UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

Interaction of Brain Derived Neurotrophic Factor and the HPA Axis Stress Response System with Neonatal d-Methamphetamine Induced Spatial Learning and Memory 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 of the University of Cincinnati

2005

by

Carrie A. Brown-Strittholt

B.A., A.A., Thomas More College, 1999

Committee Chair: Charles V. Vorhees, Ph.D.

Committee Members: Floyd R. Sallee, MD, Ph.D. James P. Herman, Ph.D. Michael T. Williams, Ph.D. Renu Sah, Ph.D.

This research was supported by NIH grant DA06733 (CVV), and training grant ES07051 (CAS). ABSTRACT

Neonatal methamphetamine (MA) exposure P11-20 or P11-15 in rats is known to produce long-term spatial learning and memory deficits. However, little is known concerning the mechanism by which this results. Data from experiments exploring behavior indicate a role for neurotrophins and corticosterone (CORT) in learning and memory processes. It is hypothesized that neonatal MA alters levels of neurotrophins and/or alters the stress/CORT response resulting in impaired spatial learning and memory deficits in the Morris water maze (MWM). The current set of experiments explored first the levels of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) during the neonatal dosing period and in adulthood. Treatment effects were observed for

BDNF P15. Sex differences in hippocampal and hypothalamic protein content for

BDNF were found at P11; higher levels occurring in male pups. Sex differences in hippocampal protein content for NGF were found at P15 and sex differences in the hypothalamus were found at P68; higher levels in females at both ages.

Behavioral tests in the MWM mimicked previous results demonstrating a clear

MA-induced spatial learning deficit. Anxiety testing demonstrated more exploratory behavior in female animals. Secondly, pharmacological and surgical methods of corticosterone regulation were explored. Metyrapone (MET) injection and bilateral adrenalectomy (ADX) approaches were successful in the adult animal. Swimming ability was impaired only in ADX animals without CORT replacement. Zero maze data indicated increased activity among animals treated neonatally with MA 20 mg/kg. Trends were apparent among MWM data

ii indicating a slight correction of the MA-induced learning and memory deficit when CORT levels were controlled; however, the MA effect was not clear among control animals to enable a definitive comparison. Overall results suggest a role for neurotrophins and CORT in the mechanism of MA’s effects on the developing brain but neither appears to be the sole source of insult.

iii ACKNOWLEDGEMENTS

I would like to acknowledge my advisor, Dr. Charles Vorhees, for his guidance and for giving me the opportunity to do this research.

I would like to acknowledge the members of my dissertation committee for their help and support throughout my candidacy.

I would like to acknowledge the members of the Vorhees’ lab, past and present, for their help, with special recognition to Ms. Mary Moran and Dr. Martha Cohen for their immeasurable help learning and interpreting statistical procedures.

I would like to acknowledge members of the Molecular and Developmental Biology Graduate Program for their support during my graduate career. I would like to extend a special thank you to the MDB secretarial staff for keeping me up to date despite my many moves within the division, the tri-state area, and even the nation.

I would like to thank the members of the Division of Infectious Diseases, where I began my graduate studies, for their continued support and friendship throughout my graduate career. I would like to thank Dr. Lawrence Stanberry for accepting me as a graduate student in the division. Also, a very special thank you to Dr. Richard Pyles; he made my education a priority in his work and remains one of the most influential teachers in my educational career as well as a friend.

I would like to acknowledge Gerald Franzen, Ph.D. for his contributions of chemical structures to this dissertation.

I would like to acknowledge my family and friends, especially my husband and parents, for their support and encouragement in all areas of my life.

I would like to dedicate this dissertation to my grandparents, Thomas and Elizabeth O’Daniel. From as early in my life as I can remember they have been pillars of support and taught me that after the presence of God and family in one’s life, that education and hard work are essential tools to success. In a growing family of 58 people that still gather for holidays and family events, this doctorate is the first and is dedicated to them with gratitude and with love.

iv TABLE OF CONTENTS Chapter 1 – Methamphetamine, Neurotrophic Factors & Stress …. p.008-092 Methamphetamine………………….……………………………… p.008-026 Chemical structures and function …………….…………… p.009-013 History and prevalence of use ………….……………….… p.014-018 Developmental processes and vulnerability to injury ….…. p.018-021 Research review ……………………………….………….. p.025-043 Human studies ……………………………………. p.025-028 Animal studies ……………………………………. p.028-043 Dose comparisons across species ………………………… p.044 Model system ……………………………………………... p.045 Neurotrophic Factors ……………………………………………… p.046-064 Nerve Growth Factor …………………………………… p.050-055 Brain Derived Neurotrophic Factor ……………………….. p.055-059 Neurotrophin-3 ……………………………………………. p.059-061 Neurotrophin Receptors …………………………………… p.061-064 Stress ………………………………………………………………. p.065-083 Hypothalamic-Pituitary-Adrenal Axis …………………….. p.069-078 Development – Stress Hyporesponsive Period ……………. p.078-083 Hypotheses and aims ……………………………………………… p.084-087

CHAPTER 2 – BDNF and NGF Protein Levels in the Brains of Rats Neonatally Treated with Methamphetamine: Implications for Spatial Learning and Memory Deficits. …………………………………………………………… p.088-117 Abstract …………………………………………………………… p.088-089 Introduction ……………………………………………………….. p.089-092 Materials and methods ……………………………………………. p.093-100 Results …………………………………………………………….. p.100-104 Discussion ………………………………………………………… p.104-110

CHAPTER 3 – MA-Induced Increased Levels of CORT During the SHRP and Neonatal MA-Induced Long Term Changes in Basal CORT in the Adult: Implications for Spatial Learning and Memory Deficits..………... p.118-193 Abstract …………………………………………………………… p.118-119 Introduction ………………………………………………………. p.119-130 Materials and methods ……………………………………………. p.132-149 Results and discussion …………………………………………….. p.149-187 Summary discussion and conclusions……………………………… p.188-193

CHAPTER 4 – Summary ……..……………………….………... p.194-203 Conclusions ………………………………………………………. p.194-199 Future Directions …………………………………………………. p.199-203

1 INDEX OF FIGURES

Chapter 1 – Methamphetamine, Neurotrophic Factors & Stress …. p.008-083 Figure 1.1: Chemical structures …………………………………. p.013 Figure 1.2: Neurotransmitter receptor subtype development….…. p.022 Figure 1.3: Brain region development – human/rat………………. p.023 Figure 1.4: Brain growth spurt – human/rat…………………..…. p.024

CHAPTER 2 – BDNF and NGF Protein Levels in the Brains of Rats Neonatally Treated with Methamphetamine: Implications for Spatial Learning and Memory Deficits. …………………………………………………………… p.088-117 Figure 2.1: MWM Annuli and direct swim diagrams ………..…. p.111 Figure 2.2: Neonatal CORT treatment effect …….……….…..…. p.111 Figure 2.3: Adult CORT treatment effect ………..……….…..…. p.112 Figure 2.4: Adult CORT sex by time interaction……………..…. p.112 Figure 2.5: Treatment effect ………………………………….…. p.113 Figure 2.6: BDNF sex effect ………………………….……....…. p.113 Figure 2.7: BDNF treatment by time interaction.…….……....…. p.114 Figure 2.8: NGF sex effect; sex by treatment interaction.….…... p.115 Figure 2.9: MWM treatment effect …………………………..…. p.116 Figure 2.10: Annuli and direct swim treatment effect ……..….…. p.117

CHAPTER 3 – MA-Induced Increased Levels of CORT During the SHRP and Neonatal MA-Induced Long Term Changes in Basal CORT in the Adult: Implications for Spatial Learning and Memory Deficits..………... p.118-193 Figure 3.1: Chemical structures …….……………….……….…. p.131 Figure 3.2: Neonatal MET injection series ……………….….…. p.152 Figure 3.3: Neonatal KTZ injection ……………..…….……..…. p.155 Figure 3.4: Adult MET injection, Preliminary CORT……….…. p.166 Figure 3.5: Adult MET injection, dosing weights...………….…. p.166 Figure 3.6: Adult MET injection, MWM AQ latency………..…. p.167 Figure 3.7: Adult MET injection, MWM RV latency………..…. p.167 Figure 3.8: Adult MET injection, MWM AQ probe trials...…. p.168 Figure 3.9: Adult ADX, Preliminary CORT.……….…..…….…. p.180 Figure 3.10: Adult ADX, post-behavioral CORT …….…………. p.180 Figure 3.11: Adult ADX, dosing weights………………..……….. p.181 Figure 3.12: Adult ADX, adult weights……………….…….……. p.182 Figure 3.13: Adult ADX, Straight channel ………………………. p.183 Figure 3.14: Adult ADX, Zero maze ……………………….……. p.184 Figure 3.15: Adult ADX, MWM Latency .…………………….…. p.185 Figure 3.16: Adult ADX, MWM learning curves...………………. p.186 Figure 3.17: Adult ADX, MWM DR probe trials……..….……. p.187

2 ABBREVIATIONS USED IN THIS DISSERTATION (alphabetized; for any abbreviation used for more than one term please refer to surrounding context)

°C Degrees Celsius 5-HT Serotonin, 5-hydroxytryptamine

5-HT2R Serotonin receptor subtype 2 5-HIAA 5-hydroxyindoleacetic acid 6-OHDA 6-hydroxydopamine aa Amino acid A-GR Glucocorticoid receptor antagonist A-MR Mineralocorticoid receptor antagonist AAALAC Association for Assessment and Accreditation of Laboratory Animal Care AChE Acetylcholinesterase ACTH Adrenocorticotropic hormone ADMX Adrenodemedullation ADX Adrenalectomy ADX-C Adrenalectomy with corticosterone replacement AMPA Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA Analysis of variance ATP Adenosine triphosphate AQ Acquisition phase of the Morris water maze AVP Argenine vasopressin BCA Bicinchoninic acid BDNF Brain derived neurotrophic factor cAMP Adenosine 3',5'-cyclic monophosphate cDNA Complimentary deoxyribonucleic acid cRNA Complimentary ribonucleic acid CNS Central nervous system CNTF Ciliary Neurotrophic Factor CORT Corticosterone

3 CRF/H Corticotropin releasing factor/hormone d-MA Dextro isomer of methamphetamine d,l-MA Dextro/levo methamphetamine racemic mixture

D2/D1 Dopamine receptor subtype 2/dopamine receptor subtype 1 D Day D/d Dextro isomer DA Dopamine DAT Dopamine transporter DEX Dexamethasone DG Dentate gyrus DMSO Dimethyldulfoxide DNA Deoxyribonucleic acid DOPAC 3,4-dihydroxyphenylacetic acid DR Double reversal phase of the Morris water maze DRG Dorsal root ganglion E East E Embryonic ECG Echoencephalograph EDTA Ethylenediaminetetraacetic Acid EIA Enzyme immunoassay ELISA Enzyme-linked immunosorbent assay EPI Epinepherine EtOH Ethyl alcohol FS Forced swim GABA Gamma aminobutyric acid GAS General adaptation syndrome GC Glucocorticoid GDNF Glial-derived neurotrophic factor GFP Green fluorescent protein GH Gonadotropin hormone GR Glucocorticoid receptor

4 FGF Fibroblast growth factor HPA Hypothalamic pituitary adrenal axis IACUC Institutional animal care and use committee I.c.v./i.c.v. Intracerebroventricular IgG Immunoglobulin G ITI Inter-trial interval KTZ Ketoconazole L/l Levo isomer LH Luteinizing hormone LIF Leukemia inhibitory factor M10 Methamphetamine 10 mg/kg M15 Methamphetamine 15 mg/kg M20 Methamphetamine 20 mg/kg MA Methamphetamine MA HCl Methamphetamine hydrochloride MAOI Monoamine oxidase inhibitor MAP Microtubule associated protein MAPK Mitogen-activated protein kinase MC Mineralocorticoid MDMA 3,4-methylenedioxymethamphetamine mdr Multidrug resistance MET Metyrapone mRNA Messenger ribonucleic acid MR Mineralocorticoid receptor MWM Morris water maze N North NAcc Nucleus accumbens NE Northeast NEPI Norepinepherine NGF Nerve growth factor NIDA National Institute for Drug Abuse

5 NMDA N-methyl-D-aspartatic acid NT-3 Neurotrophin 3 NT-4/5 Neurotrophin 4/5 NT-6 Neurotrophin 6 NW Northwest P Postnatal P-2-P Phenyl-2-propanone PCA P-chloroamphetamine PCR Polymerase chain reaction PI-3 Phosphatidylinositol 3 PKA Protein kinase A PLC-γ Phospholipase C gamma PNS Peripheral nervous system POMC Proopiomelanocortin PVC Polyvinyl chloride PVN Paraventricular nucleus RNA Ribonucleic acid RPM Revolutions per minute RV Reversal phase of the Morris water maze S South SAL Saline s.c. Subcutaneous SCN Suprachiasmatic nucleus SE Southeast SERT Serotonin transporter SHRP Stress hyporesponsive period SMART Spontaneous motor activity recording and tracking SOS Son of sevenless SW Southwest TGF-β Transforming growth factor beta Trk Tropomyosine-related kinase

6 US United States VEH Vehicle VMAT Vesicular monoamine transporter VIP Vasoactive intestinal polypeptide W Week W West

7 CHAPTER 1 – METHAMPHETAMINE, NEUROTROPHIC FACTORS, & STRESS

Methamphetamine

Methamphetamine (MA) is a highly addictive stimulant. Registered as a schedule II drug by the United States Drug Enforcement Agency, MA abuse is an increasing problem across the nation. Once only prevalent on the west coast and in Hawaii, MA abuse has quickly spread throughout American suburbs with the rise of “meth labs.” Knowledge of MA production methods, ease of obtaining required ingredients, and high demand have contributed to the growing epidemic of abuse (Brecht et al., 2004). Additionally, statistics show the average age of first exposure to MA is during the later teenage years (Brecht et al., 2004), a time also characterized by increased sexual exploration, thus raising concern for potential of fetal drug exposure. This concern becomes even greater when considering data stating that there is increased percentage of sexual activity under the influence of drugs (Buffum, 1988). Research studies show that MA acts as a stressor on the body manipulating normal function so as to release and block reuptake of the neurotransmitter dopamine (DA). Additionally, prolonged increases in the hormone corticosterone have been shown with MA administration in animals (Williams et al., 2000). The interaction of these events is likely to have a negative impact on the development of an organism following exposure.

Few human data exist concerning the effects of prenatal methamphetamine exposure on long-term outcomes.

