * p < 0.05 from SAL
control.
145
A 10
8
6
4 DA (ng/mg tissue) DA 2
0
P16 P30
B 1.6 SAL MA 1.4 MDMA 1.2
1.0
0.8
0.6
0.4 DOPAC (ng/mg tissue) DOPAC 0.2
0.0
P16 P30
C 0.25
0.20
0.15
0.10 DOPAC/DA
0.05
0.00 P16 P30
Figure 4. Dopaminergic markers in neostriatum following P11-15 drug administration on
P16 (n = 13/group) and P30 (n = 6/group; mean ± SEM). (A) P16 concentrations
of DA and P30 DA, (B) P16 DOPAC and P30 DOPAC, (C) P16 DOPAC/DA
ratio and P30 DOPAC/DA ratio in the neostriatum on P16 following
administration of MDMA or MA. No changes in any dopaminergic markers were
observed following 5 days of drug treatment at the 24 h time-point or on P30.
146
A 0.5
0.4
0.3 * 0.2 * 5-HT (ng/mg tissue) 0.1
0.0
P21 P30
SAL B 0.6 MA MDMA 0.5
0.4 **
0.3
0.2 5-HIAA (ng/mg tissue) (ng/mg 5-HIAA 0.1
0.0
P21 P30
C 3.0
2.5
2.0
1.5
1.0 5-HIAA/5-HT ratio 5-HIAA/5-HT
0.5
0.0 P21 P30
Figure 5. Serotonergic markers in the neostriatum following P11-20 drug administration
on P21 (n = 16/group) and P30 (n = 8/group; mean ± SEM). (A) P21
concentrations of 5-HT and P30 5-HT, (B) P21 5-HIAA and P30 5-HIAA, (C)
P21 5-HIAA/5-HT ratio and P30 5-HIAA/5-HT ratio in the neostriatum of
animals dosed for 10 days with MDMA or MA. On P21, 5-HT and 5-HIAA
levels were decreased following both MDMA and MA treatments. No other
differences were noted in the neostriatum of P11-20 treated animals. *p < 0.05
relative to saline controls.
147
A 10
8
6
4 DA (ng/mg tissue) (ng/mg DA 2
0
P21 P30
B 2.5 SAL * MA 2.0 MDMA
1.5 *
1.0
DOPAC (ng/mg tissue) 0.5
0.0
P21 P30
C 0.4 *
0.3 *
0.2
DOPAC/DA ratio 0.1
0.0 P21 P30
Figure 6. Dopaminergic markers in neostriatum following P11-20 drug administration on
P21 (n = 16/group) and P30 (n = 8/group; mean ± SEM). (A) P21 concentrations
of DA and P30 DA, (B) P21 DOPAC and P30 DOPAC, (C) P21 DOPAC/DA
ratio and P30 DOPAC/DA ratio in the neostriatum on P21 following
administration of MDMA or MA. No changes in DA were observed following 10
days of drug administration. DOPAC and the DOPAC/DA ratio were increased
following MDMA and MA compared to SAL treatment on P21. No differences
in dopaminergic markers were noted on P30. *p < 0.05 relative to saline controls.
148
A 0.6
0.5
0.4
0.3 #
0.2 * 5-HT (ng/mg tissue) 0.1 *
0.0
P16 P30
B 0.6 Sal MA 0.5 MDMA
0.4 # * * 0.3 * 0.2 *
5-HIAA (ng/mg tissue) 5-HIAA 0.1
0.0
P16 P30
C 2.0
1.5
1.0 * * 5-HIAA/5-HT
0.5
0.0 P16 P30
Figure 7. Serotonergic markers in the hippocampus following P11-15 drug
administration on P16 (n = 13/group) and P30 (n = 6/group; mean ± SEM). (A)
P16 concentrations of 5-HT and P30 5-HT, (B) P16 5-HIAA and P30 5-HIAA,
(C) P16 5-HIAA/5-HT ratio and P30 5-HIAA/5-HT ratio in the hippocampus of
animals dosed for 5 days with MDMA or MA. Decreases in 5-HT were observed
following MDMA and MA treatment at the 24 h time-point on P16. MDMA
produced the greatest decrease in hippocampal 5-HT which was significantly
different from SAL and MA. 5-HIAA levels were also decreased on P16 and on
149
P30 in both the MDMA- and MA- treated groups with MDMA producing the greatest 5-HIAA decreases. The 5-HIAA/5-HT ratio was not affected on P16 however, on P30 the ratio was lower in the MDMA and MA groups when compared to SAL. *p < 0.05 from SAL control; # = MDMA and MA are different.
150
A 0.6
0.5
0.4 # 0.3 * * 0.2 5-HT (ng/mg tissue) 0.1
0.0
P21 P30
SAL B 0.6 MA MDMA 0.5 0.4 * * 0.3 *
0.2 5-HIAA (ng/mg tissue) 0.1
0.0
P21 P30
C 1.6
1.4
1.2
1.0
0.8 *
0.6
5-HIAA/5-HT ratio 0.4
0.2
0.0 P21 P30
Figure 8. Serotonergic markers in the hippocampus following P11-20 drug
administration on P21 (n = 16/group) and P30 (n = 8/group; mean ± SEM). (A)
P21 concentrations of 5-HT and P30 5-HT, (B) P21 5-HIAA and P30 5-HIAA,
(C) P21 5-HIAA/5-HT ratio and P30 5-HIAA/5-HT ratio in the hippocampus of
animals dosed for 10 days with MDMA or MA. Decreases in 5-HT and 5-HIAA
were observed following MDMA and MA treatment at the 24 h time-point on
P21. MDMA produced the greatest decrease in hippocampal 5-HT which was
significantly different from SAL and MA. On P30, 5-HIAA and the 5-HIAA/5-
HT ratio were significantly decreased compared to SAL and MDMA treatments.
*p < 0.05 from SAL control; # = MDMA and MA are different.
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500 A * 400 * SAL MA MDMA 300
200 * CORT (percent control) 100
0 12 16 21
120 B
100
80 * * * 60 * * 40 5-HT (percent control)
20
0 12 16 21 Age (days)
Figure 9. Time-course of CORT and hippocampal 5-HT changes 24 h following 1, 5, and
10 days of exposure. Data are presented as percent of SAL control and the P11
data from a previous experiment (Schaefer et al., 2006). (A) CORT and B) 5-HT
levels following MA and MDMA exposure. Following MA, CORT levels are
elevated from P12 through P16 and 5-HT levels are decreased by at least P16,
remain depleted through P21. After MDMA exposure CORT levels are elevated
only on P12 while 5-HT levels are decreased on P12, 16 and 21. *p < 0.05
relative to saline controls.
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CHAPTER 5:
Alterations to learning and memory following neonatal exposure to 5-HT altering drugs:
individual and combined effects of MDMA and citalopram
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ABSTRACT
Developmental exposure to 3,4-methylenedioxymethamphetamine (MDMA) produces spatial and path integration learning deficits (Skelton et al. 2009;Vorhees et al.
2007;Williams et al. 2003b). The underlying mechanism that alters cognitive function is unknown however, may involve perturbations to the serotonergic system during the time of drug exposure. During development 5-HT is though to act a neurotrophic factor and is involved in neurogenesis, synaptogenesis, migration, and the development of target regions (Whitaker-Azmitia et al. 1996). We have previously shown postnatal day (P) 11-
20 MDMA exposure (4x10 mg/kg/day), a time analogous to human late second – third trimester exposure, to produce ~50% decrease in 5-HT throughout the exposure period
(Schaefer et al. 2006;Schaefer et al. 2008;Williams et al. 2005). It is proposed that these
5-HT decreases are altering the normal progression of brain development and result in cognitive deficits. To test this hypothesis, we administered citalopram (CIT), a selective serotonin reuptake inhibitor (SSRI), in conjunction with MDMA from P11-20. We found that 5 mg/kg CIT administered two times daily in addition to MDMA on P12 (after P11 exposure), 16 (after Pll-15 exposure) and P21 (after P11-20exposure) attenuated 5-HT depletions such that hippocampal, striatal, and entorhinal cortex 5-HT tissue concentrations were significantly higher than animals treated with only MDMA. We observed increases in CORT and hippocampal NGF on P16 and P21, respectively.
Behavioral analysis of these animals as adults revealed no cognitive improvement as a result of the CIT treatment and 5-HT attenuation in MDMA exposed rats and CIT treatment alone produced path integration learning deficits in the Cincinnati water maze
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with out any significant depletion of 5-HT. This may suggest that the 5-HT depletion produced by MDMA may be secondary to a more detrimental alteration to the 5-HT system during this critical period. Neonatal MDMA or CIT exposure may result in a substantial and prolonged release of 5-HT and overstimulation of various 5-HT receptors.
More data is needed to determine the mechanism resulting in decreased learning and memory ability following developmental exposure to 5-HT releasing drugs such as
MDMA, CIT, or other SSRIs.
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Introduction
Over the past decade 3,4-methylenedioxymethamphetamine (MDMA) has
become a popular drug of abuse especially with adolescents and younger adults of whom
3-12% report use within a given year (Johnston 2008). Adult human exposure to MDMA
has been shown to disrupt verbal recall and spatial associative learning (Zakzanis and
Campbell 2006). Similarly, adult rats that were administered a serotonin (5-HT) depleting regimen of MDMA demonstrated reference memory deficits in the Morris water maze (MWM) and learning disruptions in the Cincinnati water maze (CWM) when tested under low light conditions (Able et al. 2006;Sprague et al. 2003). Congruent to adult MDMA abuse in humans, women who abuse MDMA and are pregnant expose their fetus to MDMA (Ho et al. 2001;McElhatton et al. 1999). The effects of developmental exposure have not been well investigated, although there are reports of increased incidences of cardiac malformations and club-foot (McElhatton et al. 1999;McElhatton
1997;van Tonningen-van Driel MM et al. 1999). Nonetheless, no prospective studies exist examining cognitive and neurological outcomes of children exposed in utero to
MDMA. The neonatal rat has been used to model human late second through third
trimester brain development. MDMA exposure from postnatal day (P)11-20 produces
deficits in spatial and path integration learning when these animals are tested as adults.
The mechanism(s) by which developmental MDMA produces these long-term cognitive
deficits is unknown.
MDMA is thought to exert many of its effects via the serotonergic system by
binding to the serotonin transporter (SERT) and inducing an initial release of 5-HT. At
higher doses, protracted release results in depletion of 5-HT (reviewed by (Green et al.
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2003)). Neurotransmitter depletions during development can be detrimental since 5-HT
acts as a neurotrophic factor that supports the development of 5-HT neurons as well as
neurons in various target regions during early development. Following developmental
PCA- or PCPA-induced 5-HT depletions, delays in neuronal proliferation, decreased
spine density, and slowed migration have been observed as well as deficits in spatial
learning in the radial arm maze (Lauder and Krebs 1978;Lauder 1990;Mazer et al.
1997;Whitaker-Azmitia et al. 1996;Yan et al. 1997). The hippocampus, striatum, and
entorhinal cortex are known to be innervated by serotonergic fibers and are important in
learning and memory (Sodhi and Sanders-Bush 2004). Further, the hippocampal granular
cells are proliferating at a high rate later in human gestation, a period of brain
development analogous to the neonatal period in the rat (Bayer et al. 1993;Clancy et al.