8 Chemical Structures & Function

Amphetamines belong to a class of molecules with a substituted phenylethylamine structure. The structure of amphetamine is very similar to the naturally occurring substances norepinephrine and dopamine. These are key monoamines which act as neurotransmitters in the brain. Due to the similarity in structure, amphetamine has profound effect on the central nervous system (CNS).

Amphetamine is a lipid soluble substance and is thus readily absorbed into the body and distributed rapidly to organs and tissues. Furthermore, the drug has the ability to cross the blood brain barrier. It has been reported that brain levels of drug can reach ten times that of drug concentration in the blood (Radcliffe et al.,

1985). This property gives some explanation for its array of effects on the CNS.

The activation of dopamine and norepinephrine results in the rush or speed effect of amphetamine from which its most recognized slang name was derived. The official chemical name of amphetamine is 1-phenyl-2-aminopropane indicating the type and position of the molecular groups attached to the main carbon chain.

The compound has 2 enantiomers, the dextro (D) and the levo (L) form. These are named depending on the rotation of the amino group and hydrogen about the chiral center of the compound. Carbon #2, (as pictured in Figure 1.1), is the chiral center of the amphetamine compound. A racemic mixture or the D enantiomer alone is most widely used in research studies; amphetamine sulfate is also widely used in research studies. The LD50 (dose amount that will kill 50% of

acutely treated) of amphetamine in rats is 180 mg/kg administered

subcutaneously. The oral LD50 of amphetamine sulfate in mice and rats is 24.2

9 and 55 mg/kg, respectively. Amphetamine is listed as chemical compound #616

in the Eleventh Edition of the Merck Index (Budavari et al., 1989).

Methamphetamine (MA) differs from its parent compound by the addition

of a methyl group as indicated by its name. Due to the similarity in structure to

amphetamine, MA has many of the same physiological effects with the exception

of its more potent stimulant effects on the CNS giving it increased potential for

abuse. The official chemical name for MA is 1-phenyl-2-methylaminopropane

indicating the type and position of molecular groups attached to the main carbon

chain. The methyl group that distinguishes MA from amphetamine is attached to

the amino group linked to carbon #2 of the main chain (see Figure 1.1). Like

amphetamine, MA also has 2 enantiomers, a D and an L form. Carbon #2 is again

the chiral center of the compound. The D oriented enantiomer is the most

commonly used form in research studies. The LD50 of MA-HCl in mice is 70 mg/kg administered intraperitonally. MA is listed as chemical compound #5859 in the Eleventh Edition of the Merck Index (Budavari et al., 1989).

d-A and d-MA elicit effects through four main actions. Both compounds are monoamine transport inhibitors resulting in the reduction of presynaptic reuptake of dopamine (DA), serotonin (5-HT), and norepinepherine (NEPI)

(Kuzenski and Segal, 1994). Additionally, amphetamines increase monoamine release by reversing the normal action of the DA transporter (DAT). Interference with the intracellular vesicular transporter (VMAT2) mechanism also occurs

creating a higher concentration of DA in the cellular cytoplasm (Seiden et al.,

1993) creating additional DA for the DAT to mobilize. Evidence of action as

10 monoamine oxidase inhibitors (MAOI) has also been reported (Kuzenski and

Segal, 1994). Strong emphasis is placed on the reaction of amphetamines with

DA as the resulting effects on the exposed system generally arise from interaction with this monoamine more so than from the others, 5-HT and NEPI.

The neurotoxic properties of MA are well documented. MA induces its effects on the brain primarily through induction of monoamine release and inhibition of monoamine reuptake. The most severe effects are depletion of DA and the DA transporters (DAT) specifically in the region of the striatum; but also showing long-lasting depletion of forebrain 5-HT and 5-HT receptors (SERT)

(Kuzenski and Segal, 1994;Seiden et al., 1993;Wagner et al., 1981). Long-term

NEPI depletion is also seen in monkeys. Evidence of astrogliosis, silver degeneration, and Fluro-Jade staining following MA administration have also been demonstrated as further evidence of neurotoxicity (O'Callaghan and Miller,

2002;Pu and Vorhees, 1993).

Amphetamines are sympathomimetic amines because they act on peripheral NEPI, DA and epinephrine (Nichols, 1994;Seiden et al., 1993).

Amphetamine and MA increase heart rate, blood pressure and respiration (Seiden et al., 1993), and cause adrenal release of corticosterone or cortisol. In the CNS, amphetamine and MA heighten attention, reduce fatigue, increase motor activity, suppress appetite, induce euphoria, and adrenocorticoid receptors (Lowy,

1990;Lowy and Novotney, 1994) and body temperature (Bowyer and Holson,

1995;Ricaurte et al., 1994;Seiden et al., 1993). Hyperthermia is often the proximate cause of emergency room visits. When taken repeatedly, responses to

11 amphetamines change over time. For example, tolerance is seen for the positive

or rewarding effects of the drugs and dysphoric symptoms appear during

withdrawal (Segal and Kuzenski, 1994). The withdrawal effects contribute to

usage relapse and progressive increases in dose over time. Repetitive use also

induces progressive increases in some effects, termed sensitization (Segal and

Kuzenski, 1994). Sensitization has been documented in animals for locomotor

and stereotypic movements induced by psychostimulants (Kalivas and Stewart,

1991;Robinson and Becker, 1986;Robinson and Berridge, 1993) and confirmed in

humans (Strakowski and Sax, 1998). At higher doses, amphetamines sometimes

induce aggressive, delusional, and paranoid symptoms.

MA can be injected, smoked, swallowed, or snorted. Timing of noticeable

effects differs with route of drug administration with almost immediate effects

following injection and smoking. Effects after snorting the drug may take several minutes until detected and oral ingestion results in the longest wait with drug effects appearing after approximately 20 minutes (Anglin et al., 2000). Common slang names for the drug include crank, crystal, glass, speed, meth, and ice; ice refers specifically to the form of the drug that is smoked. Smoking and injecting appear to be the most preferred routes of drug administration. Several reasons have been stated in the literature as motivators for use of MA. These reasons include the following: 1) substitute for another stimulant drug due to availability and cost effectiveness; 2) use as a “crutch” to enable feelings of “normalcy” during times of illness or distress; 3) stay awake; 4) enhance sexual performance; and 5) weight loss (Buffum, 1982;von Mayhauser et al., 2002). MA is currently

12 available by prescription under the name Desoxyn for conditions including

narcolepsy, attention-deficit disorder and depression.

Chemical Structures

1 2 3 1 2 3 1 2 3 CH2CHCH3 CH2CHCH3 CH2CHCH3 NHCH3 NH2 NHCH3 O O CH2

A. Amphetamine B. Methamphetamine C. MDMA

1 2 12 CH CH NH 1 2 2 2 2 HO CHCH2NHCH3 4 HO CHCH2NH2 3 4 4 3 3 2 2 2 1 OH OH 1 1 OH OH OH OH

D. Dopamine E. Epinephrine F. Norepinephrine

7 1 6 NH 2 5 HO 3 4 CH2CH2NH2 12 G. Serotonin

Figure 1.1: Chemical structure of the amphetamines most widely abused (amphetamine, methamphetamine and MDMA). Note structural similarity between the amphetamines and the neurotransmitters dopamine and norepinephrine, and to a lesser extent, serotonin (5-HT).

13 History and Prevalence of Use

Methamphetamine was discovered in Japan early in the 20th century

(Suwaki, 1991) and remains the country’s most widely abused drug (Matsumoto

et al., 2002). Its parent compound amphetamine was synthesized late in the 19th

century and prescribed over the next several decades for various conditions

including asthma and weight loss (Anglin et al., 2000). The initial wide scale use

for MA came during World War II at which point the drug was used

intravenously by Japanese troops in efforts to stay awake and alert. Post WWII

MA use in Japan hit epidemic proportion when supplies no longer needed for troops were made available to the public (Anglin et al., 2000;Suwaki, 1991).

Legally manufactured MA tablets became available in the United States in the

1950’s under the name Methadrine. Individuals in sleep deprived professions or high demand jobs were the first groups to abuse the drug and increase its spread of use. Widespread use of MA for conditions ranging from weight-control to treatment of depression continued until the 1970 when the Controlled Substance

Act was passed regulating the use of the drug.

Despite regulation, MA production and use has continued to spread across the United States. The advent of “meth labs” and the smokable form of MA known as “ice,” “crystal,” or “glass” have resulted in increasing spread of the drug into the south, midwest, and eastern areas of the country when initially the problem was central to areas of California and Hawaii. Clandestine production of

MA is a very dangerous procedure. The two most common methods include the ephedrine/pseudophedrine reduction method and the phenyl-2-propanone (P-2-P)

14 method. As P-2-P is prohibited in the United States as a Schedule II controlled substance, the ephedrine reduction method is almost exclusively used for MA production. Pseudoephedrine can be found in many in over-the-counter cold medicines; other ingredients can be found in match books, batteries, engine starting fluid, drain cleaners, and other common household items. A recent study interviewed 350 MA users and of those that manufactured the drug, 70% stated that the chemicals needed were easily obtainable (Brecht et al., 2004;von

Mayhauser et al., 2002). Furthermore, the locations most used for production included houses, motels, and mobile vehicles. These locations are not only dangerous to the individuals involved in the drug making activity but also to anyone in the surrounding area as the production involves emission of toxic fumes and can be explosive and even lethal if not handled correctly (Irvine and Chin,

1991). Also, a wider public health problem can occur as a result of improper disposal and/or storage of the hazardous chemicals used in the drug making process. Often chemicals and waste are disposed of in sewers or simply dumped on the ground resulting in contamination of nearby waterways (Irvine and Chin,

1991).

Increased spread of production is indicative of increased demand for the drug. Common sociodemographics for users indicate individuals who are

2 primarily characterized as white (non-hispanic) in origin with /3 majority having less than a college education and a median household income of $14,000 (Brecht et al., 2004). These individuals also tend to have a history of substance abuse within their families and high incidences of physical abuse and trouble with law

15 enforcement. Age of first MA use is indicated as being in the late teenage years

with “regular” use being reported as occurring 2 years following drug

introduction (Brecht et al., 2004). These statistics are alarming in that these

individuals are also at an age of increased sexual exploration.

The National Institute on Drug Abuse's (NIDA) "Epidemiologic Trends in

Drug Abuse" provides data on emergency department mentions of recreationally

used drugs. The 2001 report (Anonymous, 2001) shows that drug use varies

widely within the US. In cities such as Miami, emergency department mentions

of drugs of abuse use exceed 50% of all admissions, whereas in cities such as

Minneapolis they are mentioned around 20% of the time. In many major US

cities (New York, Atlanta, Washington, Philadelphia, Miami, Detroit, Chicago,

St. Louis, New Orleans, Dallas, Denver, and Los Angeles) cocaine is the most

frequently mentioned drug in emergency departments followed by heroin. In

other cities (San Diego and Honolulu), MA is the most frequently mentioned

drug. Overall, the report finds that emergency department mentions of MA use

show a rising trend in recent years (Anonymous, 2001).

NIDA also reports drug prevalence through the "Monitoring the Future"

surveys (Johnston et al., 2004;Johnston et al., 2002) of secondary school and

college students, as well as adult surveys (up to age 40). For the secondary

school, sampling is done on 8th, 10th and 12th graders and includes about 45,000 students. All amphetamines were reported at just under 11% use, with nearly 4% reporting use of MA (2.5% specifically reporting use of ‘ice’ during the preceding

12 months.

16 There are gender differences is the use of psychostimulants. The NIDA

survey data show that among high school seniors, college students and young

adults, males use illicit drugs at higher rates than females. However, there are

exceptions. Among younger age groups (8th and 10th graders) gender differences are small.

Annual prevalence of drug use for the last 20 years show peak usage of cocaine among all age groups during the 1980s, with highest rates among those

18-24 years (as high as 20%). Rates declined throughout the early 1990s

(reaching under 5% for all 18-24 year-olds) and today are 5-6%. MDMA use remained relatively steady until 1995-1996, when it rose gradually from around

2% to around 4% among 18-24 year-olds, and rose sharply from 1998 to 2001.

By contrast, amphetamines show a different pattern. They show peak annual prevalence of ~25% in 1981, then show a steady decline until 1992 when prevalence levels off at <5% for 19-24 year-olds and at about 8% for 18 year- olds. After 1992, annual amphetamine prevalence shows a slow rise to the present. MA prevalence was not separately reported until 1990. From 1990-2001

MA use shows a slow rising trend with highest rates among 18 year-olds at ~4%.

The 2003 survey (Johnston et al., 2004) reveals that the annual prevalence for amphetamines is 6.1% for males and 4.7% for females age 19-30 years. For MA, the rates are 3.3% and 1.8%, respectively, for males and females. For crystal MA, called “ice”, the rates are 1.3% and 0.9%, respectively, for males and females.

For reference, the comparable prevalence for cocaine among males and females of the same age range is 8.2% and 5.0%, respectively.

17 These data provide little information concerning use among pregnant women. The last survey from NIDA on pregnancy was the "National Pregnancy and Health Survey: Drug Use Among Women Delivering Livebirths: 1992"

(Anonymous, 1996). The use of MA, however, was not prevalent when this survey was completed. It is worth noting that among 15-44 year-old women, the survey found a 6.8% prevalence rate for illicit drug use. Women age 18-34 deliver 85% of all live births. Research also indicates increased sexual contact when individuals are under the influence of drugs (Buffum, 1988). Therefore, the ages when illicit drugs are most likely to be taken are also the years of greatest likelihood of giving birth creating increased risk of MA exposed fetuses. The survey found that irrespective of age, 5.5% of women reported at least one use of an illicit drug during pregnancy. If this prevalence remains similar today, it suggests that there are 250,000 births per year to women who took an illicit drug during pregnancy. Unfortunately, this gives little information concerning which drugs are used by these women, how much is taken, or when during pregnancy use occurred.

Developmental Processes and Vulnerability to Injury

Although development includes everything from conception to senescence, the focus of the experiments contained in this dissertation is on early development. It is evident that all effects on the embryo and fetus are dependent on when exposures occur during development (Wilson, 1973). This is because different organs and tissues develop at different developmental ages.

Organogenesis is the process of organ formation and occurs during

18 embryogenesis. Vulnerability to the induction of malformations occurs only during organogenesis (Wilson, 1973). Thus, for the induction of cardiac defects, cleft palate, and other birth defects by exogenous agents, exposure must occur during this time window. This is also the stage of gestation when women are least likely to be aware of their pregnancy and in some cases to fully acknowledge it.

Organogenesis ends with closure of the hard palate, which is embryonic (E) day

15-16 in rats and week (W) 8 post-conception in humans (Shepard, 1986). In terms of gross malformations of the brain and spinal cord, organogenesis is the period when such effects are induced (Schardein, 1993). However, the CNS is different from other organs in that cell division (neurogenesis and gliogenesis) extends beyond the period of organogenesis. The CNS has a protracted period of development. Cell proliferation in the brain lasts throughout gestation in humans and synaptogenesis extends into postnatal development in humans. Taken together, functional changes in the CNS can occur even after organogenesis.

Comparative neuroanatomy and brain growth data provide the most data for developing models of prenatal brain development. It would be desirable to have precise timetables of human-rodent development for neurotransmitters, receptors and other signaling proteins, but only limited data are available

(Yamamoto et al., 1998). What data are available in humans is summarized in

Figure 1.2.

The rat-human neuroanatomical evidence is based on structural landmarks from human fetal brain. Data from rats are a combination of neuroanatomical landmarks and 3H-thymidine incorporation as compiled by Bayer et al. (Bayer et

19 al., 1993). For purposes of this discussion development of selected forebrain regions and the cerebellum are illustrated in Figure 1.3 (adapted from Bayer et al.

(Bayer et al., 1993). Brain growth as a fraction of adult brain weight data are depicted in Figure 1.4, adapted from Dobbing and Sands (Dobbing and Sands,

1979). Note that in order to model all stages of intrauterine human brain development, it is necessary to consider the interval from E6 to ~ P20 in rats.

As noted above, the nature of the defect is dependent on the stage of development when the exposure occurs. Furthermore, the occurrence of an effect depends on whether the teratogen acts via the peak concentration or the time- averaged (area under the concentration-time curve) concentration or body burden of the agent. Some agents, such as lead, methylmercury, and alcohol, produce cumulative effects, therefore, area under the concentration-time curve are generally the best predictors of effect, but for drugs, peak concentration is often critical. The classic example is valproic acid, in which daily dosing during the critical period induces malformations, whereas constant release of the same total dose does not (Nau et al., 1981). Current evidence supports this pattern for agents that cause CNS injury manifest as changes in behavior (Vorhees, 1986;Vorhees,

1992;Vorhees, 1994). Amphetamines also produce differential effects that are dependent upon the dosing regimen in adults. For this reason, studies exposing animals to a drug throughout brain development can be less revealing than those that focus on critical intervals of CNS development. Another factor that limits the value of chronic exposure models is that with chronic administration, the tolerance of the mother for the drug becomes the rate limiting factor. In order to

20 avoid maternal toxicity, the dose must be reduced compared to what can be taken during a short exposure period. Since people seldom take drugs steadily, chronic exposure models have limited applicability. This is particularly true for drugs of abuse that are most commonly taken episodically (binges).

21 Prenatal Period in Months (Human)

0-3 4 5 6 7 8 9 neocortex NMDA + cerebellum Non-NMDA putamen

kainate hippocamopus AMPA entorhinal cortex

NMDA

D1/D2 basal ganglia

muscarinic reticular formation

muscarinic frontal cortex

nicotinic reticular formation

5-HT1A cerebellum

5-HT1A brainstem hippocampus striate cortex basal ganglia 5-HT1D cerebellum

5-HT1D frontal cortex

Figure 1.2: Estimated pattern of brain neurotransmitter receptor subtype development in different human fetal brain regions (adapted from Herschkowitz et al., 1997). Note how neurotransmitter receptors develop primarily during the later stages of human gestation (as shown) and even extend into postnatal period (not shown, but see Herschkowitz et al., 1997).

22

Human development – Experimentally determined time in weeks 3.5- 4.1- 5.3- 5.8- 6.7- 7.1- 7.5- 8.0- 10- 12- 15- 19- 24- 28- 32- 36- 4.0 5.2 5.7 6.6 7.0 7.4 7.9 9.9 11.9 14.9 18.9 23.9 27.9 31.9 35.9 40.0 Rat development – Experimentally determined time in days E21 P0- P4- P8- P12 P16 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 -22 3 7 11 -15 -19

Cerebellum

Thalamus

Hypothalamus

Pallidum

Striatum

Amygdala Neocortex & Limbic cortex Pirifrom cortex Entorhinal cortex Hippocampus CA1-3 Dentate granule cells

Figure 1.3: Developmental ages at which different regions of the brain develop in humans and rats (adapted by Bayer et al., 1993). Shaded areas are the days when neurons of varying types are proliferating in each region of the brain in humans (estimated from anatomical landmarks) and rats (3H-thymidine incorporation).

23 Figure 1.4: Comparative brain growth spurt (as a percentage of adult brain mass) in human and rats (adapted from Dobbing and Sands, 1979). Note especially the fact that the rat brain growth spurt is shifted to the right compared to humans, such that more of early brain growth that is prenatal in humans occurs neonatally in rats.

24 Research

Human Studies

Limited data has been published concerning the effects of neonatal exposure to amphetamine or MA in humans. The data that exist are complex due to the multitude of variables involved. In human studies it is often very difficult to discern the effect of a particular drug due to the fact that there are usually multiple drugs that have been used by the subject; additionally, the primary source of information is the user themselves which must be considered when determining the validity of the information provided. Despite these impediments, the information that has been reported is informative and useful.

Gestational amphetamine

A report of prenatal exposure to amphetamine in 1971 by Levin describes several cases of biliary atresia associated with amphetamine abuse. The study reports that 5 of 11 mothers of infants with biliary atresia admittedly took amphetamine during pregnancy, 4 were exposed in the first trimester and one

(likely an irrelevant case) in the third trimester. Of the fifty non-exposed control infants only 3 had biliary atresia (Levin, 1971). This observation although not a definitive result of amphetamine use alone raised concern of a possible association between the disorder and drug use. A series of studies concerning prenatal amphetamine exposure was performed by Eriksson and others in the late

1970’s and into 80’s. The initial report consisted of data from 23 mothers who had taken amphetamine, of whom 17 continued to take amphetamine throughout their pregnancy. Few complications were reported; however the following was

25 observed: one infant stillborn with myelomeningocele, 6 preterm deliveries, 3

“small” infants, and 2 infants determined to be drowsy and in need of tube feeding. Most adverse outcomes or complications were reported among the mothers who continued drug use throughout gestation (Eriksson et al., 1978). The second study reported a 25% incidence of preterm delivery and a higher perinatal mortality rate (7.5%) among children of the chronic amphetamine subgroup.

Other observations among exposed offspring included tachypnea, hypotonicity, feeding difficulties, and the need for tube feeding. Observations among mothers included increased complications at delivery, anemia, poor weight gain, proteinuria, postpartum hemorrhage, and retained placenta (Eriksson et al., 1981).

The follow-up study of 66 prenatally exposed children showed normal somatic and psychmotor development 12 months after birth. Despite this finding some children who were removed from their mothers at birth (where the mothers continued drug use), were hospitalized due to failure to thrive, or because of suspected physical and/or emotional abuse (Billing et al., 1980). Gillogey et al presented data on gestational exposure to cocaine, amphetamine, and opiate use in which offspring had low birth weight and reduced head circumference from women who tested positive for cocaine or amphetamine alone among 168 cases

(Gillogley et al., 1990). Other investigators (Slutsker et al., 1993) obtained reports from birth attendants on amphetamine use among their patients. Common results among amphetamine users indicate growth and developmental abnormalities among exposed children.

It is difficult to determine the exact form, route, or dose of drug taken in

26 human studies nor can it be definitively determined if these effects were due to the ingestion of amphetamine alone. Nevertheless, concern and further study is warranted for infants of mothers who use amphetamine during pregnancy.

Gestational methamphetamine

Methamphetamine use became more popular during the 1980’s in some parts of the United States. Oro and Dixon published a study in 1987 on prenatal cocaine and MA exposure. Results demonstrated increased occurrence of bradycardia, anemia, and prematurity (28%, p<0.05) among exposed children.

Additionally, 8.7% of children demonstrated growth retardation, 17% reduced head circumference, 81% abnormal sleep patterns, 71% tremors, 58% poor feeding, 52% hypertonia, 51% vomiting, 41% sneezing, 42% had high pitched cry, 42% demonstrated frantic fist sucking, 19% tachypnea, 16% loose stool, 16% fever, 12.9% increased yawning, and 1.5% hyperreflexia as well as other symptoms (Oro and Dixon, 1987). These symptoms were seen among the infants whose mothers took cocaine, MA, or both drugs prenatally, but not in offspring from normal control mothers or mothers who took opiates. Common trends of growth retardation and reduced head circumference seen in MA exposed infants are reinforced by another study. Little et al. (Little et al., 1988) reported significant reduction in body weight, length, and head circumference among children exposed prenatally to MA. Dixon (Dixon, 1989) also reported that women who used MA during pregnancy had increased occurrences of postpartum hemorrhage, increased use of alcohol by the mother, and higher mortality among their infants. Results from echoencephalographic (ECG) tests performed on these

27 infants showed a 35.1% incidence of cranial abnormalities vs. 5.3% in controls.

Furthermore, the report stated specifically that 12.5 % of the MA exposed children had increased incidence of white matter abnormalities in the brain, indicative of hemorrhage or necrosis (Dixon and Bejar, 1989a;Dixon and Bejar,

1989b).

One of the most recent human studies examined brain proton magnetic resonance spectroscopy infants exposed to MA in utero. An increase in the marker for creatine, suggestive of increased metabolic activity, was found in the striatum in the absence of changes in neuronal or glial markers (Smith et al.,

2001). The creatine changes are similar to those reported previously in infants after intrauterine cocaine exposure. The authors suggest that like cocaine, prenatal MA exposure does not induce structural changes in the brain, but rather biochemical alterations.

Animal Studies

Animal studies provide the opportunity to test for specific effects of a substance due to the ability to manipulate and control variables such as agent, amount, and time of exposure, all of which are important factors to the outcome of the exposure. Animals also provide a variety of models that imitate specific periods of human development. These advantages allow for the most complete testing of specific drugs and their effects on multiple aspects of an organism.

Data obtained can be applied to potential effects in humans and used to educate people on the immediate, long lasting, and mechanistic actions of the drug.

28 Amphetamine

Animal studies on the effects of prenatal amphetamine exposure are extensive. These studies cover five decades of research and use a variety of animal species and drug concentrations. The first report appeared in Lancet in

1965. Nora et al (Nora et al., 1965) administered d-amphetamine, a drug then commonly prescribed as a weight loss agent, to pregnant A/Jax mice at a concentration of 50 mg/kg via intraperitoneal (i.p.) injection on E8. Fetuses were removed 1-2 days before term. Treated dams had 55% resorptions vs. 8% in control animals. d-Amphetamine exposed offspring had a higher percentage of malformations (overall 38% vs. 7%, cardiac anomalies 12% vs. 0%, cleft lip 18% vs. 4%, eye anomalies 8% vs. 3%, and other anomalies including exencephaly).

Using the same treatment protocol noted above, these authors tested 50 mg/kg of d-amphetamine given to A./Jax or C57BL/6 mice (Nora et al., 1968) and again discovered an increase in malformations. Cleft lip, micropthalmia, and atrial septal defects were observed in the A/Jax strain while ventral septal defects were observed in the C57BL/6 strain. The results from these studies spurred increased interest. Clark and collegues (Clark et al., 1970) reported reduced locomotor activity in pre-weanling animals following prenatal exposure to 1 mg/kg d- amphetamine from E12-15; this deficit was no longer apparent in these animals in adulthood. No changes in T-maze learning and reversal or operant learning were found. Seliger (Seliger, 1973;Seliger, 1975) reported results from two studies concerning prenatal d-amphetamine exposure in rat offspring. d-Amphetamine was administered by subcutaneous injection (s.c.) to the dam at doses of 0, 5, or

29 10 mg/kg from either E5-9 or E12-16. Increased activity levels were seen in all drug exposed animals but were most pronounced in the E5-9 group having received 5 mg/kg drug. The 5 mg/kg group also showed reduced passive avoidance learning. The second report showed reduced running in shallow water, in the E5-9 exposure group. A study in C57BL/6J mice exploring effects of exposure to 5 mg/kg d-amphetamine during the last third of gestation (Middaugh et al., 1974) resulted in pups with decreased birth weight, increased locomotor activity scores in adulthood, and increased concentrations of NE on P21 and P30 and increased DA on P21 (Middaugh et al., 1974). Nasello et al (Nasello et al.,

1974) reported that offspring from rats given s.c. injections of 0.5 mg/kg d- amphetamine daily throughout gestation displayed functional deficits as a result of having a lower threshold for seizure in the region of the hippocampus vs. control animals despite these animals having no evidence of growth retardation or gross malformations. Hitzemann et al (Hitzemann et al., 1976) measured locomotion and monoamine content in offspring of rats exposed to 1 or 3 mg/kg d-amphetamine by s.c. injection from E5-22. Results showed reduced locomotor habituation at 90 days as well as decreased NEPI on P35 by 21% and at P84 by

18% in the diencephelon and brain stem. Dopamine was also decreased (by 21%) at P84 in the brainstem. A similar study looked at altered brain catecholamine levels in offspring of drug treated pregnant rats. Nasello et al (Nasello and

Ramirez, 1978) examined brain catecholamine metabolism in rat offspring from dams exposed to s.c. injections of 0.5 mg/g d-l-amphetamine throughout

30 gestation; these offspring demonstrated increased tyrosine hydroxylase activity in adulthood.

Prenatal amphetamine exposure studies in the 1980’s focused heavily on functional effects and behavioral studies. Monder (Monder, 1981) reported developmental deficits in rats after exposure to 2 or 5 mg/kg d-amphetamine throughout gestation administered in the drinking water. Pups showed a delay in righting latency on P1 and P3 in addition to having delayed eye and vaginal opening. Others have shown deficits in escape learning in a Y water maze and increased sensitivity to drug challenge after prenatal exposure to 0.5, 1, or 2 mg/kg d-amphetamine via s.c. injection on E12-15 (Adams et al., 1982).