2007). The serotonergic system is known to be disrupted by MDMA exposure during the rat neonatal period. These disruptions include protracted decreases in hippocampal and neostriatal 5-HT and 5-HIAA levels from P11-20. These early 5-HT depletions may contribute to the learning and memory deficits induced by early MDMA exposure, although direct evidence is lacking. The purpose of this study was to determine if blocking the 5-HT transporter (SERT) with a selective serotonin reuptake inhibitor
(SSRI) would attenuate 5-HT depletions in the hippocampus, neostriatum, and entorhinal cortex caused by MDMA exposure. If MDMA-induced 5-HT reductions can be signficiantly attenuated by SSRI pretreatment, we wanted to determine if this would attenuate MDMA-induced the learning deficits that emerge later in life.
The SSRI chosen for this purpose was citalopram (CIT) because it selective and has a high affinity for SERT and has the least effect on cytochrome P450 activity, several
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of which are important for MDMA metabolism (de la Torre et al. 2004;Hemeryck and
Belpaire 2002). In order to identify a dose of CIT pretreatment that would be the most effective at blocking or attenuating MDMA-induced 5-HT reductions in the hippocampus, we examined single versus twice dosing regimens of CIT. 5-HT levels were considered acceptable in MDMA-treated animals with CIT pretreatment if they were not significantly reduced compared to saline (SAL) controls or were significantly
greater compared to animals receiving MDMA alone. Two pretreatment doses of 5
mg/kg of CIT in combination with MDMA were determined to be the most effective
during the P11-20 exposure period. We subsequently examined the biochemical changes
of this dosing regimen following P11 only and after P11-15 MDMA exposure to
determine its effectiveness at early and intermediate stages of the MDMA treatment
regimen (P11-20). Plasma corticosterone (CORT) and NGF and BDNF were also
determined to further characterize the changes induced by MDMA, CIT, and the
combination. The 2 x 5 CIT dosing regimen was then used to determine if CIT prevented
any of the later behavioral effects of MDMA.
The following predictions were made based on previous data . First, animals
pretreated with SAL and treated with MDMA would be affected the same way as
observed in past experiments, i.e., show less spontaneous locomotor activity, have increased latency, errors, and start returns in the Cincinnati water maze (CWM), and have
increased path lengths, latency, and cumulative distance in the Morris water maze
(MWM) compared to animals only receiving SAL pretreatment and SAL treatment
(Broening et al. 2001;Morford et al. 2002;Schaefer et al. 2006;Skelton et al.
2009;Vorhees et al. 2004;Vorhees et al. 2007;Williams et al. 2003b). Second, we
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predicted that the CIT pretreatment combined with MDMA treatment would lead to attenuated effects, and therefore would show no change in spontaneous locomotor activity, reduced deficits in learning in the MWM and CWM compared to SAL-MDMA treated animals. Third, while no evidence exists that P11-20 exposure to CIT alone produces cognitive deficits, there are data showing that developmental exposure to CIT at similar doses to those used here can affect locomotor and sexual behavior (Maciag et al.
2006a;Maciag et al. 2006c;Maciag et al. 2006b). For this group, we opted to posit the null hypothesis, that CIT would not alter later learning. Finally, in order to test whether
MDMA, CIT or the combination altered anxiety, animals were tested in the elevated-zero maze and ligh-dark test, but no a priori predictions were made as no preliminary data for these were available.
Methods
Animals and housing
Nulliparous female Sprague-Dawley CD, International Genetic Strain, rats were obtained from Charles River Laboratories (Raleigh, NC) and mated with males from the same breeder. Pups from these matings were used as subjects. Rats were housed in a 22
± 1°C environment at 50 ± 10% humidity with a 14/10 h light/dark cycle (lights on at 600 h). Prior to the animals being mated, a period of at least 1 week ensued to allow the animals to habituate to the conditions of the facility. Each polycarbonate cage (46 x 24 x
20 cm) contained wood chip bedding, ad libitum food and water, and was equipped with a stainless steel enclosure for environmental enrichment (Vorhees et al. 2008). The
Cincinnati Children’s Research Foundation’s Institutional Animal Care and Use
Committee approved all protocols and the vivarium was fully accredited by the
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Association for the Assessment and Accreditation of Laboratory Animal Care
(AAALAC). The day of birth was considered P0, and on P1, litters were culled to the
appropriate number of males (8-10). The experiments described here were split-litter
studies. Pups were individually identified by ear punch on P7 and weighed prior to every
injection. Pups used for the behavioral study were also weighed weekly.
Only males were used for these studies for several reasons. Firstly, we have
previously shown only subtle, if any, drug x sex interactions on learning and memory
following neonatal MDMA administration in rats (Broening et al. 2001;Skelton et al.
2006;Skelton et al. 2009;Vorhees et al. 2004;Vorhees et al. 2007;Williams et al. 2003b).
Secondly, our model produces no significant differences between males and females in
CORT or monoamine levels after neonatal MDMA exposure (Williams et al. 2005).
Thirdly, we have observed that between 1.25 and 10 mg/kg CIT treatment alone or in combination with MDMA on P11 does not produce sex differences in hippocampal 5-HT levels (unpublished observations). Lastly, we wanted to maintain similar litter sizes between the current study and previous experiments, while using the split-litter design for behavioral assessment which required more pups per litter than a sex-stratified design would permit.
Determination of citalopram regimen
For biochemical determinations, 7 litters consisting of 10 male pups each were injected from P11-20 with a 1x or 2x pretreatments of 5 or 10 mg/kg R,S-citalopram hydrobromide (CIT) (Sigma, St. Louis, MO) or isotonic saline (SAL) 30 min prior to the
first and fourth administration of 10 mg/kg (±)-3,4-methylenedioxymethamphetamine
HCl (MDMA; National Institute on Drug Abuse) or SAL. MDMA or SAL was injected
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4x/day with a 2 h interdose interval for a total of 6 injections each day (2 pretreatments
plus 4 drug treatments). CIT and MDMA were expressed as the freebase (purity >95%)
and dissolved in isotonic SAL in a volume of 3 ml/kg. Treatments represented within
each litter were as follows: 1) pretreatment: 2x SAL, drug: 4x SAL (SAL+SAL); 2)
pretreatment: 2x SAL, drug: 4x10 mg/kg MDMA (SAL+MDMA); 3) pretreatment: 1x5
mg/kg CIT, drug 4x SAL (1x5 CIT+SAL); 4) pretreatment: 1x10 mg/kg CIT, drug: 4x
SAL (1x10 CIT+SAL); 5) pretreatment: 1x5 mg/kg CIT, drug 4x10 mg/kg MDMA (1x5
CIT+MDMA); 6) pretreatment: 1x10 mg/kg CIT, drug: 4x10 mg/kg MDMA (1x10
CIT+MDMA); 7) pretreatment: 2x5 mg/kg CIT, drug: 4x SAL (2x5 CIT+SAL); 8)
pretreatment: 2x10 mg/kg CIT, drug: 4x SAL (2x10 CIT+SAL); 9) pretreatment: 2x5
mg/kg CIT, drug: 4x10 mg/kg MDMA (2x5 CIT+MDMA); 10) pretreatment: 2x10
mg/kg CIT, drug: 4x10 mg/kg MDMA (2x10 CIT+MDMA). Animals that received the
1x CIT pretreatments were injected with SAL for the second pretreatment dose (0.5 h
prior to the last drug dose) to maintain a total of 6 injections/day for all animals. Twenty- four hours following the first MDMA dose on P20 animals were sacrificed (i.e. P21).
It was determined that the best pretreatment dosing regimen for attenuating the reductions in 5-HT after 10 days of MDMA administration was 2x5 CIT. Therefore, an additional 8 litters were dosed using the same daily 6 injection dosing regimen with 2 pups from each litter receiving SAL+SAL, SAL+MDMA, 2x5 CIT+SAL, or 2x5
CIT+MDMA. One pup from each treatment was dosed on P11 only and the remaining 4 pups were dosed from P11-15. Twenty-four hours after the first drug dose on P11 or P15 animals were killed (P12 or P16, respectively). These 3 exposure periods (P11, P11-16, and P11-20) were chosen to ensure that the pretreatment doses of 5 mg/kg CIT attenuated
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MDMA induced 5-HT reductions at the beginning and half-way through the dosing
regimen, as well as after the full 10-day exposure period. The 24 h time-point was used
following each dosing regimen (P11, P11-15, and P11-20) because we have previously
shown the most dramatic decrease in hippocampal 5-HT following P11 MDMA exposure
occurs at this time with continued decreases on P16 and P21 (Schaefer et al.
2006;Schaefer et al. 2008;Williams et al. 2005).
Serotonin, 5-HIAA, and the 5-HIAA/5-HT ratio were determined for each brain region. For the SAL+SAL, SAL+MDMA, 2x5 CIT+SAL, and the 2x5 CIT+MDMA groups at P12, 16, and 21, neostriatal DA, DOPAC, and the DOPAC/DA ratios were
determined as well as neostriatal and hippocampal nerve growth factor (NGF) and brain- derived neurotrophic factor (BDNF) and corticosterone in plasma (CORT). Neurotrophic
factors were analyzed because they regulate development, are important in learning and
memory, and have been shown to be affected by MDMA and CIT exposure (Huang and
Reichardt 2001;Koprich et al. 2003;Russo-Neustadt et al. 2004).
Blood and Tissue Collection
At the designated time point, each animal was decapitated and blood was
collected in tubes containing 2% EDTA (0.05 ml) and centrifuged (1399 RCF) for 25 min
at 4°C. Plasma was collected and stored at –80°C until assayed for CORT.
The brain was simultaneously removed and placed in a chilled brain block (Zivic-
Miller, Pittsburgh, PA) to aid in dissection of the neostriatum and hippocampus as described previously (Williams et al. 2007). For the neostriatum, a coronal cut was made at the optic chiasm and another 2 mm rostral to the first. The neostriatum (caudate- putamen) was dissected from this 2 mm section. The entorhinal cortex was dissected by
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making a coronal cut at the posterior extent of the mammillary body and another 2 mm posterior to the first. From this 2 mm section, the entorhinal cortex was removed bilaterally by making a cut at the rhinal fissure and removing the cortical tissue inferior to this cut to the tip of the corpus callosum. Hippocampi were removed from the remaining tissue. Tissues were immediately frozen on dry ice and stored at –80°C until monoamines were quantified by high-pressure liquid chromatography with electrochemical detection.
Monoamine determinations
The tissue concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid
(DOPAC), serotonin (5-HT), and 5-hydroxyindolacetic acid (5-HIAA) in the neostriatum and 5-HT and 5-HIAA in the hippocampus and entorhinal cortex were quantified using high-pressure liquid chromatography with electrochemical detection (Schaefer et al.
2006). Tissue weights were determined prior to homogenization in 50 volumes of 0.2 N perchloric acid and centrifuged for 6 min at RCF: 10,000 x g. Aliquots of 20 μl were injected onto a C18-column (MD-150, 3x150mm; ESA, Chelmsford, MA) connected to a
Coulochem detector (25A, Chemsford, MA) and an integrator recorded the peak heights for each injection. The potentials of the E1 and E2 on the analytical cell (model 5014B) of the Coulochem were –150 and 160 mV, respectively. The mobile phase consisted of
35 mM citric acid, 54 mM sodium acetate, 50 mg/l of disodium ethylenediamine tetraacetate, 70 mg/l of octanesulfonic acid sodium salt, 6% (v/v) methanol, 6% (v/v) acetonitrile, pH 4.0, and pumped at a flow rate of 0.4 ml/min. Quantification of analytes was calculated on the basis of standards. Retention times for DOPAC, DA, 5-HIAA, and
5-HT were approximately 6, 8, 11, and 17 min, respectively.
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NGF and BDNF assessment
The concentrations of NGF and BDNF in the hippocampus and neostriatum were
determined on P12, P16, and P21 using the Emax ImmunoAssay System (Promega Corp,
Madison WI). The samples were homogenized in lysis buffer (1 ml) according to kit
instructions and hippocampal samples were further diluted 1:2 and the neostriatal
samples 1:10 prior to assay. All samples were assayed in duplicate according to the manufacturer’s instructions and levels were expressed against total protein (i.e., pg/mg protein). Protein was assayed using a BCA protein assay kit (Pierce Biotechnology,
Rockford, IL). Optical densities were measured on a SpectraMax Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA).
Corticosterone assessment in plasma
Plasma was diluted 3:1 in supplied assay buffer and CORT levels (ng/ml) were assayed in duplicate using a commercially available EIA (Immunodiagnostic Systems
Inc., Fountain Hills, AZ) that was read on a SpectraMax Plus (Molecular Devices,
Sunnyvale, CA). The CORT EIA has little cross-reactivity with other hormones or precursors (< 1.4%) with the minor exceptions of 11-dehydrocorticosterone and 11- deoxycorticosterone (< 6.7%).
Behavioral Analysis
The CIT dosing paradigm that blunted the MDMA induced 5-HT depletions on
P12, P16, and P21 (i.e., 2 x 5 mg/kg CIT; 30 min prior to the first and fourth MDMA dose) was used for the behavioral experiment. A total of 20 litters were prepared. Two male pups from each litter received one of the following dosing regimens with all pretreatments injected 30 min prior to the first and fourth MDMA dose and all MDMA
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doses injected 4x/day at 2 h intervals from P11-20: 1) SAL+SAL; 2) SAL+MDMA; 3)
2x5 CIT+SAL; 4) 2x5 CIT+MDMA. One pup from each pair of the 4 treatment groups was put through one of two behavioral tracts (A or B) and testing began on P60. Each test was performed in the order presented for each tract.
Behavior Tract A
Light-Dark test (L/D test). On day 1 of testing, animals were placed in an infrared photocell activity chamber composed of a clear acrylic box (41 x 41 cm;
Accuscan Electronics, Columbus, OH) fitted with a dark enclosure (1/2 the size of the chamber) placed against one wall. To begine, rats were placed in the corner of the open
(lighted) side. Number of transitions from light to dark (dark entries), latency to the dark side, and total time spent in the light side of the chamber were recorded for 10 min
(Crawley and Goodwin 1980). The room was illuminated with overhead fluorescent lights. The chambers were cleaned with a 70% ethanol solution in between animals.
Straight Channel. On P61, animals were tested in a 15 x 244 cm straight swimming channel with a platform submerged 1.5 cm below the surface of the water at one end. Each rat received four consecutive trials on the test day. On each trial, the rats were placed at one end facing the wall and timed until they climbed on the platform at the opposite end (maximum time = 2 min/trial).
Cincinnati water maze (CWM). The CWM, a test of path integration, began on
P62. The maze, as described elsewhere (Vorhees 1987), consists of a series of nine Ts that branch from a central circuitous channel. The width of the Ts and channel were 15 cm with walls 51 cm high and filled half-way with water. Water was drained and refilled at least twice a week and allowed to equilibrate overnight to room temperature (21 ± 1
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°C). Testing was conducted in complete darkness using infrared light emitters and a camera placed above the maze that was connected to a closed circuit TV monitor located in an adjacent room where the experimenter watched and recorded performance.
Administering the test under infrared light was used to eliminate extramaze cues and prevent animals from using distal cues to spatially navigate. The animals were started facing the wall from the point originally described as position-B (Vorhees 1987) and were allowed a maximum of 5 min per trial to find the escape platform with a 5 min intertrial interval if an animal failed to locate the goal the first trial of the day. Animals were given two daily trials for 18 days. Errors, latency to escape, and number of returns to the start were recorded. An error was counted when an animal digressed from the center channel into a stem and when they continued into a dead-end portion of a T.
Perseverations within a T were counted as individual errors. Start return errors were counted separately. Occasionally an animal did not find the escape after completing all trials and a few stopped exploring. This lack of exploration left them with relatively few errors compared to animals that were escaping the maze in under 5 min. To adjust for this, error scores for these animals were assigned a value of 1 more than the score of the worst performing animal in the experiment.
Behavior Tract B
Elevated zero maze (EZM). The first test administered on P60 was the elevated zero maze (Shepherd et al. 1994) with minor modification (Williams et al. 2003a). The test room was dimly lit and each animal was brought directly from its home cage in the housing room and placed in a closed quadrant of the apparatus at which point the experimenter exited the room. Sessions were scored in real-time via an overhead camera
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connected to a monitor that was located outside the testing room. The dependent
variables were time in open, number of head dips, number of open arm entries, and
latency to first enter an open quadrant and recorded with a computer program (ODLog,
Macropod Software).
Spontaneous locomotor activity. Locomotor activity was assessed on P61 in infrared photocell activity chambers (41 x 41 cm; Accuscan Electronics, Columbus, OH) for 1 h. Total distance, margin distance (i.e., within 1 cm from the wall), and center distance were analyzed in 5 min intervals.
Straight Channel. Straight channel was assessed on P62 as described above in tract A.
Morris water maze (MWM). Spatial learning and memory were assessed in the
MWM using procedures described previously (Vorhees and Williams 2006). Testing began on P63 and was performed in a 210 cm circular tank. Animals were tested in 3 phases of the hidden platform version of the MWM: acquisition (platform in SW
position), reversal (platform in NE position), and shift (platform in NW position).
Briefly, animals received 4 trials per day for 6 days followed by a 30 s probe trial on day
7 with the platform removed. Each phase used a platform of a different size (10, 7, and 5
cm diameter, respectively). Following the three hidden phases, a cued version of the
maze was performed to ensure that the rats could swim normally and see well enough to
be able to locate a visible platform by providing proximal cues. For this procedure, the
submerged 10 cm platform was used with the addition of a plastic ball attached to a brass
rod that protruded above the surface of the water to mark the platform’s location.
Curtains were drawn around the maze to minimize extramaze cues and the animals were
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given four trials per day for two days with the platform and start positions changed for
every trial so spatial strategies to solve the task would be ineffective. During hidden
platform trials, a video camera and tracking software were used to map the animals’
performance (AnyMaze, Stoelting Company,Wood Dale, IL). On platform trials, latency, cumulative distance, path length, and speed were analyzed. For probe trials, platform site crossovers, speed, and average distance from the target were analyzed. For the cued phase, latency to reach the visible platform was recorded.
Statistical Analysis.
Monoamine, CORT, BDNF, and NGF were analyzed using Dunnett’s test after
each dosing regimen and significance was reported for treatments that were different
from SAL+SAL or SAL+MDMA treatments. All other data were analyzed using mixed
linear ANOVA models (SAS Proc Mixed, SAS Institute, Cary, NC). Litter was
considered a randomized factor to control for litter effects. For the light-dark test and
elevated zero maze, the covariance matrix for each data set was checked using best fit statistics. In most cases the best fit was to the autoregressive-1 (AR(1)) covariance structure. Proc Mixed calculates adjusted degrees of freedom using the Kenward-Rogers method, and therefore do not match those obtained from general linear model ANOVAs and can be fractional. Measures taken repetitively on the same animal, such as trial, day, or test interval, were repeated measure factors. Planned comparisons were performed for spontaneous locomotor, CWM, and MWM tests based on the specific predictions outlined above based on prior data. Significance was set at p ≤ 0.05 and trends at p <
0.10. Data are presented as least square (LS) mean ± LS SEM.
Results
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Hippocampal monoamines P21
Hippocampal 5-HT levels on P21 in animals dosed from P11-20 with 1x or 2x, 0,
5, or 10 mg/kg CIT prior to MDMA or SAL were decreased in the SAL+MDMA, 1x5
CIT+MDMA, and 2x10 CIT+MDMA treatments compared to the SAL+SAL treatment, whereas all other treatments were not different from SAL+SAL. The SAL+SAL, 1x5
CIT+SAL, 2x5 CIT+SAL, 1x10 CIT+SAL, 2x10 CIT+SAL, 2x5 CIT+MDMA, and 1x10
CIT+MDMA treatments had significantly greater 5-HT levels than the SAL+MDMA
treatment (Fig.1, 2C).
These data indicated that the 2x5 dose was sufficient for the intended purpose.
Given this, additional groups were prepared and monoamines examined in additional
brain regions and on different days for SAL+SAL, SAL+MDMA, 2x5 CIT+SAL, and
2x5 CIT+MDMA treatments. From this point forward, animals treated with the 2x5
mg/kg citalopram treatment will be referred to as CIT. Hippocampal 5-HIAA levels on
P21 were significantly decreased in the SAL+MDMA-treated animals and increased in the CIT+SAL-treated animals compared to SAL+SAL-treated animals (Table 1). No difference was noted between the SAL+SAL and CIT+MDMA treatments.
The ratio of 5-HIAA/5-HT in the hippocampus was significantly increased by
CIT+SAL treatment compared to SAL+SAL treatment after P11-20 exposure. No other
differences were noted for 5-HIAA/5-HT on P21 in the hippocampus (Table 1).
Hippocampal monoamines P12
Serotonin (Fig. 2A) and 5-HIAA (Table 1) levels following P11 dosing (sacrificed on P12) were significantly decreased in SAL+MDMA-, CIT+SAL-, and CIT+MDMA- treated animals compared to SAL+SAL-treated animals. However, the CIT+SAL- and
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CIT+MDMA-treated animals had significantly greater 5-HT levels than SAL+MDMA- treated animals (Fig. 2A). No other differences were observed for hippocampal 5-HIAA on P12.
On P12, the ratio of hippocampal 5-HIAA/5-HT was significantly increased by
SAL+MDMA exposure compared to SAL+SAL treatment and decreased by
CIT+MDMA treatment when compared to both SAL+SAL and SAL+MDMA. The
CIT+SAL treatment also showed a smaller 5-HIAA/5-HT ratio compared to
SAL+MDMA treatment (Table 1). No other differences were noted.
Hippocampal monoamines P16
Following P11-15 dosing and sacrifice on P16, hippocampal 5-HT levels were significantly decreased in the SAL+MDMA and CIT+MDMA-treated rats compared to
SAL+SAL-treated rats. Nonetheless, the CIT+SAL- and CIT+MDMA-treated animals had significantly higher levels of 5-HT compared to SAL+MDMA-treated animals (Fig.
2B).
Hippocampal 5-HIAA levels were decreased in SAL+MDMA-treated animals compared to SAL+SAL-treated animals. The CIT+SAL-treated animals had greater 5-
HIAA levels compared to SAL+MDMA-treated animals (Table 1). No other differences were noted.
The 5-HIAA/5-HT ratio in the hippocampus was significantly increased in the
SAL+MDMA- and CIT+SAL-treated animals but not in the CIT+MDMA-treated animals compared to SAL+SAL-treated animals (Table 1). No other differences were noted between the SAL+MDMA and other treatments.
Entorhinal cortex monoamines
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5-HT levels in the entorhinal cortex were significantly decreased in animals treated with SAL+MDMA following P11 exposure (Fig. 2D) and after P11-15 exposure levels were decreased in the SAL+MDMA, CIT+SAL, and CIT+MDMA (Fig. 2E) compared to SAL+SAL treated animals. On P21 SAL+MDMA- and CIT+MDMA- treated animals had decreased 5-HT levels compared to SAL+SAL-treated animals (Fig.