However, in a larger follow-up study, the same dose and exposure period of prenatal d-amphetamine administration produced no changes in a large battery of neurobehavioral tests (Buelke-Sam et al., 1985). A parallel study using littermates of these animals but a different set of neurobehavioral tests, also showed no effects of prenatal d-amphetamine exposure in the offspring, although a delay in eye opening of exposed offspring was seen (Vorhees, 1985). Holson et al (Holson et al., 1985) showed no significant effects of d-amphetamine when administered on E12-15 at a dose of 3 mg/kg. Fein et al (Fein et al., 1987) showed a 58% increase in resorption with 50 or 100 mg/kg d-amphetamine given

E 9-11 in ICR mice. There was also a 15% incidence of gross malformations including microphthalmia, exencephaly, and cleft lip in surviving pups in addition to ECG results suggesting developmental delay in heart function.

Together the results reviewed thus far suggest that prenatal d-

31 amphetamine has weak teratogenic effects at high doses in mice, but not in rats.

However, there are neurobehavioral changes after prenatal or pre- and postnatal d- and d,l-amphetamine exposure similar to human findings. Concurrent with the neurobehavioral data, the animal data show changes in monoamine neurotransmitters.

Day (D) 14 in ovo chick development has been correlated to third trimester human development; furthermore, the chicken allows direct application of drug to the fetus and observation of development. Doses of 8, 16, or 32 mg/kg were applied to D14 eggs and a challenge dose was administered i.p. on P1 at 4,

8, 16, or 24 mg/kg. All doses of d-amphetamine challenge caused distress vocalization, wing extension, tremor, flat body posture, loss of righting reflex, and convulsant kicking at a higher rate in prenatally exposed chicks (Bronson et al.,

1994). A rat study with 2 or 5 mg/kg exposure on E11-14 showed reductions in exploratory activity and locomotor hyperactivity in the offspring on P20-30. Late juvenile behavior showed increased time spent eating and increased latency in reversal learning (Lyon and McClure, 1994). Tavares and Silva (Tavares and

Silva, 1996) showed that rats dosed with 5 mg/kg d-amphetamine via s.c. injection twice daily from E8-10 gained less weight than controls and had delayed cerebellar growth relative to body growth. Yamamoto et al (Yamamoto et al.,

1998) exposed E10.5 explanted rat embryos to d-amphetamine at concentrations of 0.1, 0.4, 0.8, 1.2, or 1.6-mM for 24 hours. This design allowed for direct application of the drug to the embryo. Viability was not affected; however, the

1.2 and 1.6 mM dose produced reductions in yolk sac diameter, crown rump

32 length, and somite number as well as an increase in malformations that included

microencephaly, neural tube defect, and incomplete rotation of the body axis and

twisted spinal cords.

Nasif et al (Nasif et al., 1999) showed no difference in birth defects or

growth patterns following prenatal exposure to 4 mg/kg d-amphetamine from E8-

21; however, an increase in NEPI levels was seen in the prefrontal cortex of these

animals in adulthood.

Crawford et al. (Crawford et al., 2000) dosed rat pups from P11-17 with

2.5 mg/kg d-amphetamine by i.p. injection as a model of third trimester brain

development. Long-term reductions in DA content and PKA activity in the

striatum resulted as well as an up-regulation of D2-like binding sites when examined at P90.

Prenatal amphetamine studies cover a range of doses, dosing regimens, animal species, and test batteries. Results remain mixed and often contradictory.

A review published by Middaugh (Middaugh, 1989) of the pre-1990 data concluded that no clear picture of the effects of prenatal d-amphetamine was apparent. However, it was noted that what evidence was available pointed in the direction of long-term neurobehavioral and possibly monoamine transmitter changes in rodents after prenatal d-amphetamine exposure. Another review of the pre-1990 prenatal d-amphetamine literature (Buelke-Sam, 1986) concluded that the most consistently reported effect was increased reactivity among exposed offspring. By contrast, the post-1990 data support the idea that there are lasting neurochemical effects from early d-amphetamine exposure, especially when the

33 exposure occurs during later stages of brain development (Crawford et al., 2000).

These later stages are when neurotransmitter systems are undergoing rapid development and may be the most susceptible to interference. Unfortunately, no data were found that described the morphology of the neurons and synapses following d-amphetamine exposure. Taken together, studies have shown evidence of a range of developmental changes from minimal growth retardation to long lasting functional deficits to severe gross malformations and even death at very high doses; these results support the need for further investigation.

Methamphetamine

With the early evidence of potential teratogenic effects of prenatal amphetamine exposure, many researchers began to explore the teratogenic potential of its analogue methamphetamine (MA). A study by Courtney and

Valerio (Courtney and Valerio, 1968) investigated the effects of 0.5 mg/kg/day

MA administered throughout gestation to 5 gravid Macaca mulatta monkeys; this is the only non-human primate study on record of its kind. Four normal young and one stillborn resulted from the pregnancies. The stillborn ratio was not statistically different from the colony norm and all live infants had normal organ growth and development. Kasirsky and Tansy (Kasirsky and Tansy, 1971) used

CF-1 mice and rabbits and administered MA (unspecified isomer) intravenously at doses of 5 or 10 mg/kg for 3, 4, or all 7 days on E9-15 in mice and at a dose of

1.5 mg/kg from E12-30 in rabbits. Exposed mouse fetus malformation rates were

12% (10 mg/kg on E12-15), 13.6% (10 mg/kg on E9-15), and 2% (5 mg/kg on

E9-15). Rabbit offspring showed a 15.5% malformation rate. All malformation

34 percentages were above control rates but no statistical analysis was provided.

Tonge (Tonge, 1973) administered MA (unspecified isomer) in drinking water to

gravid rats throughout gestation (ingested dose unspecified). Exposed pups

showed increased 5-HT and 5-HIAA in the cortex and hippocampus; and,

decreased 5-HT levels in the hypothalamus, pons, and medulla.

Martin et al explored neurobehavioral effects of prenatal d,l-MA in a

series of studies. Locomotor activity in aging Sprague Dawley rats prenatally

exposed to MA was examined to determine if drug exposure had long term effects

on behavior. Pregnant dams were exposed to 5 mg/kg s.c. twice daily throughout

gestation and lactation; offspring were tested for locomotor activity once per

month from 3 to 39 months of age. Rats exposed prenatally to drug were

significantly more active in 29 of the 35 sessions tested (Martin and Martin,

1981). Related studies provided similar but less dramatic behavioral results

following prenatal d,l-MA exposure (Martin, 1975;Martin et al., 1976;Martin et al., 1979;Martin et al., 1983). Behavioral and neurochemical changes were observed by other researchers as well (Sato and Fujiwara, 1986). Gravid rats were administered 2 mg/kg MA (unspecified isomer) s.c. throughout gestation.

Decreased locomotor activity with increased vertical activity was seen in MA- exposed offspring. Locomotor reactivity to sound was also altered, but there were no changes in circadian patterns or induction of locomotor sensitization after repeated MA challenge doses. There was also an increase in 3H-spiperone

binding in the frontal cortex of prenatal MA-exposed offspring as adults,

suggesting upregulation of DA receptors. Cho et al (Cho et al., 1991)

35 administered MA 1-4.5 mg/kg (unspecified isomer) to rats from E7-20. Results showed differential dose effects. Doses above 2 mg/kg resulted in decreased maternal weight gain. Doses of 3 mg/kg and higher resulted in multiple pup anomalies including decreased growth rate, delayed reflex, late incisor eruption, and delayed eyelid opening and testes descent. Treatment with 2 mg/kg resulted in decreased locomotor activity but enhanced conditioned avoidance response in male offspring.

More recently, there was a report that prenatal d,l-MA exposure induces anophthalmia in rats treated on E7-12 but not when treated on E13-18 (50 mg/kg

2x/day) (Vorhees and Acuff-Smith, 1990). A follow-up study provided additional detail concerning MA-induced anopthalmia and also reported behavioral deficits.

This study followed the same dosing paradigm, d,l-MA 50 mg/kg 2x/day E7-12.

The control group was pair-fed to the treated group. The drug exposed offspring showed significantly lower olfactory orientation scores P9-13 and hyperactivity as adults. Eye defects include anophthalmia, microphthalmia, and folded retina

(Acuff-Smith et al., 1992). A third study by Acuff-Smith et al (Acuff-Smith et al., 1996) in rats treated with 5, 10, or 20 mg/kg 2x/day d-MA on E7-12 or E13-

18 showed anophthalmia after E7-12 exposure while folded retinas were observed after E13-18 exposure. E13-18 exposure also resulted in increased maternal and offspring mortality as well as a decrease in 5-HT in the nuclear accumbens following exposure to the 20 mg/kg dose among surviving offspring. E7-12 exposure also produced delays on several tests of early reflex development and

36 impaired probe trial performance in the Morris water maze (MWM), a test of spatial memory, in the 20 mg/kg dose group (Acuff-Smith et al., 1996).

In another study, d,l-MA was given twice daily at a dose of 2 or 10 mg/kg by s.c. injection prior to and throughout gestation to rats (Weissman and

Caldecott-Hazard, 1993). The 10 mg/kg dose significantly decreased open-field locomotor activity and increased 5-HT and DA uptake sites in the brain whereas the 2 mg/kg dose produced decreased uptake of both neurotransmitters

(Weissman and Caldecott-Hazard, 1993). In another study, 2 or 10 mg/kg d-MA was administered s.c. throughout gestation and monoamine changes were noted in adulthood. At the 2 mg/kg dose, a 38% decrease in NE was observed in posterior cortex, 45% decrease in DA in the striatum, and 5-HT uptake reductions of 18% in the hypothalamus, 32% in the striatum, and 42% in the hippocampus. At the

10 mg/kg dose there was a 41% increase in NE uptake in the midbrain, 27% increase in NE uptake in the medulla-pons, 25% increase in 5-HT uptake in the hippocampus, and 33% increase in the medulla-pons. Serotonin and its metabolite, 5-hydroxyindolacetic acid (5-HIAA) were decreased across all regions examined (Weissman and Caldecott-Hazard, 1995). Cabrera et al

(Cabrera et al., 1993) administered d,l-MA at 5 mg/kg s.c. twice daily from E13-

20. No differences were observed in adrenocorticotropin (ACTH), corticosterone

(CORT), or prolactin responses to the 5-HT releaser p-chloroamphetamine (PCA) when administered as a challenge at P30 (females) or P70 (males). However, long-term decreases in plasma renin were observed in response to PCA. No changes in basal hormone concentrations or cortical density of 5-HT or 5-HT2R

37 were reported. A study done in fetal primary neuronal culture demonstrated a decrease in monoamines in a dose dependant manner after d-MA exposure (0.1-

100 µmol/L); these doses also interrupted normal monoaminergic neuronal morphology (Heller et al., 1995).

In subsequent studies using postnatal exposure as a model of late human gestation, neonatal rats were administered 30 mg/kg d-MA twice daily during either P1-10 or 11-20 and tested for behavioral effects as adults. Both exposure age groups showed increased acoustic startle reactivity. Compared to controls, the P11-20 group was also significantly impaired at learning the location of a hidden platform in the MWM task; this effect was not seen in the P1-10 exposure age group. These data indicate that MA administration during later postnatal development affects the brain differently than during earlier exposure periods.

Further, rats tested at P30, 45, and 60 for locomotor activity showed that offspring exposed to d-MA were less active than saline-treated littermate controls. The effect was most notable at P30 but was evident at P45 and P60 for males. Some animals were also administered a drug challenge with 10 mg/kg fluoxetine or 2 mg/kg of d-MA. The males exposed to d-MA from P1-10 showed prolonged suppression of activity under fluoxetine challenge (Vorhees et al., 1994a;Vorhees et al., 1994b). These experiments showed that d-MA has age-dependent and selective effects on brain development and behavior. Furthermore, these data suggest that events in late gestation may represent a particularly sensitive stage of brain development to the effects of d-MA exposure.

A later study confirmed that P1-10 d-MA treatment with 20 mg/kg twice

38 daily persistently augmented acoustic startle response amplitude (Vorhees et al.,

1996). These investigators also looked at genetic differences in P450 metabolism following developmental d-MA exposure in another study (Vorhees et al., 1998).

The CYP2D6 gene in humans is homologous to CYP2D2 in rats and confers a polymorphism for poor vs. extensive metabolizers of the debrisoquine/sparteine group of drugs that include the amphetamines. Females of the ACI rat strain are poor CYP2D2 metabolizers and Sprague-Dawley rats are extensive metabolizers.

Rats of each strain were administered 30 mg/kg of d-MA subcutaneously twice daily on P11-20. Results showed higher offspring mortality in ACI rats indicative of lower metabolism leading to toxicity and lethality. Offspring of both strains showed Morris maze spatial learning impairment, but the effects were no worse in the ACI females compared to ACI males (Vorhees et al., 1998), or Sprague-

Dawley males or females. This may be because the dose of MA used induced a maximal effect. Subsequently, the Dark Agouti strain of poor metabolizer

CYP2D2 animals became available. In this study, rats were exposed from P11-20 to d-MA, but this time the dose was reduced to 15 mg/kg d-MA given s.c. twice daily. In this experiment, both strains of d-MA treated offspring showed MWM spatial learning impairments, but the Dark Agouti females showed a more severe spatial learning deficit (Vorhees et al., 1999). Since the slow metabolizers of d-

MA were the most affected, these data suggest that in developing animals, the effects of d-MA are most likely due to the parent compound rather than to an active metabolite.

Williams et al (Williams et al., 2000) examined hypothalamic-pituitary-

39 adrenal (HPA) axis responses following neonatal d-MA treatment in rats.

Offspring were administered 15 mg/kg d-MA given 4 times per day on P11, P11-

15, or P11-20. Animals were sacrificed on the final day of dosing and blood collected from which hormone levels were assessed. Results showed a decrease in body weight but brain weight remained unchanged. The P11-20 group showed increases in ACTH and CORT. The P11-15 group showed larger increases in

CORT, while the P11 group animals showed the largest and most prolonged

CORT increases accompanied by increased ACTH. Vorhees et al (Vorhees et al.,

2000) showed long-term learning and memory deficits in rats following neonatal d-MA exposure. Rat pups were administered either 10 mg/kg d-MA 4 times per day or 20 mg/kg d-MA 2 times per day from P11-20. Animals were tested as adults. Treated animals showed no changes in swimming a straight channel, learning a complex multiple-T maze or finding a visible platform on cued trials in the MWM. Significant spatial learning and memory deficits were seen among the

MA exposed group as compared to controls when tested in the reference-memory, spatial learning version of the MWM. Latency to the target, path length, and cumulative distance from the target were all impaired (Vorhees et al., 2000).

These data indicate spatial learning deficits are specific to neonatal MA exposure, whereas learning a complex multiple-T maze (sequential task) was not.