2F). On P16 (Fig. 2E) and P21 (Fig. 2F) the CIT+SAL and CIT+MDMA treatments produced 5-HT levels that were elevated compared to SAL+MDMA treatment. No other differences were noted on P12 (Fig. 2D).
5-HIAA levels in the entorhinal cortex on P12 were significantly decreased in all treatments compared to the SAL+SAL group. Animals pretreated with CIT did not differ from the SAL+MDMA treated animals. Following P11-15 exposure 5-HIAA levels in the entorhinal cortex were significantly decreased in both MDMA groups compared to
SAL+SAL treatment. In comparison with the SAL+MDMA-treated animals the
CIT+MDMA group had higher levels of 5-HIAA. All treatments on P21 significantly increased 5-HIAA levels in the entorhinal cortex compared to SAL+SAL treatment, but
CIT pretreated animals were not different from SAL+MDMA-treated animals (Table 1).
5-HIAA/5-HT ratio in the entorhinal cortex on P12, P16, and P21 was increased in the
SAL+MDMA-treated animals compared to SAL+SAL-treated animals. No other differences on any of the days were observed for the other treatments. All CIT treatments produced lower ratios than the SAL+MDMA treatment on P12 and P16, but not on P21 when no differences were observed (Table 1).
Neostriatum monoamines
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In the neostriatum, 5-HT levels were significantly decreased following P11 exposure to SAL+MDMA and CIT+SAL, but not CIT+MDMA, compared to SAL+SAL exposure. The CIT+MDMA-treated animals had elevated 5-HT compared to
SAL+MDMA (Fig. 2G). After P11-15 exposure, SAL+MDMA treatment produced a decrease in 5-HT compared to SAL+SAL, whereas CIT pretreatment did not differ from
SAL+SAL. The CIT pretreatment did block the 5-HT reductions compared to
SAL+MDMA exposure (Fig. 2H). After P11-20 exposure, the CIT+MDMA treatment produced neostriatal 5-HT levels that were increased compared to both SAL+SAL and
SAL+MDMA exposure (Fig. 2I). No other differences were noted on any of the days tested.
Neostriatal 5-HIAA levels on P12 were decreased in all 3 treatments compared to
SAL+SAL treatment, however no differences were noted between the CIT pretreatments and the SAL+MDMA treatment. On P16, SAL+MDMA treatment, but not the CIT pretreatments, produced a decrease in neostriatal 5-HIAA compared to SAL+SAL treatment. The CIT pretreated animals also had greater levels of 5-HIAA compared to the SAL+MDMA treated animals. Following P11-20 exposure, CIT+SAL treatment increased neostriatal 5-HIAA compared to SAL+SAL, whereas CIT pretreatment, regardless of MDMA, produced greater 5-HIAA levels compared to SAL+MDMA treatment (Table 1).
In the neostriatum on P12, SAL+MDMA treatment produced an increase in 5-
HIAA/5-HT compared to SAL+SAL treatment. All groups had lower 5-HIAA/5-HT ratios compared to SAL+MDMA treatment. No differences in 5-HIAA/5-HT ratios were observed following P11-15 or P11-20 exposure (Table 1).
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Neostriatal DA was not altered by any treatments at the 24 h time-point following any dosing regimen when compared to SAL+SAL treatment. The CIT+SAL treatment produced significantly decreased DA levels compared to SAL+MDMA treatment on P12 only (Table 1).
DOPAC levels in the neostriatum on P12 were significantly decreased in the
MDMA-treated animals, regardless of pretreatment, compared to SAL+SAL treatment.
The CIT+SAL levels were significantly higher than SAL+MDMA. On P16, following
P11-15 exposure, neostriatal DOPAC levels were not different between any groups.
After P11-20 exposure, neostriatal DOPAC levels were significantly increased in the
MDMA treatments, regardless of pretreatment, compared to SAL+SAL treatment and the
CIT+SAL treated animals had significantly greater levels than the SAL+MDMA treated animals (Table 1). No other differences were noted.
The ratio of neostriatal DOPAC/DA was decreased by MDMA, regardless of pretreatment, compared to SAL+SAL on P12 and P21, and the ratio in the CIT+SAL group was more than the SAL+MDMA group on P12 and less on P21. No differences in
DOPAC/DA were observed on P16 in the neostriatum (Table 1).
Overall, CIT pretreatment in combination with MDMA attenuated 5-HT reductions significantly such that levels were maintained similarly to SAL+SAL levels or higher than SAL+MDMA in all 3 brain regions on all 3 days analyzed. Few effects were observed for the dopaminergic system.
NGF and BDNF
Twenty-four hours following P11, P11-15, and P11-20 administration of the treatment regimens, NGF and BDNF were quantified in the hippocampus and
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neostriatum. Treatment with CIT+MDMA produced a significant increase in
hippocampal NGF compared to both SAL+SAL and SAL+MDMA treatments on P16.
No other differences in BDNF or NGF were observed at any other time-point or in the
neostriatum (Table 1).
Corticosterone
There were no differences in CORT levels 24 h following P11 or P11-15
exposure. On P21, following P11-20 exposure, MDMA-treated animals, regardless of
pretreatment, had significantly elevated CORT levels compared to SAL+SAL-treated
animals. The CIT+SAL treatment produced a decrease in CORT compared to
SAL+MDMA treatment (Table 1).
Body weights
For the dosing weights of animals in the behavioral study, the mean of the first
and last drug dose each day was used for analysis. There was a Pretreatment x Drug x
Day effect (F(9,1357) = 4.82, p < 0.0001) such that on P12 SAL+MDMA- and
CIT+MDMA-treated animals weighed significantly less than SAL+SAL controls. On
P13 through P20 SAL+MDMA-, CIT+SAL-, and CIT+MDMA-treated animals weighed
less than SAL+SAL-treated animals (Fig. 3).
Light-Dark test
There was a main effect of Pretreatment for number of dark entries (F(1,54) =
4.05, p < 0.05) in which the animals pretreated with CIT, regardless of MDMA treatment,
entered the dark side of the box more often than animals that received SAL pretreatment.
There was a significant interaction of Pretreatment x Drug for time in the light (F(1,54) =
4.38, p < 0.04) and latency to the dark (F(1,54) = 6.72, p < 0.01). Slice effects showed
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only tendencies for SAL+MDMA and SAL+SAL to be different for time in the light (p <
0.1). For latency to enter the dark there were trends for SAL+SAL vs. SAL+MDMA,
SAL+MDMA vs. CIT+MDMA, and CIT+SAL vs. SAL+SAL to be different (p < 0.1)
(Table 2).
Elevated Zero Maze
There was a main effect of Drug (F(1,55) = 6.20, p < 0.02) and a trend for a
Pretreatment x Drug interaction for number of open arm entries (p < 0.1) . MDMA-
treated animals, regardless of pretreatment, entered the open arms of the maze more than
all SAL-treated animals. There were no differences observed for time in open, latency to enter the open, or number of head dips (Table 2).
Spontaneous Locomotor
Preplanned comparisons based on a priori hypotheses were analyzed for total distance. SAL+MDMA-treated animals traveled significantly less during interval 2 and 6 and there was a trend for decreased activity during interval 8 compared to SAL+SAL- treated animals (Fig. 4A). Distance traveled was decreased during interval 6 and 8 in the animals exposed to CIT+SAL compared to SAL+SAL-treated animals (Fig 4B).
Comparison of the two MDMA groups showed that animals treated with 2x5
CIT+MDMA traveled less during the first interval compared to SAL+MDMA (Fig. 4C).
Distance traveled near the perimeter showed a similar pattern (not shown).
Straight Channel
Animals from tract A and B were combined for this analysis and there were no effects of Pretreatment, Drug, or Tract for latency to find the escape platform on the last trial (Table 2).
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Cincinnati water maze
Preplanned comparisons were analyzed for days 11-18. For latency to find the
escape SAL+MDMA-treated animals took significantly longer to find the platform on
days 14 and 16 and there was a trend for them to have longer latencies on days 11, 17,
and 18 compared to SAL+SAL-treated animals (Fig. 5A). SAL+MDMA treatment also
produced significantly more errors on days 11, 14, 17, and 18 and there was a trend for
these animals to commit more errors on days 12, 13, 15, and 16 compared to SAL+SAL
treatment (Fig. 5B). Start returns were also increased in the SAL+MDMA group compared to the SAL+SAL group on day 14 with trends toward an increase on days 11 and 16 (Fig 5C). When comparing the CIT+SAL and SAL+SAL animals, we observed
significant increases in latency on days 14 and 16 and a trend toward an increase on day
11 in the CIT+SAL (Fig. 5D). Errors were increased on days 11 and 14 and there was a
trend toward increase on days 12 and 13 in the CIT+SAL compared to the SAL+SAL
animals (Fig. 5E). The CIT+SAL treatment produced an increase in start returns on days
11, 12, and 14 and similar trends on days 13, 16, and 18 compared to SAL+SAL
treatment (Fig. 5F). Compared to the SAL+MDMA treatment, the CIT+MDMA
treatment did not alter the effects of MDMA for latency (Fig. 5G), errors (Fig. 5H), or
start returns (Fig. 5I).
Morris water maze
Preplanned comparisons for the comparison of the SAL+MDMA- to SAL+SAL-
treated animals, the SAL+MDMA treatment produced a significant increase in path
length on day 3 and a similar trend on day 4 in the acquisition phase (Fig. 6A). During
reversal SAL+MDMA treatment produced significant increases in path length on days 3
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and 6 and trends on days 4 and 5 compared to SAL+SAL treatment (Fig. 6B). On days 4 and 6 in the shift phase, SAL+MDMA treatment showed increased path length and a trend toward an increase on day 3 compared to SAL+SAL treatment (Fig. 6C).
For the comparison of the CIT+SAL and SAL+SAL treatments, the CIT+SAL animals had no differences on acquisition (Fig. 6D) or shift (Fig. 6F) and only a trend toward increased path lengths on days 1 and 3 in reversal (Fig. 6E). The comparison of the CIT+MDMA and SAL+MDMA treatments showed that path length during acquisition was significantly decreased in the CIT+MDMA animals on days 3 and 4 (Fig.
6G). During reversal, the CIT+MDMA treatment increased path length on days 1 and 2
(Fig. 6H) and during shift, showed increases on day 2 with a trend on day 1 (Fig. 6I).
On probe trials after acquisition, SAL+MDMA animals had fewer platform crossings than SAL+SAL animals (0.32±0.28 and 1.20±0.27, respectively) with no differences for average distance from the target or speed. Comparison of the CIT+SAL and SAL+ MDMA treatments demonstrated that animals pretreated with CIT crossed the platform position more (0.84±0.28 and 0.32±0.28, respectively) and had a decreased average distance from the target (0.53±0.04 and 0.56±0.04, respectively) compared to the
SAL+MDMA with no differences in speed. No differences were observed for platform crossings, average distance from the target, or speed between the CIT+SAL and
SAL+SAL treatments.
During the reversal probe trial SAL+MDMA-treated animals crossed the platform less (0.61± and 1.1±, respectively) and there was a trend for an increase in average distance from the target compared to SAL+SAL-treated animals (0.68±0.05 and
0.60±0.05, respectively). For the comparison of the two MDMA drug treated groups, the
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CIT+MDMA-treated animals crossed the platform significantly more (0.85±0.21 and
0.61±0.21, respectively) and had a trend to swim slower than SAL+MDMA-treated
animals (0.21±0.01 and 0.23±0.01, respectively) with no differences observed for average distance to the target. No differences were noted between the CIT+SAL and
SAL+SAL for platform crossings, average distance from the target, or speed.
During the shift probe, platform crossings were significantly decreased and
average distance to the target increased in the SAL+MDMA- (0.55±0.21 and 0.7±0.04,
respectively) and CIT+SAL-treated animals (0.67±0.21 and 0.56±0.04, respectively) compared to SAL+SAL-treated animals (1.05±0.2 and 0.48±0.04, respectively). No
differences between the two MDMA groups were observed for platform crossings or
average distance to the target and no differences in speed were observed.
During the cued phase of the MWM, there were no effects of Pretreatment or
Drug or any interactions on latency to locate the platform.
Discussion
MDMA administration (4x10 mg/kg) from P11-20 is sufficient to produce
significant spatial and path integration deficits in rats as adults. We have previously
shown that this dosing regimen, during a time analogous to late second through third trimester human brain development, produces 5-HT depletions in the hippocampus and neostriatum on P12, P16, and P21. The neostriatum, hippocampus, and entorhinal cortex in particular are known to be involved in learning and memory. We proposed that the 5-
HT depletions produced by MDMA during this time-period may be playing a role in the cognitive dysfunction following P11-20 MDMA exposure. In order to test this hypothesis, we investigated the ability of citalopram to block or attenuate the MDMA-
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induced 5-HT reductions without affecting neurotrophic factors or HPA axis functions.
Subsequently, with a dosing regimen of citalopram that was effective for this purpose, we investigated the impact on learning and memory.
Determination of an effective citalopram dose
We determined that the 2x5 CIT pretreatment was the most effective dose at attenuating the P11-20 MDMA-induced 5-HT depletions in the hippocampus on P21, 24 h after dosing (Fig. 1). The P11-20 period of exposure to MDMA is known to induce learning and memory deficits. Although complete attenuation was not achieved with this dose, the higher dose of 2x10 CIT in combination with MDMA produced a paradoxical decrease in 5-HT levels. This decrease may be the result of saturation of monoamine oxidase, which is inhibited by MDMA treatment (Leonardi and Azmitia 1994), and is primarily responsible for the metabolism of citalopram (Kosel et al. 2002) and 5-HT
(Borue et al. 2007). If correct, such saturation would result in an increased outflow of 5-
HT potentially further exacerbating CIT inhibiting effects on tryptophan hydroxylase
(Maciag et al. 2006b) and the synthesis of 5-HT resulting in the observed 5-HT depletions however, more testing would be required to prove this.
We replicated the previously published MDMA-induced 5-HT depletions
(SAL+MDMA group versus the SAL+SAL group) in the hippocampus and neostriatum
(Schaefer et al. 2006;Schaefer et al. 2008;Williams et al. 2005) and this is the first study to show similar decreases in the entorhinal cortex on P11, 16, and 21. All regions showed a greater than 50% decrease in 5-HT on P12, P16, and P21 except in the neostriatum on P21 (Fig 2). Both the 2x5 and 1x10 CIT pretreatments were successful at attenuating the MDMA-induced 5-HT depletions in the hippocampus on P21, however
179
we decided to use two doses of 5 mg/kg CIT because citalopram has a half-life of 3 h
(Melzacka et al. 1984;Overo 1982) and therefore 2 half-lives would occur during the 6 h dosing period. We know that the half-life of MDMA in neonates is ~4 h (Williams et al.
2004) and we wanted to ensure that the levels of citalopram would be maintained even after the fourth MDMA injection of each day. The 2x5 CIT+SAL treatment did not alter
5-HT levels in any region throughout dosing except in the striatum on P12 suggesting minimal effects of the citalopram pretreatment on 5-HT levels during the times analyzed.
Learning and Memory
As expected, SAL+MDMA treatment produced increases in latency, errors, and start returns in the CWM and path length in the MWM. It was predicted that pretreatment with CIT would lessen the severity of such learning deficits in both the
CWM and MWM because the 5-HT depletion in the CIT+MDMA animals was not as severe as in the SAL+MDMA animals during dosing. This prediction was based on the fact that there appears to be a dose-response relationship with 5-HT reductions. For example, even greater 5-HT depletions with fenfluramine treatment during this same
P11-20 period results in even greater cognitive deficits than in MDMA-exposed animals
(Morford et al. 2002;Schaefer et al. 2006). Nonetheless, pretreatment with CIT prior to
MDMA treatment failed to moderate any of these indices of learning. In fact, the
CIT+SAL animals had path integration learning deficits.
The current study is the first to report path integration learning deficits induced by
CIT (2x5 CIT+SAL group) and warrants some concern for women considering taking citalopram while pregnant. The dose of citalopram administered (2x5 mg/kg) produces serum and brain drug/metabolite levels in the neonates similar to those achieved in adult
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treatment of the antidepressant in rats and serum levels are consistent with those achieved in humans at therapeutic doses of the drug (Maciag et al. 2006b). Others have identified biochemical changes induced by the same dose used in the present study when administered from P8-21 including reduced tryptophan hydroxylse (TPH) immunoreactivity in the dorsal and median raphe and reduced SERT immunoreactive fibers in cortical areas in the absence of cell death, suggesting the currently observed
CIT-induced CWM learning deficits are probably not attributable to early neuronal loss
(Maciag et al. 2006b).
As with the CWM, in the MWM, attenuation of 5-HT depletions produced by administration of CIT+MDMA treatment did not appear to influence spatial learning since only early and sporadic differences were observed between this group and the
SAL+MDMA group during the hidden phases of the task (Fig. 5G, H, I).
It has been previously reported that 4x10 MDMA administration from P11-20 and
P8-21 administration of CIT produce a decrease in weight gain therefore it was not surprising that the SAL+MDMA, CIT+SAL, and the CIT+MDMA animals gained less weight than the SAL+SAL control animals (Broening et al. 2001;Deiro et al. 2004).
Importantly, we have previously shown that these decreases in body weight gain in the absence of MDMA administration can not account for learning and memory deficits alone (Williams et al. 2003b).
This study further supports that learning in the MWM and CWM involves different mechanisms and/or brain regions. This is based on the observation that
CIT+SAL associated deficits were seen in the CWM but not in the MWM, which suggests that early 5-HT development is sensitive to path integration changes but not
181
necessarily to spatial learning. Spatial learning in the MWM requires the use of distal cues in order to navigate to the hidden platform whereas the CWM relies on self- movement cues as distal cues are eliminated when testing is under infrared lighting. Path integration learning involves presubiculum head-direction cells, entorhinal cortex grid and border cells (Solstad et al. 2008), and subsets of hippocampal place cell (Fuhs and
Touretzky 2006;McNaughton et al. 2006;Rondi-Reig et al. 2006;Sargolini et al.
2006;Whishaw et al. 1997;Witter and Moser 2006) that together constitute what is currently known about egocentric-learning circuitry. Spatial learning in the MWM has been shown to require hippocampal function (Morris et al. 1982) as well as other regions
(Eichenbaum and Lipton 2008;Mura and Feldon 2003). It is possible that the differential innervation of 5-HT target regions by various raphe nuclei, which differentially express
SERT and 5-HT1A (Invernizzi et al. 1997), may play a role in acquiring spatial versus path integration learning.
Since MDMA and citalopram independently bind SERT and interfere with the normal method of removing 5-HT from the synapse, it is likely that overstimulation of various 5-HT receptors is occurring in both the CIT+SAL animals as well as in the
SAL+MDMA and CIT+MDMA animals regardless of the tissue concentrations of 5-HT.
In adults, both MDMA and citalopram have been shown to release extracellular 5-HT
(Baumann et al. 2007;Ceglia et al. 2004). It has been shown using SERT knockout mice that severe depletions in tissue 5-HT levels can occur in combination with large increases in extracellular 5-HT levels (Kim et al. 2005). This suggests that the degree of 5-HT depletion may be unrelated to the cognitive effects in the CIT- or MDMA-treated animals, especially considering that substantial increases in released 5-HT may be
182
occurring. Interestingly, P11-20 MDMA administration has been shown to increase 5-
HT1A sensitivity in adults (Crawford et al. 2006), further supporting the idea that drugs
that alter 5-HT during development also affect 5-HT receptors.
Locomotor and anxiety behavior
We have previously shown that P11-20 administration of MDMA results in relatively mild but long lasting hypolocomotion. Administration of CIT from P8-21 has been shown to produce an increase in spontaneous locomotor activity in adult rats
(Maciag et al. 2006c;Maciag et al. 2006b) although in the current study we did not show any differences between animals treated with SAL+SAL and CIT+SAL. This discrepancy may be due to procedural differences. For example, in the previous study,
CIT was administered for an additional 4 days and the increased locomotion was observed during the dark phase of the light/dark cycle whereas in the current study, all testing was performed during the light phase.
It has been suggested that the increase in extracellular 5-HT, as a result of developmental SSRI treatment, would lead to an altered anxiety phenotype (Borue et al.
2007). In the light/dark test, an increase in the number of dark entries together with decreased latency to enter the dark and increased time spent in the dark are generally regarded as reflecting increased anxiety. Although the last measure was not affected by
CIT, dark entries were significantly increased and there was a trend toward a decrease in latency to enter the dark, suggesting that anxiety may be increased following developmental CIT exposure. This is the first study to examine animals exposed to developmental MDMA treatment in the light/dark test and it appears that these animals, similarly to CIT-treated animals, have an increase in anxiety since there were trends
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towards decreased time spent in the light and latency to enter the dark. These results are complicated by the finding that MDMA treatment, regardless of pretreatment, caused an increase in open arm entries in the elevated zero maze which suggests decreaseed anxiety. The basis of this discrepancy is not currently known.
CORT and growth factors
In addition to 5-HT alterations, other biochemical changes that occur after neonatal MDMA treatment may also contribute to the cognitive deficits. The P21 increase in CORT observed after 10 days of MDMA exposure, regardless of pretreatment, was not previously observed (Schaefer et al. 2008), but may provide an explanation for the lack of behavioral improvement in the CIT+MDMA group compared to the SAL+MDMA group. Fluctuations in CORT are known to interfere with the proliferation of neurons during development, especially in the hippocampus (Gould et al.
1991;Liu et al. 2003;Woolley et al. 1990) and we have previously shown MDMA increases CORT on P11 1 h after the first MDMA dose and this continues for at least 24 h (Williams et al. 2005). It has also been demonstrated that citalopram exposure in adult rats produces increased HPA axis activation following acute treatment with a suppression of CORT following chronic treatment (Jensen et al. 1999). Chronic human fetal exposure results in a significant decrease in cord blood cortisol levels at birth (Davidson et al. 2009). It is possible that in the current study, MDMA- or CIT-treated animals may be experiencing fluctuations in CORT prior to the 24 h time-point, however, no differences were noted on P12 or P16 after MDMA or CIT exposure or on P21 after CIT treatment. Further investigation is required to determine the effect of altered CORT levels on cognitive ability after neonatal MDMA or CIT exposure.
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The neurotrophins, NGF and BDNF, are thought to be important maintenance
factors for learning and memory (Chen et al. 1997;Kesslak et al. 1998;Linnarsson et al.
1997;Mizuno et al. 2000). They peak at different times during development and have
been shown to be influenced by stress, changes in CORT levels, and changes in 5-HT
(Roskoden et al. 2005;Schaaf et al. 1997;Smith et al. 1995;Zetterstrom et al. 1999). In
the current study it appears that hippocampal NGF reaches its peak between P12 and 21 and striatal NGF remains constant which is consistent with previous reports (Grace et al.