In another study, rats administered 0 (saline), 5, 10, or 15 mg/kg d-MA four times daily from P11-20 were again found to have MWM performance impairments. As compared to control groups, all MA dose groups showed deficits in the acquisition phase, as well as on reference memory probe trials. The

40 MA-treated groups also showed impaired reversal learning and larger deficits

when the platform was moved for a second time and a smaller platform used. The

same rats showed no impairment in cued learning in the same apparatus to a

visible platform and no changes in a working memory version of the maze

(Williams et al., 2002). The data indicate that the effects of early d-MA treatment

are dose-dependent and selective for reference memory-based spatial abilities.

The critical dosing period for producing MA-induced long-term learning and

memory deficits was refined from P11-20 to P11-15 (Williams et al., 2003b).

Animals exposed to MA from P11-15 demonstrated deficits in spatial learning

and memory while animals exposed to MA from P16-20 did not show this effect.

Heller et al (Heller et al., 2001b) examined the susceptibility of prenatally

exposed mice to a neurotoxic regimen of MA challenge as adults. Pregnant

C57BL/6 mice were dosed on E7-18 with 40 mg/kg d-MA twice daily. At 11

weeks the offspring were challenged with a dose of 5, 10, 15, or 20 mg/kg.

Enhanced neurotoxicity was observed in adults that had been prenatally exposed

to the drug in terms of reduced striatal DA. The enhanced neurotoxicity occurred

in male offspring.

Three recent studies published provide information concerning drug concentrations in the brain following prenatal or neonatal d-MA exposure. Two studies by the same group use C57BL/6 mice to examine fetal and maternal brain

MA concentrations. The first used 40 mg/kg d-MA administered on E14. Results indicate peak MA concentration in fetal brain at 122 ng/mg total brain protein one hour following drug administration to the mother (Won et al., 2001). The second

41 study used the same dose of d-MA administered from E7-13 or 7-15. Results showed comparable MA concentrations as in the previous report as well as increased DA levels in MA groups in the rostral mesencephalon and rostral striatum when assessed on E16; levels were elevated by 25-60% over controls

(Heller et al., 2001a). The MA concentrations reported were similar to those found in post mortem case reports of early deaths of human infants born to MA users. A study in neonatal rats (Cappon and Vorhees, 2001) reported plasma and brain MA concentrations following neonatal exposure to 15 mg/kg s.c. once, 20 mg/kg twice, or 10 mg/kg 4 times on either P1 or P11. Not surprisingly, the results showed peak plasma and brain drug concentrations higher in neonates administered MA directly than in fetal mice with transplacental exposure as reported above (Heller et al., 2001a). The concentrations, however, were within the range predicted for heavy users based on human pharmacokinetic data (see

(Cappon and Vorhees, 2001) for details). Furthermore, the plasma and brain MA concentrations were similar in the P1 vs. P11 treated animals. This is intriguing given that only P11-20 MA exposure induces later learning and memory impairments, indicating that internal dose by itself does not predict later cognitive effects (Cappon and Vorhees, 2001).

Experimental results reported within the last two years further address

MA-induced toxicity and long-term learning and memory deficits. Increased concentration of cleaved microtubule-associated protein (MAP)-tau in the striatum, dorsal hippocampus, and prefrontal cortex was reported following four injections of MA 10 mg/kg in the adult rat supporting MA-induced axonal

42 degeneration (Wallace et al., 2003). Crawford et al (Crawford et al., 2003)

reported decreases in protein kinase A (PKA) activity, DA and DOPAC content,

and decreased density of D2-like binding sites in the dorsal striata of adult rats

(P90) treated neonatally, P11-20, with MA 10mg/kg. MA administration P11-20 was shown to result in morphological changes in key brain regions in the adult rat

(Williams et al., 2004a) displayed by reduced dendritic length in the nucleus accumbens (NAcc), increased dendritic length in the parietal cortex, and decreased spine density in both the NAcc and dentate gyrus (DG). A 2003 study by Williams et al (Williams et al., 2003c) reiterates MA-induced deficits in spatial learning and reference memory ability while further stating that the drug treatment does not effect working memory. Evidence of spatial learning and memory parameters as well as growth effects were seen as a result of MA treatment at concentrations as low as 0.625 mg/kg administered four times daily from P11-20 substantiating concern for the outcome of fetuses exposed to any concentration or duration of MA during gestation (Williams et al., 2004b).

Spatial learning deficits resulting from neonatal MA treatment were shown in an aversive version of the Barnes maze (Barnes, 1979), a non swimming maze task

(Williams et al., 2003a). Animals treated neonatal with MA in this study also displayed an attenuated CORT response to a forced swim (FS) task suggesting possible malfunction of the HPA axis stress response system.

43 Dose Comparisons Across Species

There is no simple, direct method of comparing the doses of drugs across

species unless one has detailed pharmacokinetic data in two or more species; such

data cannot be obtained for most drugs of abuse. The most basic approach,

expressing dose on a body mass-adjusted basis (mg/kg) is the most universal but

is also known to be inaccurate. Two other approaches to interspecies scaling have

been proposed. These are allometric scaling and comparisons based on

physiological pharmacokinetic modeling (Martin, 1975). In order to use

physiological pharmacokinetic modeling, pharmacokinetic data in both humans

and animals at comparable developmental ages would be required; but, obtaining

such data from pregnant MA users is not feasible. Since this is not available,

allometric scaling may be applied using the formula of Dosehuman = Doseanimal

0.7 (Weighthuman/Weightanimal ) (Mordenti and Chappell, 1989). Consider the highest dose used in the study by Broening et al. (Broening et al., 2001) of 20 mg/kg of MDMA in neonatal rats. Based on body weight of P20 rats in that study, the dose of 20 mg/kg in young rats would be scaled to a dose of 2.4 mg/kg in a 50 kg woman. The same dose in P11 rats would scale by this method to a dose of 0.9 mg/kg in a 50 kg woman. Since human doses of drugs of abuse are not often expressed on a weight adjusted basis, 2.4 mg/kg for a 50 kg woman would be 120 mg of MDMA, and the dose of 0.9 mg/kg would be 45 mg. These are doses that are higher than most 'average' users would probably take at a time, but are not unreasonable for regular users. Therefore, these extrapolations suggest that the rat doses are within the range that humans actually use for MA.

44 Model System

The rat model system of neonatal exposure to MA used by the Vorhees lab is used in all studies contained in this dissertation (Vorhees et al., 1994a;Vorhees et al., 2000;Williams et al., 2002;Williams et al., 2003b). The period of rat brain development equivalent to third trimester human brain development continues through postnatal day 19 (Bayer et al., 1993). This period of development is of interest due to the continued robust development of the hippocampus (Rice and

Barone S Jr, 2000), and also overlaps the stress hyporesponsive period (SHRP),

P4-14, in the rat (Levine, 1994;Sapolsky and Meaney, 1986).

45 Neurotrophic Factors

Normal development of vertebrate nervous systems depends on the pluripotent characteristic of neuroblasts and on occurrence of programmed cell death (Bell, 1996). Members of a family of proteins called neurotrophins are critical for both aspects of developmental processes. Cell death during development may appear counterproductive; however, it is critical that the body dispose of neurons having redundant properties in a normally occurring and selective process.

Key members of the neurotrophin family include nerve growth factor

(NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3).

These three neurotrophins are derived from a single gene family, they exhibit a high degree – approximately 50% - protein homology, each have approximately

120 amino acids (aa), similar molecular weight and isoelectric points (Bell,

1996;Leibrock et al., 1989;Rush et al., 1996;Tapia-Arancibia et al., 2004).

Neurotrophin-6 (NT-6) and neurotrophin-4/5 (NT-4/5) have also been identified.

NT-6 has only been isolated in fish and is reported to act on similar populations of cells as NGF; NT-4/5 has been isolated in most mammalian species, except in birds, and is reported to almost entirely overlap functional properties of BDNF

(Gotz et al., 1994;Reichardt and Farinas, 1999;Rocamora and Arenas, 1996).

The neurotrophic factor hypothesis (Levi-Montalcini, 1987) states as its central concept that target organs produce only limited amounts of survival factors in order to regulate balance between size of the innervating population of neurons and the size of the target territory. Therefore, the primary developmental

46 function of the neurotrophin family members is to promote neuron survival; however, each member supports survival of distinct neuronal populations. In addition to their primary function, neurotrophins also affect cell proliferation, cell differentiation, and neuronal function (Bell, 1996).

The ability of neurotrophin proteins to act on different neuronal populations is a primary means of differentiation between the family members.

All three neurotrophins promote survival of sensory neurons of the PNS; however, only those derived from neural crest cells are responsive to NGF while neural crest and neural placode derived sensory cells respond to BDNF (sensory neurons have two distinct embryologic sources - neural crest and neural placode populations) (Barde, 1990;Lindsay et al., 1985a;Lindsay et al., 1985b;Reichardt and Farinas, 1999) thus demonstrating differential action in subpopulations of sensory neurons. Additionally, NGF and NT-3 act on sympathetic neuronal populations and NT-3 also acts on enteric populations in the PNS. CNS neuronal populations supported by neurotrophins include the following: basal forebrain cholinergic, striatal cholinergic, and cerebellar purkinje neuron populations by

NGF; basal forebrain cholinergic, trigeminal mesencephalic, substantia nigra dopaminergic, cortical, hippocampal, cerebellar granule, retinal ganglion, and motor neuron populations by BDNF; basal forebrain cholinergic, locus coeruleus adrenergic, and motor neuron populations by NT-3 (Reichardt and Farinas, 1999).

Within the CNS, the hippocampus is the area of greatest expression of both mRNA and protein for all three neurotrophins in the adult animal (Das et al.,

2001;Maisonpierre et al., 1990a); however, in comparison, BDNF may have

47 concentrations as much as fifty times greater than those present for NGF or NT-3

(Barde, 1990). Specifically, BDNF concentrations are high in the cornu ammonus-2 (CA2) region of the hippocampus as well as in the cerebral cortex.

BDNF is also expressed in the cerebellum, striatum, and spinal cord (Barde,

1990;Hofer et al., 1990). NGF expression in the CNS is similar to the expression of BDNF but is expressed at lower levels in these areas; NT-3 is expressed in the cerebellum in addition to the hippocampus. Neurotrophin expression has also been observed in areas outside the CNS in areas including the dorsal root ganglion (DRG, BDNF), testis (NGF), and ovaries and kidney (NT-3) indicating that these proteins may function outside of the nervous system (Barde, 1990).

In a detailed study, developmental neurotrophin expression in the CNS was examined; mRNA expression and protein concentrations were observed in the neocortex, hippocampus, and cerebellum on P1, 7, 14, 21 and 92 in the rat

(Das et al., 2001). Neocortical mRNA expression of NGF and BDNF increased with age reaching plateau levels by P14 while NT-3 did not demonstrate significant change with age. Corresponding protein levels in the neocortex showed peak concentration at P14 for both NGF and BDNF and at P1 for NT-3.

Hippocampal mRNA expression of all three neutotrophins increased with age reaching plateau levels by P14; protein levels of NGF peaked at P7, BNDF at P14 and NT-3 at P1. In the cerebellum, mRNA for NGF did not show significant change, BDNF and NT-3 mRNA expression increased with age (NT-3 showed a peak at P14). Protein levels in the cerebellum displayed peak levels for NGF at

P7, mixed levels for BDNF and peak levels for NT-3 on P1. Authors concluded

48 that there were no consistent relationships regarding mRNA and protein expression for any of the neurotrophins between brain regions examined (Das et al., 2001) and caution that both mRNA and protein levels should be addressed when characterizing neurotrophin expression. Results supported high expression levels of each neurotrophin in the hippocampus and perhaps even higher than as seen in the adult (Barde, 1990;Maisonpierre et al., 1990a) and further demonstrate both regional and temporal expression patterns for mRNA and for protein throughout development (Das et al., 2001).

Related to neurotrophin proteins are other neurotrophic factors that are also secreted by target cells to promote survival of innervating cells. These factors include glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), transforming growth factor beta (TGF-β), and other cytokines (Reichardt and

Farinas, 1999). GDNF is reported to be distantly related to TGF-β and to act on neuronal populations including sympathetic, sensory, and enteric neurons of the peripheral nervous system (PNS) and substantia nigra dopaminergic, locus coeruleus adrenergic and motor neuron populations of the central nervous system

(CNS) (Moore et al., 1996;Pichel et al., 1996;Reichardt and Farinas,

1999;Sanchez et al., 1996). CNTF, a relative of LIF and other cytokines, acts on sympathetic, sensory, and parasympathetic PNS neuronal populations and on striatal cholinergic and motor CNS neuronal populations (Bazan, 1991;Ernsberger et al., 1989;Reichardt and Farinas, 1999). Most of these same populations of

49 neurons are acted on by members of the NGF neutrophin family indicating some

redundant activity in functional responsibility among neurotrophic factors.

Nerve Growth Factor (NGF)

Nerve growth factor (NGF) is perhaps the most widely known and recognized neurotrophin protein and was the first to be characterized.

Experiments leading to the discovery of NGF were first performed in the late

1940’s when Elmer Bueker (Bueker, 1948) observed enlarged dorsal root ganglia

(DRG) and fiber extension as a result of transplanting mouse sarcoma 180 into chick embryos (Levi-Montalcini, 1982). Interestingly, motor neurons growth was not stimulated (Bueker, 1948). Growth of the ganglia, and even more so, differential growth of ganglia, led to further experiments using a more detailed silver-staining technique to better observe nerve fibers. Transplanted mouse sarcoma 180 or 37 resulted in enlargement of sensory and sympathetic ganglia (as much as six times larger than control) and observation of the newly formed nerve fibers innervating the tumor without making synaptic connections (Levi-

Montalcini and Hamburger, 1951). Levi-Montalcini also observed that these tumor-induced fibers invaded other targets in addition to the tumor itself, leading to the hypothesis that the tumor released a factor that gained access to the ganglia via the circulatory system; and furthermore, that all sympathetic ganglia were enlarged and not only those near the transplant (Levi-Montalcini, 1952). Levi-

Montalcini then traveled to Rio de Janeiro to perform cell culture studies with these tumor cells and saw that in vitro, both sensory and sympathetic ganglia

50 formed a “halo” of nerve fibers within 12 to 24 hours of exposure to the tumor cells (Levi-Montalcini et al., 1954).

The growth promoting factor was identified by Stanley Cohen in collaboration with Levi-Montalcini in 1953 (Cohen et al., 1954) and named

“nerve growth promoting factor” (the name was later shortened to nerve growth factor) (Levi-Montalcini, 1982). For this accomplishment, in addition to those experiments that followed, Levi-Montalcini and Cohen were awarded the 1986

Nobel Prize for achievement in Physiology or Medicine (Marx, 1986). Continued experimentation of NGF led to discovery of large amounts of NGF in the submaxillary salivary glands of mice (Cohen, 1958;Cohen, 1960) and the ability to purify NGF from this source. Cohen was able to determine the identity of NGF as a protein (vs. a nucleic acid) due to experimentation with snake venom and his discovery that venom, which breaks down nucleic acids via phosphodiesterase enzyme, also contained NGF (Cohen and Levi-Montalcini, 1956).

Despite identification having occurred in the late 1950’s, the structure of the NGF protein was not discovered until 1971 (Angeletti and Bradshaw, 1971).

NGF was then described as two monomers, each consisting of 118 aa, held together by non-covalent bonds (Angeletti and Bradshaw, 1971;Levi-Montalcini,

1982). It was described earlier that there were two forms of NGF examined for biological activity, a high molecular weight form and a low molecular weight form, differing only in additional sequence to the larger and not in activity

(Angeletti et al., 1967;Bocchini and Angeletti, 1969). These findings led to later discovery that the bioactive form of NGF is processed from a larger molecular

51 weight precursor (Levi-Montalcini, 1982). Subsequent experiments by Selby et al. (Selby et al., 1987) actually described four different complimentary DNA

(cDNA) transcripts for NGF with differences occurring in the pre-coding region.

This finding suggested differential processing and expression of the gene. The four primary transcripts are differentially expressed within the body; however, regardless of the primary sequence, the bioactive protein which results is identical due to proteolytic cleavage at N and C termini (Edwards et al., 1988b;Rush et al.,

1996).

It was discovered that NGF undergoes retrograde axonal transport as a means of providing information to the innervating neuronal cell body (Hendry et al., 1974). During early axonal cell growth, only some axons reach the specified target and are then able to transport the neurotrophin back to the innervating cell body via retrograde motion to support survival; axons not reaching the correct target do not transport the neurotrophin and these cells then undergo programmed cell death (Hendry, 1996). More specifically, it is hypothesized that NGF does not act as the message by itself but rather forms a factor-receptor complex that is transported via retrograde motion to the region of the cell body and produces a secondary product that acts as the mediator of the message (Hendry, 1996). In vivo evidence supporting this transport mechanism is shown by results that up to

50% of neurons at the L5 (lumbar) DRG location die following spinal cord disruption via rhizotomy performed after birth in the rat thus effectively blocking the ability of NGF to be transported from a primary site of synthesis in the target to the neuron population; furthermore, death of these cells can be prevented by

52 treatment with exogenous NGF (Yip and Johnson, Jr., 1984). Also, observation

of retrograde transport of 125I-NGF from the spinal cord to the DRG via the dorsal roots was reported (Yip and Johnson, Jr., 1984).

Experiments of chronic NGF administration to neonatal mice (daily s.c.

injections for 14 days, 0.05 ml/g body weight) produced six fold increases in

sympathetic ganglia volume vs. control littermates (Levi-Montalcini and Booker,

1960b). Later studies showed this increase was due to increased cellular

differentiation, increased production of microtubules, neurofilaments, and

microfilaments (used to build axons), and prevention of programmed cell death

(Levi-Montalcini et al., 1968;Levi-Montalcini, 1982). In contrast, injection of an

antiserum (s.c. injection 0.05 ml/ 1.5 g body weight) developed from rabbits by

Cohen (Cohen, 1960) resulted in large-scale destruction of up to 99% of neural

crest derived sympathetic nerve cells after eight days exposure (Barde, 1990;Levi-

Montalcini and Booker, 1960a). These results exemplify the growth promoting

aspects and critical importance of NGF for normal development.

A series of experiments were designed to address effects of exogenous

NGF on its ability to rescue injured cells; 6-hydroxydopamine (6-OHDA),

surgical axotomy, and vinblastine were utilized to injure cells and exogenous

NGF was administered to examine rescue effects. Results from daily treatment,

for 14 to 31 days, with NGF (10 µg/g) and 6-OHDA (100 µg/g) showed

functional protection of sympathetic neurons and even an increase in ganglia

volume while treatment with 6-OHDA alone in the neonate is known to destroy

nerve terminals and produce lesions in cell bodies of adrenergic neurons of the

53 sympathetic ganglia (Angeletti and Levi-Montalcini, 1970;Levi-Montalcini et al.,

1975). The exogenous source of NGF allows greater concentration to reach the

cell body than would normally via retrograde transport (Hendry et al., 1974) thus

allowing for additional production of axonal fibers to counteract those destroyed

by 6-OHDA (Levi-Montalcini, 1982). Likewise, surgical axotomy results in

large-scale destruction of immature sympathetic neurons preventing NGF from

reaching the cell body; however, this too is rescued by exogenous NGF (Aloe and

Levi-Montalcini, 1979;Levi-Montalcini, 1982). Vinblastine administration

destroys microtubule assembly thus interrupting endogenous NGF signaling; and,

is rescued by exogenous NGF administration (Johnson, Jr., 1978). These

experiments demonstrate the protective effects of NGF.

Tropic action of NGF has been observed using an in vitro chamber system

(Campenot, 1977). Sympathetic nerve cells were cultured in a central chamber with ample NGF. A fluid impermeable matrix was placed between the central and two lateral chambers, one containing NGF-enriched media and another with control media. Neurite growth penetrated through the fluid impermeable matrix toward only the chamber containing NGF-enriched media; furthermore, when

NGF was removed from distal portions of neurite growth, growth stopped and

degeneration began, this did not occur with removal of NGF from the proximal

portion of neurite outgrowth nor with removal from the location of the cell bodies

as long as NGF was maintained near distal outgrowth regions (Campenot, 1977).

In addition to its trophic and tropic properties, NGF also displays

transforming ability as exemplified by production of normal nerve cells following

54 exposure of tumor cells to NGF for 7 days (Greene and Tischler, 1976;Tischler

and Greene, 1975). Pheochromocytoma cells obtained from rats were

transformed into cells undistinguishable from normal sympathetic neurons in

morphology and bioactivity following exposure to NGF; this cell line was

designated PC12 (Greene and Tischler, 1976). PC12 cells do produce and store

both dopamine (DA) and norepinephrine (NEPI) but not epinephrine (EPI).

Brain Derived Neurotrophic Factor (BDNF)

The discovery and subsequent characterization of NGF led to the search for other neurotrophic factors able to support survival of additional neuronal populations; more than twenty-five years passed before identification and characterization of the second neurotrophin, brain-derived neurotrophic factor

(BDNF) (Barde et al., 1982). Evidence of a factor, other than NGF, able to

support sensory neuron survival was originally observed in vitro in glioma cell

cultures (Barde et al., 1978); subsequently, similar activity was observed with rat

brain extract (Barde et al., 1980). Additionally, not only did the glial-cell

conditioned medium and rat brain extract support neuronal survival alone, but

each displayed additive survival ability when NGF was added to the cultures

(Barde et al., 1980). Pig brain homogenate was used for purification of this factor

that was later named brain-derived neurotrophic factor (BDNF) (Barde et al.,

1982).

The complete sequence of BDNF was not reported until seven years

following discovery of the neurotrophin (Leibrock et al., 1989) although partial sequences had been previously determined. Primers were designed using one of

55 the pre-existing sequence fragments and used to screen a genomic pig template via PCR; the product obtained from these reactions was then used to design more specific primers that were used to screen cDNA (resulting from reverse transcriptase of total RNA isolated from the optic lobe as BDNF was known to support survival of retinal cells) again via PCR (Leibrock et al., 1989). The resulting product displayed a protein sequence of 252 aa resulting in a mature protein of 119 aa (Leibrock et al., 1989). It was noted that this sequence had very high homology with the sequence of NGF sharing 51 aa. Each displayed six cystine residues in the same relative positions; furthermore, the BDNF sequence retained (in number and general location) three trytophan, two phenylalanine, and most valine and aspartic acid residues seen in the NGF sequence (Leibrock et al.,

1989).

BDNF was observed to support survival of approximately 20% of cultured

DRG neurons from E10 chick embryos (Barde et al., 1982); the survival percentage was enhanced when NGF was added to the culture and was not effected by addition of NGF antiserum clearly identifying this factor as a new neurotrophin. A higher percentage of DRG neuron survival, 60-70%, was observed a few years later with BDNF in cultures performed using a different substrate for cell attachment (Lindsay et al., 1985b). BDNF had no effect on sympathetic or parasympathetic neurons in vitro (Lindsay et al., 1985b).

Differential activity between the two neurotrophins was observed with exposure of nodose ganglion (placode derived sensory neurons) as BDNF supported neurite outgrowth from 40-50% of plated neurons from E6-12 chick

56 embryos and NGF had no notable effect on this population (Lindsay et al.,

1985b). BDNF was also shown to stimulate neurite outgrowth of cultured retinal neurons from fetal rats in a dose-dependant manner (Turner et al., 1982); NGF was not effective in stimulating neurite outgrowth from these cells alone or in combination with BDNF.

Specific activity of the complete protein was examined following discovery of the complete sequence (Leibrock et al., 1989) using transfected COS cells and chick DRG neurons; results upheld previous finding stating BDNF support of sensory neuron survival. Expression sites for BDNF were determined using a cRNA probe utilized on mouse tissue via northern blot; results indicated expression limited to the brain and spinal cord (Leibrock et al., 1989) however other subsequent studies discovered expression outside of the CNS in heart, lung, and platelets (Bailey and K.A., 1996;Maisonpierre et al., 1990a).

Evidence of in vivo requirement of BDNF for cell survival was demonstrated by gene targeting experiments and subsequent production of BDNF knockout mice (Ernfors et al., 1994;Jones et al., 1994) that show severe degeneration in sensory ganglia. Administration of exogenous BDNF (1µg/day) to quail embryos E3-7 resulted in increased numbers of surviving neurons from nodose and DRG ganglion (Hofer and Barde, 1988). Reduction in motor neuron death was also observed with exogenous BDNF administration in chick embryo

(Oppenheim et al., 1992).

In addition to its cell survival effects, BDNF has also been shown to have a role in maturation of sensory neurons from pluripotent neural crest cell

57 precursors (Sieber-Blum, 1991). Neural crest primary cultures were established with a known number of cells; with the addition of BDNF to the culture the number of cells expressing the sensory neuron lineage marker (stage-specific embryonic atigen-1+/dopamine-β-hydroxylase-) increased up to 21-fold (Sieber-

Blum, 1991). This increase was a result of a decrease in number of undifferentiated cells and not due to an increase in total cell number; NGF did not produce these effects alone but did have some additive effect to increasing the percentage of sensory neurons when present with BDNF (Sieber-Blum, 1991).

In vitro experiments with granule cell populations from the cerebellum have demonstrated neurite outgrowth with addition of BDNF to culture, however this effect has been preferentially observed for early granule cells and not for more mature neurons (Lindholm et al., 1993); mature granule cells of the cerebellum are known to respond to NT-3 for continuing development (Bailey and K.A., 1996;Segal et al., 1992). These results indicate BDNF is involved in survival of these cells but not in maturation/differentiation; the opposite pattern has been observed for hippocampal neuronal cultures that do not respond to

BDNF with neurite outgrowth but rather with an increase in cells expressing acetylcholinesterase (AChE), an enzyme involved in information transfer across the synaptic cleft (Ip et al., 1993). These results exemplify temporal and differential effects of BDNF.

Like NGF, BDNF has also been observed to undergo retrograde axonal transport as a means of providing information to the innervating neuronal cell body and regulating gene expression (DiStefano et al., 1992). Neurotrophin high

58 affinity receptors serve as carriers for retrograde signal via vesicular transport;

green fluorescent protein (GFP) tagged BDNF receptors were observed moving

from neuritis to cell bodies thus triggering nuclear responses including CREB

phosphorylation or c-fos induction (Bhattacharyya et al., 1997;Watson et al.,

1999). Evidence for anterograde transport of BDNF has also been demonstrated

(Conner et al., 1997) by BDNF immunostaining and observation of BDNF

immunoreacticity in a diffuse pattern closely associated with cell populations

containing BDNF mRNA vs. a robust punctuate pattern of NGF expression

observed in basal forebrain cholinergic neurons (Conner and Varon, 1992).

Neurotrophin-3

Neurotrophin-3 (NT-3) was the third protein to be identified and characterized as a member of the neurotrophin family (Hohn et al.,

1990;Maisonpierre et al., 1990b). Hohn and collegues (Hohn et al., 1990) developed primers from identical stretches of sequence (2 stretches of 6 aa each) from NGF and BDNF and used them in polymerase chain reactions (PCR) on complimentary DNA (cDNA) templates obtained from murine brain, liver, and muscle tissue which revealed a new protein. Maisonpierre et al. (Maisonpierre et al., 1990b) also utilized conserved sequence of NGF and BDNF to develop primers for PCR on genomic DNA; subsequent screening of the PCR products by

Southern blotting revealed novel sequences in the DNA. The product obtained from both groups was subsequently sequenced and named NT-3 (Hohn et al.,

1990;Maisonpierre et al., 1990b).

59 Overall structure of NT-3 was found to be very similar to the two existing neurotrophins enabling generalizations to be created concerning neurotrophin structures. Each begins with a signal sequence followed by a prosequence, a single N-glycosylation site, and cleavage site respectively (Hohn et al., 1990).

Mature NT-3 consists of 119 aa and retains all six cysteine residues in the same relative location as is observed within both NGF and BDNF (Barde, 1990;Hohn et al., 1990;Maisonpierre et al., 1990b). Comparison of the three mature protein sequences also reveals four variable regions of 7-11 aa each that are thought to confer neuronal specificity (Hohn et al., 1990). Mature rat NT-3 shares 57% and

58% aa homology with NGF and BDNF, respectively; 57 aa residues are common to all three neurotrophin proteins (Maisonpierre et al., 1990b). Also, like NGF and BDNF, NT-3 has also been observed to undergo retrograde axonal transport

(DiStefano et al., 1992).

NT-3 was expressed in all tissues originally examined but in differing concentrations; these tissues included heart, skin, gut, skeletal muscle, brain, liver, spleen, lung (Hohn et al., 1990), thymus and kidney (Maisonpierre et al., 1990b).