2008). BDNF did not appear to fluctuate over the 10 days analyzed in the current study
although others have shown that between P7 and 14 BDNF levels increase (Das et al.
2001). Increases in hippocampal NGF in the animals that received both CIT and MDMA
suggests additional neuronal stress although this does not appear to have resulted in
increased cognitive deficits. It was previously shown that BDNF levels in the
hippocampus and striatum were increased following P11-20 administration of MDMA
(2x20 mg/kg/day) (Koprich et al. 2003) however, we did not show any changes in BDNF
at any time-point analyzed.
Finally, one caveat to the interpretation of the inefficacy of CIT at preventing
MDMA-induced cognitive deficits is that the deficits seen here in the SAL-MDMA group
were smaller than in our previous experiments. The effect of this was to make the range
of difference for demonstrating an attenuation effect of CIT narrower than we predicted.
The reason for this is unknown. One possibility is that the extra injections and handling
required in order to administer CIT may have altered the manifestation of the learning
deficits, however, this seems unlikely given that adding two injections per day to animals
already receiving 4, would not in any obvious way appear to be a major additional factor.
185
Based on our prior experience with neonatal MDMA and many other drugs given developmentally, is that we have observed that the magnitude of such effect varies some over time. The basis for such fluctuations or ‘noise’ in bioassays remains unknown, but that they occur is certain. We have since completed another experiment with neonatal
MDMA and found learning deficits more similar to earlier experiments, suggesting that the present experiment is more likely than not, a slight deviation from the norm.
In conclusion, CIT-attenuated 5-HT depletions induced by MDMA does not block the effects of MDMA on allocentric or egocentric learning or on locomotor behavior.
CIT treatment alone produced significant path integration deficits in the CWM while spatial learning in the MWM was unaffected. Even though 5-HT depletions do not appear to be directly influencing learning in the CWM or MWM, it is still likely that other alterations involving the 5-HT system following MDMA or CIT may contribute to the cognitive deficits observed. Further investigation into the effects of MDMA and 5-
HT, including 5-HT receptors on later cognitive function are warranted to try to disentangle how MDMA causes these persistent effects.
Acknowledgments
Supported by National Institutes of Health grants DA014269 (MTW) and
DA006733 (CVV), DA007427 (GAG) and training grant ES007051 (TLS and MRS).
186
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Table 1. 5-HIAA, 5-HIAA/5-HT ratio, DA, DOPAC, DOPAC/DA ratio, NGF, BDNF, and CORT on P12, 16, and 21 P12 P12 P12 P12 SAL+SAL SAL+MDMA 2x5 CIT+SAL 2x5 CIT+MDMA 5-HIAA (ng/mg tissue) Hippocampus 0.20 ± 0.01 0.10 ± 0.01 * 0.13 ± .02 * 0.09 ± 0.014 * Entorhinal cortex 0.08 ± 0.01 0.05 ± 0.01 * 0.04 ± 0.01 * 0.03 ± 0.01 * Neostriatum 0.29 ± 0.02 0.14 ± 0.02 * 0.17 ± 0.02 * 0.17 ± 0.02 * 5-HIAA/5-HT Hippocampus 0.96 ± 0.09 1.5 ± 0.09 * 0.87 ± 0.09 # 0.64 ± 0.09 * # Entorhinal cortex 0.61 ± 0.07 0.91 ± 0.07 * 0.48 ± 0.07 # 0.44 ± 0.07 # Neostriatum 2.15 ± 0.51 4.34 ± 0.51 * 2.28 ± 0.51 # 1.76 ± 0.51 # DA (ng/mg tissue) Striatum 4.03 ± 0.28 4.73 ± 0.28 3.92 ± 0.28 # 4.52 ± 0.28 DOPAC (ng/mg tissue) Striatum 0.77 ± 0.05 0.59 ± 0.05 * 0.77 ± 0.05 # 0.57 ± 0.05 * DOPAC/DA Striatum 0.19 ± 0.01 0.13 ± 0.01 * 0.20 ± 0.01 # 0.13 ± 0.01 * NGF (pg/mg protein) Hippocampus 89 ± 20 79 ± 20 72 ± 20 100 ± 20 Striatum 16 ± 4 18 ± 4 16 ± 4 22 ± 4 BDNF (pg/mg protein) Hippocampus 45 ± 2 41 ± 2 47 ± 2 43 ± 2 Striatum 30 ± 10 37 ± 10 35 ± 10 37 ± 10 CORT (ng/ml) 7.04 ± 0.82 8.13 ± 0.82 6.79 ± 0.82 6.32 ± 0.82 P16 P16 P16 P16 SAL+SAL SAL+MDMA 2x5 CIT+SAL 2x5 CIT+MDMA 5-HIAA (ng/mg tissue) Hippocampus 0.19 ± 0.02 0.09 ± 0.02 * 0.23 ± 0.02 # 0.14 ± 0.02 Entorhinal cortex 0.23 ± 0.03 0.13 ± 0.03 * 0.22 ± 0.03 0.17 ± 0.03 * # Neostriatum 0.5 ± 0.04 0.27 ± 0.04 * 0.54 ± 0.04 # 0.44 ± 0.04 # 5-HIAA/5-HT Hippocampus 0.94 ± 0.1 1.3 ± 0.1 * 1.26 ± 0.1 * 1.09 ± 0.1 Entorhinal cortex 1.04 ± 0.23 1.96 ± 0.23 * 1.02 ± 0.23 # 1.02 ± 0.23 # Neostriatum 2.21 ± 0.83 3.49 ± 0.83 2.21 ± 0.83 1.51 ± 0.83 DA (ng/mg tissue) Striatum 6.36 ± 0.48 5.65 ± 0.48 5.54 ± 0.48 5.43 ± 0.48 DOPAC (ng/mg tissue) Striatum 1.25 ± 0.1 1.16 ± 0.1 0.99 ± 0.1 1.04 ± 0.1 DOPAC/DA Striatum 0.20 ± 0.01 0.20 ± 0.01 0.19 ± 0.01 0.19 ± 0.01 NGF (pg/mg protein)
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Hippocampus 160 ± 28 180 ± 28 220 ± 28 240 ± 28 * # Striatum 16 ± 5 17 ± 5 24 ± 5 22 ± 5 BDNF (pg/mg protein) Hippocampus 48 ± 3 53 ± 3 48 ± 3 47 ± 3 Striatum 20 ± 8 24 ± 8 25 ± 8 29 ± 8 CORT (ng/ml) 18.35 ± 3.27 26.77 ± 3.27 18.27 ± 3.46 17.27 ± 3.27 P21 P21 P21 P21 SAL+SAL SAL+MDMA 2x5 CIT+SAL 2x5 CIT+MDMA 5-HIAA (ng/mg tissue) Hippocampus 0.43 ± 0.04 0.29 ± 0.04 * 0.54 ± 0.04 * 0.41 ± 0.04 Entorhinal cortex 0.23 ± 0.02 0.13 ± 0.022 * 0.25 ± 0.02 * 0.20 ± 0.02 * Neostriatum 0.5 ± 0.06 0.44 ± 0.06 0.79 ± 0.06 * # 0.66 ± 0.06 # 5-HIAA/5-HT Hippocampus 1.21 ± 0.09 1.31 ± 0.09 1.59 ± 0.09 * 1.27 ± 0.09 Entorhinal cortex 0.61 ± 0.11 1.03 ± 0.12 * 0.91 ± 0.12 0.85 ± 0.11 Neostriatum 2.40 ± 0.27 2.36 ± 0.27 2.77 ± 0.27 1.98 ± 0.27 DA (ng/mg tissue) Striatum 5.36 ± 0.43 5.64 ± 0.43 5.7 ± 0.43 4.97 ± 0.43 DOPAC (ng/mg tissue) Striatum 1.13 ± 0.12 1.71 ± 0.12 * 1.19 ± 0.12 # 1.55 ± 0.12 * DOPAC/DA Striatum 0.22 ± 0.02 0.31 ± 0.02 * 0.21 ± 0.02 # 0.31 ± 0.02 * NGF (pg/mg protein) Hippocampus 130 ± 32 130 ± 32 190 ± 32 160 ± 32 Striatum 24 ± 9 25 ± 9 26 ± 9 25 ± 9 BDNF (pg/mg protein) Hippocampus 51 ± 13 68 ± 13 50 ± 13 63 ± 13 Striatum 50 ± 14 37 ± 9 29 ± 9 51 ± 8 CORT (ng/ml) 15.15 ± 7.28 60.22 ± 7.28 * 11.4 ± 7.28 # 43.38 ± 7.28 *
*different from SAL+SAL; #different from SAL+MDMA (p ≤ 0.5)
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Table 2. Light-Dark test, Elevated zero maze, and Straight channel SAL+SAL SAL+MDMA 2x5 CIT+SAL 2x5 CIT+MDMA
L/D test: Dark entries 25.7 ± 3.49 26.8 ± 3.49 32.5 ± 3.4 @ 33.5 ± 3.49 @ Time in light (s) 221.0 ± 24.11 170.7 ± 24.11 † 184.0 ± 23.6 218.7 ± 24.11 Latency to dark (s) 69.6 ± 16.26 28.6 ± 16.26 † 31.5 ± 16.87 † 67.6 ± 16.26 A EZM: Open entries 7.1 ± 0.83 10.3 ± 0.85 $ 8.8 ± 0.85 9.3 ± 0.83 $ Time in open (s) 79.0 ± 9.98 80.2 ± 10.19 86.1 ± 10.19 89.3 ± 9.98 Latency to open (s) 16.8 ± 3.31 15.4 ± 3.31 8.9 ± 3.4 16.8 ± 3.23 Head dips 8.0 ± 1.01 8.4 ± 1.04 7.9 ± 1.04 8.8 ± 1.01 Straight Channel: 9.68 ± 0.6 9.63 ± 0.6 11.0 ± 0.6 10.3 ± 0.6 latency of last trial (s)
$main effect (p ≤ 0.05) of Drug; @main effect of Pretreatment; † trend to be different from SAL+SAL (p < 0.1); A trend to be different from SAL+MDMA (p < 0.1)
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Figures
0.5
# # # 0.4 # # # #
0.3 * * *
0.2 *
0.1 Hippocampal 5-HT (ng/mg tissue) Hippocampal 5-HT
0.0 L A L L L A A A A M AL A A A A M M M S D S +S +S +S DM D D D L+ M IT+ IT IT IT M +M +M +M SA L+ C C C C T+ IT IT IT SA 5 x5 10 10 CI C C C 1X 2 1x 2x 5 x5 10 10 1X 2 1x 2x
Treatment
Figure 1. Hippocampal 5-HT: P11-20 expsosure. *p < 0.05 different from SAL+SAL;
#p < 0.05 different from SAL+MDMA
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A P12 B P16 C P21 0.5 SAL+SAL 0.4 SAL+MDMA # CIT+SAL # CIT+MDMA 0.3 * # # # 0.2 # * * * 0.1 * *
Hippocampal 5-HT (ng/mg tissue) (ng/mg 5-HT Hippocampal 0.0
D EF 0.5
0.4 # # # 0.3 * * # 0.2 * *
0.1 ***
0.0 Entorhinal Cortex 5-HT (ng/mg tissue)
G 0.5 HI #
0.4 * # # 0.3
0.2 # * 0.1 * * Neostriatal 5-HT (ng/mg tissue) (ng/mg 5-HT Neostriatal 0.0
Figure 2. P12, 16, and P21 5-HT levels: P11 killed on P12 (left column), P11-15 killed on P16 (center column), P11-20 killed P21 (right column) exposure. *p < 0.05 different from SAL+SAL; #p < 0.05 different from SAL+MDMA
203
60
55
SAL+SAL SAL+MDMA 50 CIT+SAL CIT+MDMA
45 *
40 * Body weights (g) weights Body * * 35 * * * # 30 *
25 12 14 16 18 20
Age (days)
Figure 3. Body weights during dosing: #p < 0.05 SAL+MDMA and CIT+MDMA vs.
SAL+SAL; *p < 0.05 SAL+MDMA, CIT+MDMA, and CIT+SAL vs. SAL+SAL
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A B C 1000 1000 1000 * SAL+SAL SAL+SAL SAL+MDMA 800 800 800 SAL+MDMA CIT+SAL CIT+MDMA * 600 600 600
400 400 400 †
Total Distance (cm) 200 * 200 * * 200
0 0 0 0123456789101112 0123456789101112 0123456789101112 Day Day Day
Figure 4. Total distance: *p < 0.05; †p < 0.1
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A D G 350 350 350
300 SAL+SAL 300 SAL+SAL 300 SAL+MDMA SAL+MDMA CIT+SAL CIT+MDMA 250 250 250
200 200 200
150 † 150 150
Latency (s) † 100 * *†† 100 100 * * 50 50 50
0 0 0 0 2 4 6 8 1012141618 0 2 4 6 8 1012141618 0 2 4 6 8 10 12 14 16 18
B E H 100 100 100
80 80 80
60 60 60
40 40 40 Errors ††† †† † 20 * * ** 20 * * 20
0 0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
C F I 8 8 8
6 6 6
4 4 4
2 † 2 † † 2 Start returns * † ** * †
0 0 0
024681012141618 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 Day Day Day
Figure 5. Cincinnati water maze: Latency (top row), Errors (middle row), Start returns
(bottom row). (A, D, G: SAL+MDMA vs. SAL+SAL; B, E, H: 2x5 CIT+SAL vs. SAL+
SAL; C, F, I: SAL+MDMA vs. 2x5 CIT+MDMA), Errors (D, E, F). *p < 0.05; †p < 0.1
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D G A MWM Acquisition MWM Acquisition MWM Acquisition 20 20 20
SAL+SAL SAL+SAL SAL+MDMA 15 SAL+MDMA 15 CIT+SAL 15 CIT+MDMA
10 * 10 10 * † *
Path length (m) length Path 5 5 5
0 0 0 0123456 0123456 0123456
B E H 20 MWM Reversal 20 MWM Reversal 20 MWM Reversal † *
15 15 15 *
10 * † † 10 10 † *
Path length (m) Path length 5 5 5
0 0 0 0123456 0123456 0123456
C FI 16 MWM Shift 16 MWM Shift 16 † MWM Shift
14 14 14
12 12 12 * 10 † 10 10 8 * 8 8 6 * 6 6
Path length (m)Path 4 4 4
2 2 2
0 0 0 0123456 0123456 0123456 Day Day Day
Figure 6. Morris water maze: Path lengths for acquision (top row), reversal (middle row), shift (bottom row). (A, D, G: SAL+MDMA vs. SAL+SAL; B, E, H: 2x5 CIT+SAL vs. SAL+SAL; C, F, I: SAL+MDMA vs. 2x5 CIT+MDMA), Errors (D, E, F). *p < 0.05;
†p < 0.1
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CHAPTER 6: DISCUSSION
Conclusions
The results of the present studies suggest that disruption of the developing serotonergic system at critical time-points may lead to or be permissive of cognitive deficits. In chapter 2, we showed that genetic depletion of Pet-1 and an ~80% 5-HT reduction did not result in spatial deficits in the Morris water maze (MWM) or path integration deficits in the Cincinnati water maze (CWM). These findings, although surprising, may suggest that compensatory mechanisms exist that prevented cognitive dysfunction. Pet-1 knockout mice did exhibit altered behavior reminiscent of 5-HT disruption, including decreased locomotor activity and increased marble burying and startle reactivity. It is possible that because Pet-1 is missing from such an early period in brain development that other neurotransmitter systems were altered. Although the use of the constitutive Pet-1 knockout did not prove to be a useful tool for determining the importance of developmental 5-HT on learning and memory, it has become more apparent that critical periods of 5-HT development exist in which the serotonergic system is vulnerable and damage can lead to cognitive deficits. In this regard, it may be that cognitive deficits are more pronounced when the serotonergic system begins to develop normally and is then disrupted as would occur when MDMA is administered later in life.
We previously determined that postnatal day (P) 11-20 substituted amphetamine exposure in rats results in learning and memory deficits. This period in a rat is comparable to the second half of in utero human brain development (Bayer et al.
1993;Clancy et al. 2001;Clancy et al. 2007;Rice and Barone S Jr 2000). In chapter 3, we determined that neonatal administration of drugs that preferentially target the
208
serotonergic, as opposed to the dopaminergic, systems in adults were more likely to produce learning deficits in the MWM (Schaefer et al. 2006). We also showed that with a greater degree of 5-HT depletion during development a more severe learning deficit was observed. For example, fenfluramine produced the greatest decrease in developmental 5-HT content and a larger deficit in the MWM, and this was followed by
MDMA and then methamphetamine (Schaefer et al. 2006). Of the substituted amphetamines studied, MDMA is known to be abused during pregnancy (Ho et al. 2001), induces cognitive deficits when administered during a specific developmental period
(Skelton et al. 2009;Vorhees et al. 2007;Williams et al. 2003), and produces significant depletions in 5-HT and increases in CORT after a single day of neonatal exposure
(Williams et al. 2005). Therefore, we used a neonatal rat model for human second half of gestation brain development to further study the impact of 5-HT depletion following
MDMA and the role in which alterations in the developing serotonergic system affect cognition in adulthood. In chapter 4, we found that MDMA produced approximately a
50% decrease in 5-HT content in the hippocampus, neostriatum, and entorhinal cortex
(chapter 5) after exposure throughout the P11-20 exposure period (Schaefer et al.
2006;Schaefer et al. 2008). The brain regions examined receive a high degree of serotonergic innervation that develops during the first 3 weeks in the rat and are known to be important in spatial and path integration learning. In chapter 5, attenuation of these 5-
HT depletions was observed when pups were pretreated with citalopram (CIT), an SSRI that is highly selective for SERT. Although the combination of CIT and MDMA did not produce the expected improvements in cognitive ability, the finding that CIT alone was sufficient to produce at least modest path integration learning deficits suggests that
209
alterations to the 5-HT system, with or without 5-HT depletions, can affect learning and memory.
In chapter 3, it was suggested that drugs that affect both hippocampal 5-HT and
CORT (FEN and MDMA), as opposed to those that only affect CORT (MA and MPH) or that affect neither (COC), were more likely to produce CWM learning deficits following
P11-20 exposure. In Chapter 4, we showed that following additional days of dosing
(P11-15 or 11-20), MA treatment decreased 5-HT and therefore did not support our original hypothesis. However, the CWM data available at the time was run under red light conditions rather than under infrared; This left open the possibility that animals could use some kind of spatial cues and animals treated with MA were not affected.
However, we have since changed the procedure, which might explain why the CWM was not significantly affected in those original experiments. The experiment reported here was run under preventing the use of spatial cues. Under infrared lighting, the use of spatial cues in prevented and rats treated neonatally with MA did exhibit path integration
learning deficits (Vorhees et al. 2009) (See Table 1 for summary of findings). To better
determine the validity of our original hypothesis, the experiment described in Chapter 3
could be repeated using the 10 day dosing regimen and the CWM run under infrared lighting conditions.
Critical period of 5-HT development
It has been previously suggested that there are critical periods in which alterations
in serotonergic functioning can result in biochemical and behavioral alterations. The
current studies suggest that the developmental period in which 5-HT disruption begins or
ends is important for the cognitive functioning of the animal. For example, the depletion
210
of 5-HT in Pet-1 knockout mice began prior to birth and was maintained through the
same postnatal period in which MDMA induces 5-HT depletions (Hendricks et al. 2003),
however, only when 5-HT depletions began on P11 and ended prior to P30 did animals exhibit learning and memory deficits (Broening et al. 2001;Morford et al. 2002;Schaefer
et al. 2006;Skelton et al. 2009;Vorhees et al. 2004;Vorhees et al. 2007;Williams et al.
2003). In addition, others have also shown spatial learning deficits following P10-20
PCPA-induced tryptophan hydroxylase inhibition (Mazer et al. 1997). This P10-20 neonatal period appears to be vulnerable to serotonergic disruption if normal development of the 5-HT system occurred prior to this point. Further it appears that this period is more sensitive than disruption that starts earlier since MDMA administration beginning at an earlier time point, P1 and continuing until P10, or 5, 7-DHT administration on P0 (the day of birth) or on P3 did not produce learning and memory deficits (Broening et al. 2001;Hohmann et al. 2007;Ueda et al. 2005). It is likely that specific developmental events that later influence learning ability, such as synaptogenesis or neuronal pruning, are occurring during this later period and can be disrupted by alterations in 5-HT functioning.
An earlier window of 5-HT vulnerability may also exist since it was reported that
PCPA treatment from embryonic day (E) 14-17 resulted in significant spatial deficits in the MWM (Vataeva et al. 2007). Even though these PCPA-treated animals and Pet-1 knockout mice experienced 5-HT depletion beginning during a similar developmental period (5-HT appears around day E12 in the rodent), 5-HT levels returned to normal after
PCPA administration was stopped whereas in Pet-1 knockout mice, 5-HT levels were constantly low. This suggests that if compensatory mechanisms exist they may be
211
activated if very early 5-HT development is impeded and maintained at insufficient levels
of functioning. We cannot, however, fail to consider that the learning deficits observed following MDMA or PCPA administration may be the result of the effects of these drugs on other neurotransmitter systems, the hypothalamic-pituitary-adrenal axis, or other factors.
Alternative 5-HT hypothesis
We originally hypothesized that 5-HT depletions during development were important for adult learning deficits in the case of genetic manipulation and drug
exposure; however, we did not see deficits in Pet-1 knockout mice or a rectification of cognitive deficits following an attenuation of 5-HT depletions induced by P11-20 CIT and MDMA exposure. The lack of a complete attenuation of 5-HT depletions observed in the CIT and MDMA-exposed animals may account for the lack of cognitive dysfunction attenuation, however, it is also possible that increases in synaptic 5-HT levels during this time can lead to cognitive deficits. In chapter 2, we suspected that the greater
5-HT depletion produced by fenfluramine, followed in severity by MDMA, and then by methamphetamine, influenced the degree of MWM deficits; however, a similar rank order pattern of 5-HT release also occurs following these drugs. For example, fenfluramine releases 5-HT more potently than MDMA, and MDMA is a more potent 5-
HT releaser than methamphetamine (Baumann et al. 2007). MDMA also inhibits SERT
from taking up 5-HT from the extracellular space and inhibits monoamine oxidase to block the metabolism of 5-HT, therefore extending the time and concentration of 5-HT in the synaptic cleft (Baumann et al. 2007;Leonardi and Azmitia 1994). The concept of a
212
protracted decrease in 5-HT tissue content with an increase in circulating 5-HT levels, as suspected in the case of neonatal MDMA exposure, may seem counterintuitive; however this has been described previously in adult mice that lack SERT (Fabre et al. 2000).