Further examination of specific brain regions indicated NT-3 expression in the cerebellum and hippocampus (Hohn et al., 1990). More detailed analyses reveal mRNA levels of NT-3 are substantially higher in the developing brain than in the brain of the adult animal (Maisonpierre et al., 1990b).

Following its discovery, NT-3 was tested for neurotrophic action.

Transfection of cloned NT-3 protein coding sequence (plasmid pCMV or pCDM8 expression vector) into COS cells was performed and examined in vitro to

60 determine survival support of a variety of neuronal populations (Hohn et al.,

1990;Maisonpierre et al., 1990b). Sensory neurons from neural placode-derived

nodose ganglia survived when exposed to NT-3 expressing COS cells in vitro

(Hohn et al., 1990;Maisonpierre et al., 1990b); furthermore, an additive survival

effect was observed when BDNF was added with NT-3 resulting in survival of

approximately 90% of neurons (Hohn et al., 1990). Neurite outgrowth from

dorsal root ganglion (DRG) and explanted sympathetic ganglia was also observed

with COS NT-3 expressing cells in vitro (Maisonpierre et al., 1990b).

Neurotrophin Receptors

Each of the neurotrophins, NGF, BDNF, and NT-3 binds to and activates a

signal-transducing protein kinase receptor of the tropomyosine-related kinase (trk)

family. The major trks involved in processes of neurotrophin signaling include

trkA, trkB and trkC for NGF, BDNF, and NT-3 respectively; NT-3 also has some

binding capacity with trkA and trkB (Chao et al., 1998;Reichardt and Farinas,

1999). Binding of a neurotrophin to its receptor stimulates kinase activity and

thereby the phosphorylation of the receptor itself as well as proteins inside the

cell; this phosphorylation activity provides the stimulus for gene activation and

subsequent transcription controlling growth, differentiation, and survival (Kaplan,

1995).

Trk receptors have an extracellular receptor binding component, a

transmembrane domain and a conserved cytoplasmic tyrosine kinase domain

(Gilbert, 2000;Kaplan, 1995). The extracellular domain consists of two cysteine

clusters separated by a leucine motif and followed by immunoglobulin G (IgG)-

61 C2 domains that reside next to the transmembrane domain (Casaccia-Bonnefil et al., 1999). When activated by binding, the tyrosine kinase domain is capable of mediating several signaling pathways including those controlled by PLC-γ, PI-3 kinase, SHC signal transduction adaptor protein, and SNT protein (Kaplan, 1995).

Using PC12 cells (Greene and Tischler, 1976) multiple experiments were performed to determine exact signaling mechanisms activated by neurotrophin- receptor binding that resulted in growth, differentiation, and survival (Klesse and

Parada, 1999). The SHC protein contains the src-homology 2 domain (SH2) and serves as an adapter binding to Grb2 (another adapter protein) as well as binding to the son of sevenless (SOS) protein (Nakamura et al., 1996). SOS protein then recruits ras; when activated, ras initiates the MAPK signal cascade involving a series of phosphorylation events. Expression of constitutively active ras or downstream components has been observed to result in differentiation of PC12 cells without NGF present; furthermore, expression of dominant negative forms of these signal components results in blockade of neurite outgrowth that is observed during differentiation but do not inhibit neuron survival (Klesse et al.,

1999;Klesse and Parada, 1999). Therefore, these data suggest that at least for

NGF, receptor activation of the ras/MAPK pathway results in neuronal differentiation. Trk receptor activation also initiates signals through PLC-γ and the SNT protein, both are thought to be involved in neurotrophin mediated differentiation (Kaplan, 1995).

Inhibition of PI-3 kinase, another factor activated by neurotrophin- receptor binding, results in neuronal death of PC12 cells negating NGF survival

62 activity (Klesse et al., 1999). PI-3 kinase initiates formation of lipid second messengers which then activate Akt (also called Ras) protein kinase; direct stimulation factors regulating neuronal survival is unknown however action through Bcl-2 is postulated (Klesse et al., 1999).

Each of the neurotrophins can also bind the p75 receptor, a member of the tumor necrosis factor superfamily of receptors (Chao et al., 1998;Reichardt and

Farinas, 1999;Smith et al., 1994). The p75 receptor is a transmembrane glycoprotein; it binds each of the neurotrophic factors with equal affinity acting as a low-affinity receptor (vs. the trk receptors) (Ibanez et al., 1995). Despite the lack of a cytoplasmic tyrosine kinase domain the p75 receptor is able to activate

NFkB transcription factor (generally involved in immune and inflammatory response) and JNK kinase signal cascades (generally involved in growth arrest and apoptosis) (Gilbert, 2000).

Recent data reports that although p75 is generally characterized as the low affinity receptor for neurotrophins, it is actually a high affinity receptor for neurotrophins that are secreted as propeptides vs. the mature bioactive forms characterized as mature proteins (Lee et al., 2001). Propeptide neurotrophins preferentially bind the p75 receptor and mediate apoptosis; thus the authors conclude that biologic action of neurotrophins is actually mediated by proteolytic cleavage as propeptides bind p75 and induce apoptosis and mature peptides bind trk receptors and induce survival and differentiation (Lee et al., 2001). Despite this activity, other data show that when coexpressed with trk receptors the p75 receptor can bind mature neurotrophin protein and mediate enhanced binding

63 affinity to trk receptors as well as contributing to cell survival, neurite outgrowth, synaptic transmission, and cell migration (Dechant and Barde, 2002).

64 Stress

The foundation for research on stress was formed by the work of Claude

Bernard who in the latter part of the nineteenth century proposed the concept of

internal milieu to describe the internal environment of a living body (Cannon,

1935;Seiden, 1992). In his own words W.B. Cannon (Cannon, 1935), a well

known physiologist, addressed the nature of this internal milieu stating, “So long

as this personal, individual sack of salty water, in which each one of us lives and

moves and has his being, is protected from change, we are freed from serious

peril;” and, coined the term homeostasis to describe the balanced state of this internal environment.

Homeostatic maintenance is performed by collaboration of the sympathetic division of the autonomic nervous system and release of “adrenin” from the adrenal glands; Cannon called this collaboration the sympatho-adrenal system (Cannon, 1935). Activation of this system can produce biological responses including increased heart rate, constriction and dilation of blood vessels, release of energy stores via metabolization of fat and glycogen, piloerection, release of adrenaline, and shutting down of digestive, immune, and reproductive systems for the purpose of energy conservation and diversion of energy to systems responsible for returning the body to homeostasis. Cannon also describes a “critical stress” (Cannon, 1935) as an event that overwhelms the corrective ability of the sympatho-adrenal system resulting in “significant alteration” of the steady state; however, he also stated that at the time of his report

65 there were no definitive methods by which to determine this critical level and that it would be important to pursue these methods for future research.

During the same period of time as the appearance of Cannon’s report on homeostasis (Cannon, 1935), Hans Selye introduced his theory of the general adaptation syndrome (Selye, 1936) describing a series of events concerning the ability to cope with and adapt to the pressures of injury and disease that remain consistent despite the specific nature of the causative agent. The general adaptation syndrome (GAS) as described by Selye (Selye, 1936;Selye,

1950;Selye, 1952) has three stages 1) Alarm, 2) Resistance, and 3) Exhaustion.

Onset of the Alarm stage occurs 6-48 hours after initial exposure to the causative agent. Common characteristics of this stage include decrease in size of the thymus, spleen, lymph glands, and liver, as well as a decrease in fat stores, musculature tone, and body temperature (Selye, 1936). Responses of the Alarm stage are acute (Selye, 1952). The second phase of the GAS is the Resistance or adaptive stage. Enlarged adrenal glands, basophil manifestation in the pituitary gland, and cessation of growth, and reproductive functions are common characteristics of this phase (Selye, 1936). Acute responses of the previous phase have largely dissipated as adaptation to the causative agent occurs; anabolic action is increased in this stage vs. the catabolic nature of response in the Alarm stage (Selye, 1952). Characteristics of the Alarm and Resistance stages relate closely with those described by Cannon (Cannon, 1935) during activation of the sympatho-adrenal system indicating action by the body to provide an immediate

66 and prolonged defense protecting the integrity of the internal milieu through

reorganization of energy stores and recruitment of immune function.

The third stage of the GAS, Exhaustion, occurs when there is continual

action by the causative agent on the system producing a reappearance of

symptoms (that had since returned to normal) characteristic to the Alarm phase

(Selye, 1936;Selye, 1952) indicating breakdown of the organism’s defenses and

subsequent loss of resistance. This phase correlates with Cannon’s description of

a “critical stress” (Cannon, 1935) and the resulting ability of such to cause

“significant alteration” of the normal balance when the system is overwhelmed

(i.e. exhausted).

The name of “GAS” to describe this series of events was chosen to

describe actions and causes therein. The term “general” was used due to the fact

that only those agents that cause a general stress condition are able to invoke the process; “adaptive” due to the body’s habituation to the undesirable event; and

“syndrome” due to the components of the system being a distinctive complex of symptoms (Selye, 1952).

Selye’s development of the GAS is recounted in a compilation of lectures in a book by Selye entitled “The Story of the Adaptation Syndrome” (Selye,

1952). The evolution of the stress concept began when Selye was a medical student some sixteen years before the publication of the book of lectures on the topic. He personally recounts the observation of what he then referred to as the

“syndrome of just being sick” as a non-specific set of characteristics that did not define any one disease yet seemed universal to all diseases (Selye, 1952).

67 Some ten years later as an academic researcher at McGill University

(Montreal, Quebec, Can.), Selye was reminded of the characteristics of the “just sick” syndrome while studying placental physiology. Accidental observation of disturbances in reproductive cycles by non-specific stress (as he and his colleague were unable to determine a specific causative agent) triggered a recall of the “just sick” syndrome symptoms he had thought intriguing as a medical student (Selye,

1952). These observations were published as part of a manuscript on maternal placenta physiology (Selye, 1952;Selye and McKeown, 1935).

This manuscript by Selye and his student (Selye and McKeown, 1935) remains part of the foundation for current stress research as it the first account (as told by Selye himself) of Selye’s use of the word “stress” in terms relative to the connotation used today. Selye (Selye, 1952) recounts this use of the word stress to describe “non-specific tension in living matter which manifests itself by tangible morphologic changes in various organs and particularly in the endocrine glands which are under anterior pituitary control” (Selye, 1952) and gives a more precise definition for the word in a later publication as “the state manifested by a specific syndrome which consists of all the nonspecifically induced changes within a biologic system” (Selye, 1956). In his book, Selye recounts great opposition to his initial attempts to use the word “stress” (Selye, 1952) as his colleagues contested that the word was not a reality in itself but rather an abstract idea; and, furthermore was representative of both the agent (i.e. cold-stress) as well as the resulting condition. Selye’s response was to continue use of the word

“stress” to describe the condition and “stressor” to describe the agent thus

68 differentiating between the two ideas (Selye, 1952). For the advancements in the use of these terms as we continue to use them today as well as the discovery and description of the GAS, Selye is often referred to as the father of stress research.

Hypothalamic-Pituitary-Adrenal Axis

The hypothalamic-pituitary-adrenal (HPA) axis is the biological system by which the stress reaction is mediated. General action of this system involves release and reuptake of hormones and neurotransmitters with control via an end product negative feedback system. Stress triggers release of corticotrophin- releasing factor (CRF) which signals activation of adrenocorticotropin hormone

(ACTH) release and subsequent release of corticosterone (CORT) (cortisol in humans) which after reaching a certain critical level acts to shut down the system.

The HPA axis is named as such due to the key roles contributed to the system by the hypothalamus, pituitary, and adrenal glands. The hypothalamus is often considered the functional beginning point for the cascade of action to follow

(Kilts et al., 1988). Areas of the limbic system, the hippocampus and amygdale, are also involved in the system.

Corticotrophin-releasing factor (CRF) was first isolated and characterized in 1981 by Vale and colleagues (Vale et al., 1981) from hypothalamic extracts obtained from sheep following nearly thirty years of problematic attempts by various researchers (Owens and Nemeroff, 1988). Immunohistochemical methods were then employed to determine key areas of localization for this peptide.

69 The main population (most recognized and most widely studied) of CRF immunopositive neurons is located in the parvocellular region of the paraventricular nuculeus (PVN) (Carrasco and van de Kar, 2003;Owens and

Nemeroff, 1988). Primary projections of these neurons are to the median eminence of the hypothalamus (primary networking site of the hypothalamo- hypophyseal portal system) (Owens and Nemeroff, 1988). Other populations of

CRF immunopositive neurons have been observed in the amygdala, anterior hypothalamus and parabrachial nucleus of the pons and locus coeruleus with projections to areas including the bed nucleus of the stria terminalis, parabrachial nucleus, nucleus accumbens, and olfactory tubercles (Owens and Nemeroff,

1988). Highest concentrations of receptors for CRF in the rat are located in the anterior and internal lobes of the pituitary gland (Owens and Nemeroff, 1988); other binding sites include the cerebellum, olfactory bulb, neocortex, striatum, amygdale, PVN, and median eminence, and in low amounts in the medulla, midbrain, pons, thalamus, hippocampus, spinal cord, and other regions of the hypothalamus. Due to this wide distribution of CRF, it has been hypothesized that CRF function extends beyond regulatory activation of the anterior pituitary and may also play a direct role in responses of the neuroendocrine, autonomic, circulatory, and metabolic systems as well as in the behavioral response to stress

(Britton and Koob, 1988).

CRF plays the predominant role in stress-mediated increase of plasma

ACTH. Knowledge of this is supported by high concentration of CRF receptors in the anterior pituitary from where ACTH is released. Additional support is

70 provided by the finding that this stress-induced increase of ACTH can be nearly completely blocked by treatment with a CRF antagonist (Owens and Nemeroff,

1988); this blockade of CRF action also results in reversal of the stress-induced inhibitory control on release of luteinizing hormone (LH) and gonadotropin hormone (GH) and rise in plasma epinephrine (EPI) thus also suggesting a role for CRF in these areas of the stress response (Brown et al., 1985;Owens and

Nemeroff, 1988;Rivier et al., 1986;Rivier and Vale, 1985). Furthermore, blockade of EPI biosynthesis results in a significant increase in the number of immunopositive CRF neurons in the PVN (Kilts et al., 1988) substantiating the relationship between CRF and this catecholamine. CRF transportation occurs through the hypothalamo-hypophyseal portal system (Sapolsky and McEwen,

1988); receptor-mediated actions of CRF on ACTH release from the anterior pituitary in mediated by activation of adenylate cyclase and subsequent increase in cyclic AMP (cAMP, adenosine 3',5'-cyclic monophosphate) formation (Kilts et al., 1988).