Increased synaptic 5-HT levels could also account for the learning deficits observed in Chapter 5 following MDMA alone, CIT+MDMA, and CIT alone (Fig 1). In adults, MDMA has been shown to be a potent 5-HT releaser and reuptake inhibitor
(Green et al. 2003) and CIT has also been shown to release and prevent 5-HT reuptake
(Ceglia et al. 2004). Following MDMA binding to SERT and entering the cell, it has been shown that intracellular 5-HT is transported out of the cell by reversal of the normal inward-directed SERT uptake system. This effect, along with inhibition of VMAT2, results in large increases in the amount of 5-HT in the synaptic cleft. MDMA also causes a subsequent decrease in 5-HT because it has the additional effect of inhibiting TPH (Fig.
1B). Although CIT does not inhibit TPH or cause a decrease in intracellular 5-HT it is similar to MDMA in that the net result of both drugs is to trap increased amounts of 5-HT in the synaptic cleft where it has prolonged exposure to both pre- and postsynaptic 5-HT receptors (Fig 1C). In the case of CIT+MDMA, although CIT binds to SERT and prevents 5-HT reuptake, this effect is undoubtedly only partial (depending on the affinity of MDMA vs. CIT for the SERT binding site), leaving some MDMA available to bind to
SERT. When MDMA binds to SERT it enters the cell, inhibits VMAT2 and causes intracellular 5-HT to flow out into the synapse and whatever portion of MDMA that enters the cell even in the presence of CIT, will cause at least a partial inhibition TPH
(Fig. 1D) and result in some 5-HT reduction, which is exactly what we saw in the CIT-
MDMA group. This may be why we were unable to get a complete blockade of MDMA-
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induced 5-HT reductions after pretreatment with CIT. Therefore, even though we were
able to attenuate 5-HT depletion using the CIT+MDMA combination, we were not able
to prevent the increased release and hence increased exposure to 5-HT receptors. This
may explain why CIT alone as well as CIT-MDMA both had effects on later adult learning and memory in the same direction. Increased extracellular 5-HT during
development may result in aberrant activity of both pre and postsynaptic 5-HT receptors.
In the case of the Pet-1-/- mice (discussed in Chapter 2), the question is why many
behaviors were normal in these animals. It may be that the 20% remaining 5-HT
containing neurons compensated for the 80% reduction by releasing larger amounts of transmitter per release event than in WT mice thereby maintaining sufficient extracellular
5-HT levels. Or perhaps the remaining 20% of 5-HT containing cells increased their 5-
HT release some and in addition to this there was an upregulation of the number of 5-HT receptors. If this is correct, developmental 5-HT alterations may produce different outcomes depending on the nature of the 5-HT change. Low levels of 5-HT from the inception of 5-HT expression may lead to the gradual developing of some release- receptor adaptations in cortical regions, although apparently such adaptations are not universal since the Pet-1-/- had abnormalities of non-cognitive function; whereas increases in 5-HT during critical periods may be deleterious not by 5-HT depletion as we
originally hypothesized but rather by sustained release causing overstimulation of 5-HT
receptors. If correct, blocking 5-HT receptors may be one way to test this revised
interpretation of the findings.
Future Studies
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The results of these studies bring to light many questions that need future study.
The development of an inducible Pet-1 knockout may help determine if the lack of cognitive deficits observed in Chapter 2 were the result of compensatory mechanisms and/or if there are critical periods for Pet-1 expression and 5-HT functioning that when perturbed at different ages result in cognitive deficits. We could also employ the use of
TPH1/2 double knockouts which are lacking nearly 100% of the 5-HT content (Savelieva et al. 2008) to test the idea that the remaining 20% of 5-HT found in Pet-1 knock out mice was sufficient for brain development and learning and memory. We could also use in vivo microdialysis to determine the level of extracellular 5-HT in the brains of Pet-1 knockout mice to see if the release of 5-HT is different from wild type controls.
In order to further examine the mechanism by which P11-20 MDMA administration leads to cognitive deficits, the 5-HT release and receptor changes both during the developmental exposure period and in adulthood should be determined.
Although we have shown that the 5-HT depletion alone cannot fully account for learning deficits, we still cannot rule out the role of other changes to the 5-HT system, such as increased synaptic 5-HT levels, as potential mechanisms for the cognitive deficits. In vivo microdialysis during the neonatal MDMA exposure period would allow us to determine the extent that MDMA is producing 5-HT efflux in various brain regions.
Fenfluramine affects the 5-HT system more specifically by producing greater 5-HT depletion than MDMA. By comparing both MDMA and FEN in the same behavioral study, we could determine if the degree of 5-HT depletion during development could influence the severity of learning deficits. It should be noted that the use of transgenic mice would greatly facilitate our understanding of the role that developmental 5-HT plays
215
in neonatal MDMA induced cognitive impairment, however mice are differently affected
compared to rats in terms of MDMA pharmacology. For example, mice primarily
experience changes to the dopaminergic system compared to rats that show major
changes to the serotonergic system (Green et al. 2003). With this in mind, and the lack of
readily available transgenic rats, analysis of various 5-HT receptors would require the use
of pharmacological manipulation.
Since we have previously shown developmental MDMA exposure to produce an
increase in hippocampal 5-HT1A sensitivity in adults in vitro (Crawford et al. 2006), and
others have shown 5-HT1A to be present during development and suggested altered
learning as a result of non-MDMA induced 5-HT1A activation in adults (King et al.
2008), we should first examine the 5-HT1A receptors for potential alterations leading to
MDMA-induced learning deficits.
Serotonin1A presynaptic autoreceptors are densely populated in the dorsal and
median raphe nuclei, the principal source of 5-HT transmission in the brain, during
development and throughout adulthood. Activation of presynaptic 5-HT1A autoreceptors
in the dorsal and median raphe nuclei during development results in decreased
serotonergic cell firing in these neurons via down regulation of adenylyl cyclase activity
(Barnes and Sharp 1999). A decrease in serotonergic cell firing in the raphe nuclei can
lead to a decrease in 5-HT release in target regions such as the hippocampus and
entorhinal cortex which receive input from both the dorsal and median raphe nuclei (Patel
and Zhou 2005). The hippocampus and entorhinal cortex are thought to be important for
MWM and CWM learning are densely populated with 5-HT1A postsynaptic receptors in
adult animals (Barnes and Sharp 1999) and during early development nearly all
216
differentiated neurons in the hippocampus express 5-HT1A. During the early postnatal period the receptors are clustered in serotonergic target regions near the cell bodies, and as the neuron matures 5-HT1A receptor numbers are reduced and are found along the dendrites (Patel and Zhou 2005). It is possible that neonatal MDMA exposure alters serotonergic firing to an extent that 5-HT1A sensitivity is altered in the hippocampus and other brain regions.
To verify that neonatal MDMA produces an increase in 5-HT1A sensitivity in vivo we could use 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), a 5-HT1A agonist.
In rodents, 8-OH-DPAT induces hypothermia in such a way that the degree of hypothermia is directly related to the functional status of 5-HT1A (Alexandre et al.
2006;Bouali et al. 2003;El et al. 2003;Goodwin et al. 1985;Hohmann et al. 2007;Olvera-
Cortes et al. 2001). For example, if MDMA increased 5-HT1A sensitivity we would expect these animals to experience a greater drop in core body temperature compared to saline control rats following 8-OH-DPAT administration. In fact, one week after adult
MDMA treatment, increased 5-HT1A receptor binding and a potentiation of the hypothermic response induced by 8-OH-DPAT compared to SAL controls was observed
(Aguirre et al. 1997). 8-OH-DPAT-induced hypothermia has been observed in neonatal rats as young as P22 and therefore we would be able to assess the function of 5-HT1A receptors over a long period of time following neonatal MDMA exposure (Johansson-
Wallsten and Meyerson 1994). Being able to study the receptor system over time is important because in Chapter 3 we showed that P11-20 MDMA-induced 5-HT depletions recover by P30 while others have shown 5-HT depletions return at older ages (Crawford et al. 2006) suggesting that changes in receptor function may also change over time.
217
Importantly, increased sensitivity or activation of 5-HT1A receptors has been linked to a decrease in learning ability. Postsynaptic 5-HT1A stimulation with high doses
of 8-OH-DPAT impairs spatial learning in the MWM in adult rats (Luttgen et al. 2005).
Interestingly, 5-HT1A knockout mice or mice over-expressing 5-HT1A transiently during
the perinatal period show poorer learning in the MWM suggesting that there is a critical
level of developmental 5-HT1A functioning required for appropriate cognition (Bert et al.
2005). An increase in 5-HT1A expression is also thought to be related to impaired
cognition in schizophrenic patients; although it is unknown if this is the result of the underlying pathology or if 5-HT1A alterations are directly linked to altered behavior
(Sumiyoshi and Meltzer 2004). It has also recently been shown that 5-HT1A expression is
altered in various brain regions after training in an auto-shaping response task suggesting
that 5-HT1A receptors may play a role in memory formation (King et al. 2008;Luna-
Munguia et al. 2005). For example, 5-HT1A expression was decreased in the hippocampus and cingulate cortex, and increased in the amygdala, caudate-putamen, and the median and dorsal raphe nuclei following training compared to non-trained controls
(Luna-Munguia et al. 2005). Importantly, the 5-HT1A receptor is known to have
inhibitory effects on other neurotransmitter systems (glutamate and acetylcholine) known
to be important in learning and memory (King et al. 2008). Taken together, these data
suggest that the 5-HT1A receptor is an appropriate system to further investigate the
mechanisms for MDMA-induced learning and memory deficits.
At this point, it is difficult to determine if neonatal MDMA exposure from P11-20
is producing an increase or a decrease in 5-HT1A activation and if this change is
consistent in different brain regions. Therefore, it is unknown if a 5-HT1A agonist as
218
opposed to an antagonist would lessen the effects of MDMA on 5-HT1A if administered during this critical period in combination with P11-20 MDMA. However, if we assume that the increase in 5-HT1A sensitivity in the hippocampus during the time of cognitive
assessment is playing a major role in P11-20 MDMA-induced learning and memory
deficits, as suggested in the previous paragraph (disregarding, for the time being, how
this increased sensitivity developed), we could attempt to rescue this phenotype using a
5-HT1A antagonist. Chronic administration of the 5-HT1A antagonist 1-2(-
methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazin (NAN-190), which has been shown
to have high affinity for 5-HT1A receptors in the hippocampus (Glennon et al. 1988), would lessen the effect of endogenous 5-HT during learning and potentially restore cognitive ability.
In conclusion, alterations to the developing serotonergic system are likely to result in a variety of long-term changes throughout the brain either when induced by genetic disruption or drug exposure. Determination of specific critical periods and 5-HT disruptions that result in cognitive decline would aid in the understanding of various disease etiologies and even potential interventions and treatments.
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Table 1. Summary of hippocampal 5-HT, CORT, and CWM learning
FEN MDMA MA MPH COC 5-HT P12 ↑ ↓ - - - 5-HT P16 ? ↓ ↓ ? ? 5-HT P21 ? ↓ ↓ ? ? CORT P12 ↑ ↑ ↑ ↑ - CORT P16 ? - ↑ ? ? CORT P21 ? ↑ - ? ? CWM learning w/ ↓ ↓ - - - red light
CWM learning w/ ? ↓ ↓ ? ? infrared light
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Normal
A
MDMA CIT CIT+MDMA
MDMA
5-HT
CIT
BCD
http://www.humanillnesses.com
Figure 1. Schematic of 5-HT neuron following normal conditions (A), MDMA (B), CIT (C), and CIT+MDMA (D) demonstrating increased synaptic 5-HT levels. Original schematic provided by: www.humanillnesses.com
228