Although CRF is thought to be the primary modulator in stress-induced

ACTH release, other factors such as argenine vasopressin (AVP), oxytocin, angiotensin, vasoactive intestinal polypeptide (VIP), epinephrine (EPI), and norepinephrine (NEPI) are known to result in increased ACTH following stress

(Owens and Nemeroff, 1988;Sapolsky and McEwen, 1988). The four amino acid peptide sequence, Phe-His-Leu-Leu, is common to both CRF (41 aa peptide) and angiotensin (acting as the site for renin and enzymatic cleavage) indicating a potential ancestral relationship and probable cause of similar activity on

71 adrenocortical function (Owens and Nemeroff, 1988). Evidence supporting the

similar activity of EPI and NEPI to CRF relative to ACTH activation is supported

by high concentration of these two catecholamines in the PVN (relative to

concentration levels in other areas of the hypothalamus) (Kilts et al., 1988).

ACTH remains the primarily recognized hormone released from the

pituitary gland in response to stress(Rossier et al., 1980;Selye, 1950). ACTH is a

39 aa peptide derived from post-translational processing of proopiomelanocortin

(POMC) in the anterior pituitary (Kilts et al., 1988). Determining plasma ACTH

concentrations was originally reported difficult due its rapid degeneration in the

plasma of only 5-7 minutes, low circulating levels, and complex nature of plasma

components with many other plasma proteins interfering with antigen binding

(Kilts et al., 1988).

ACTH levels have been shown to increase rapidly reaching peak plasma

concentration in as little as 2 minutes following exposure to an acute stressor

(Hodges and Vernikos, 1959;Syndor and Sayers, 1954;Vernikos-Danellis and

Heybach, 1980) and returning to pre-stress levels by 20 minutes. ACTH levels in

the pituitary decrease greatly following stress; however, this decrease occurs 1-5

hours post stress (when plasma levels have returned to normal) and may not

recover for 24 hours (Fortier, 1959a;Fortier, 1959b); this decrease is greater than can be accounted for by stress-induced immediate ACTH release. Furthermore, at the time of peak stress-induced ACTH release, pituitary ACTH is largely unchanged (Vernikos-Danellis, 1963). Taken together, evidence suggests that the initial stress-induced release of ACTH is not a release of the stored ACTH in the

72 pituitary, but rather due to an increase in ACTH synthesis and subsequent release

(Vernikos-Danellis and Heybach, 1980).

Just as CRF is thought to be the primary modulator in stress-induced

ACTH release, ACTH is recognized as the primary hormone in stress-induced activation of the adrenal cortex and subsequent release of corticoids. Circulating corticosteroid levels have been recognized as popular measure of identifying the stress-response (Vernikos-Danellis and Heybach, 1980).

Activation of the adrenal cortex enables release of mineralocorticoids

(MCs) and glucocorticoids (GCs) from the zona glomerulosa and zona fasciulata respectively. MC’s encompass a group of hormones that regulate water and electrolyte balance (potassium, sodium) in the body. GCs are steroid hormones that aid in metabolism of carbohydrates, fats, and proteins. Gluconeogenesis is a very important part of carbohydrate metabolism providing glucose to the brain and muscles allowing sufficient energy to sustain and recover from the stress acting on the system in addition to providing for daily energy needs. GCs also help maintain arterial blood pressure and have anti-inflammatory properties.

Corticosterone (cortisol in humans, CORT) is a key GC in this system.

Outside of stress exposure, a normal circadian-dependant release of

(CORT) occurs with minimum levels observed at the beginning of the light period and maximum levels observed at the beginning of the dark period as controlled by the suprachiasmatic nucleus (SCN) of the hypothalamus (Vernikos-Danellis and

Heybach, 1980;Watts et al., 2004). Evidence that the HPA response to stress is under different control than that of the circadian–dependant release of CORT is

73 provided by studies indicating that a stress response to ether exposure in the neonatal rat is observed several days pre-weaning while the circadian rhythm is established 10-12 days after weaning (Allen and Kendall, 1967).

The time of peak stress-induced increase in plasma CORT concentration was determined initially at 15 minutes exposure post-stimulus as levels return to normal by 30 minutes (Guillemin et al., 1959;Levine, 1994). This time-point, although a reliable point of reference, should not be considered steadfast

(Vernikos-Danellis and Heybach, 1980). Factors important to consider when measuring stress-induced increases in CORT include stress stimulus intensity and animal condition (time of day, nutritional status) as well as having a time course sampling period established; these considerations will better enable capture of peak stress-induced plasma CORT concentration.

Biological activity of corticoids is dependant on the type of receptor activated by the hormone. Prior to characterization of the corticoid type-1 and type-2 receptors, the focus of CORT research was on determining the mechanistic site of negative feedback by CORT on the HPA system. By administration of the synthetic glucocorticoid dexamethesone, de Wied (de Wied, 1964) discovered blocking action to occur at the level of the pituitary effectively blocking stress- induced release of ACTH. This suggested a negative feedback relationship between CORT release and anterior pituitary function. Several years later it was discovered in studies with knockout mice that dexamethesone acted at this level of the cascade due to its inability to cross the blood brain barrier as a result of action by the multidrug resistance (mdr)1a-P-glycoprotein (Meijer et al., 1998);

74 (mdr1 mRNA has recently been located in regions of the hippocampus in addition

to endothelial cells of the blood brain barrier, however, function of hippocampal

P-glycoprotein has yet to be determined (Karssen et al., 2004)).

In the interim, research continued for CORT receptor localization. It was

reported that CORT was able to accumulate and was retained in regions of the

limbic system including the hippocampus (McEwen et al., 1968), leading to many

hypotheses concerning the role of CORT in processes of learning and memory.

Thus, de Kloet et al. (de Kloet et al., 1974) utilized intracerebroventricular (i.c.v.) administration of dexamethesone expecting to find accumulation in the hippocampus as shown previously with the natural GC CORT; however, results did not show retention of the synthetic GC in the hippocampus but rather in the pituitary. Despite producing expected results, this experiment provided further motivation for de Kloet and others (de Kloet et al., 1990) to elucidate information concerning CORT presence and retention in the hippocampus. It was discovered some years later (in respect to de Wied and de Kloet’s early work) that CORT retention in the hippocampus is enabled by lack of the 11β-hydroxysteriod dehydrogenase enzyme; this enzyme is responsible for conversion of CORT into inactive metabolite products and is co-localized with mineraolcorticoid receptors

(MRs) in other regions of the body (Edwards et al., 1988a).

It was not until 1982 that discovery of both mineraolcorticoid receptors

(MRs) and glucocorticoid receptors (GRs) were confirmed in the hippocampus

(Veldhuis et al., 1982) opening the door for characterization of the two receptor types. Co-localization of MRs and GRs was shown in the rat brain and evidence

75 presented for differential binding affinity of CORT to each (Reul and de Kloet,

1985;Sutanto and de Kloet, 1987;Van Eekelen et al., 1988). It was reported that

CORT would bind GRs with 10x lesser affinity than binding to MRs (de Kloet et al., 1999;de Kloet and Reul, 1987;Reul and de Kloet, 1985). The discovery of binding affinity differences of CORT lead to increasing research in the area concerning the biological implications of such.

GR antagonist (RU38486) MR antagonist (RU28318) were administered to examine the role of the receptors in both basal and stress-induced CORT sectretion in the rat (Ratka et al., 1989) I..C.V. administration (100 ng/rat) of

RU28318 produced elevations plasma CORT under basal conditions while i.c.v.

RU38486 produced a CORT reduction under conditions of stress (novel environment). Taken together, these results implicate action of MRs under normal

(non-stress) conditions and GR implication in the stress-induced negative feedback reaction (de Kloet et al., 1990;Ratka et al., 1989).

Studies addressing effects of GCs and the neurotransmitter norepinephrine

(NEPI) on hippocampal excitability revealed GR activation following stress results in an increase of the afterhyperpolarization period (depolarization is a calcium dependant potassium reaction) and NEPI administration decreases the afterhyperpolarization period (Joels and de Kloet, 1989). These results led to the conclusion that CORT activation of GRs in the hippocampus enables negative feedback though reduction of excitability caused by the presence of NEPI in response to stress (Joels and de Kloet, 1989).

76 Additional study by the same group clarified that hippocampal neuron excitability is maintained under normal conditions by CORT occupation of MRs

(enabling excitation over inhibitory γ Aminobutyric acid (GABA)) and GR occupation suppresses excitability (Joels et al., 1994;Joels and de Kloet, 1992).

The conclusion was thus reached that maximum stability is achieved when MRs are largely occupied and GRs are largely unoccupied (Joels and de Kloet, 1994).

Conditions observed in adrenalectomized (ADX) animals (both MRs and GRs unoccupied) revealed unstable electrical characteristics similar in effect to those produced by occupation of both receptor types leading to a U-shape curve in activity produced by corticoid receptor activity (Joels et al., 1994).

Activation of both receptor types was determined critical for complete information processing with MR and GR activation each having different responsibilities in the various stages (acquisition, consolidation, and retrieval) of learning and memory processes (Oitzl and de Kloet, 1992). I.C.V. administration of a GR antagonist (RU38486) resulted in impaired retention when retested 24 hours later when given immediately prior to or immediately following the first behavioral training session in the MWM but not when given prior to the retrieval task (Oitzl and de Kloet, 1992). I.C.V. administration of a MR antagonist

(spironolactone) resulted in differential swim patterns during probe trials such that the animal swam to the prior location of goal but then displayed exploratory swimming instead of remaining near the goal location (Oitzl and de Kloet, 1992).

Taken together, these results suggested that MRs have a role in determining exploratory and reactive behavior while GRs are implicated in information

77 consolidation (de Kloet et al., 1990;de Kloet et al., 1999;Oitzl and de Kloet,

1992).

As with electrical activity (discussed previously), a U-shaped curve in

receptor activation was observed relative to behavior. Specific exploration of the

role of MRs in behavioral activity via a open-field task of spatial novelty revealed

increased reactivity and exploration in the animal when devoid of CORT (ADX,

unoccupied MRs and GRs) as well as with increased levels of CORT (occupation

of MRs and GRs); administration of a low dose of CORT (50 µg/kg s.c.) to ADX

rats (a concentration that allowed primarily MR activation alone) resulted in

behavior not different than control animals (Oitzl et al., 1994). This finding

provides additional support that maximum stability as well as optimal behavioral

performance is achieved primarily under occupation of MRs alone (Joels and de

Kloet, 1994;Oitzl et al., 1994).

Development, Stress Hyporesponsive Period

Development of the HPA axis occurs in multiple stages and is affected by both intrinsic and extrinsic factors. From P4-14 during rat neonatal development a period of hyporesponsivity to stress occurs and is characterized by low steady levels of CORT and a non-responsive nature to stress stimuli (in regard to CORT release) (Levine, 1994;Sapolsky and Meaney, 1986). By P15, the animal is capable of releasing CORT in response to a stress stimulus; however, circadian control of hormonal release as well as the HPA feedback regulatory system are not established until after P20 in the rat (approx P22 and P26 respectively) (Allen and Kendall, 1967;Levin and Levine, 1975).

78 Maintenance of GC levels during development at a low and steady

concentration is critical for normal development. Administration of GC to

neonatal rats has been shown to result in decreased mitosis, decreased mylenation,

(Bohn, 1980) and abnormal adult behavior (Bohn et al., 1984). Another study

(Erskine et al., 1979) demonstrated reduction in DNA content, reduced brain size, decreased pituitary stress response at P20-25 and decreased adrenocortical response to stress at P45-48. Due to evidence suggesting excess GCs are harmful during development and the presence of the stress hyporesponsive period (SHRP), downregulation via the SHRP in development is thought to provide protection from high stress-induced GC levels enabling proper development. Intrinsic downregulators of HPA activity include low developmental levels of CRF, low levels of AVP, deficient processing of POMC, and differential steroid biosynthesis and metabolism as compared to the adult animal (Levine, 1994).

SHRP maintenance is regulated by maternal behavior as an extrinsic mechanism of downregulation of HPA (Rosenfeld et al., 1992). Behaviors of maternal regulation on physiological development are called “hidden regulators,” a term coined by Hofer (Hofer, 1978). Key characteristics of “hidden regulators” include slow development of the response (more than 8 hours of separation are needed) and restoration of the effect with replacement of the specific regulator. It was determined that effects on the SHRP as a result of maternal deprivation met the criteria defined by Hofer and maternal factors could thus be considered

“hidden regulators” of the SHRP (Levine et al., 1991). Previous experiments examining maternal behavior had already determined that the neonatal HPA axis

79 was responsive to stress following a period of deprivation from its mother despite the separation period occurring during the SHRP (Levine et al., 1988;Stanton et al., 1987;Stanton et al., 1988). Furthermore, only presence of a lactating dam, not a littermate nor an adult male nor a non-lactating female, was able to significantly suppress maternal deprivation stress-induced CORT increase in the neonates

(Stanton et al., 1988).

Several experiments followed exploring maternal behaviors more specifically. Due to the observation of CORT suppression by presence of a lactating dam, a subsequent experiment was designed to study specific contributions of nutritional contact between dam and pup relative to SHRP maintenance (Levine, 1994;Rosenfeld et al., 1992). Neonates, P7, P11, or P15 days, maintained contact with their mother under normal conditions or were placed with a dam unable to lactate (nipples had been removed) for a period of 24 hours. Both basal and stress (exposure to a novel environment) levels of CORT were obtained. Results showed high CORT levels, comparable to levels seen previously in maternally deprived pups, in the group of pups that were placed with the non-lactating dam. To examine the contribution of food specifically, another experiment utilized pups manipulated with tongue implanted cannulae, thus preventing them from feeding, maintained with their mother (Rosenfeld et al., 1993a). Results showed CORT elevation in response to food restriction and exposure to a novel environment that was unable to be compensated for by contact with the lactating dam while cannulated. Feeding ability was therefore

80 determined to be a specific “hidden regulator” of maternal behavior on

HPA/SHRP during development.

To determine the specific point of regulatory action of feeding regulation, neonatal rats were administered milk via cheek implanted cannula in absence of their mother (deprivation); CORT levels were observed at basal conditions as well as after exposure to a stressor (saline injection) (Suchecki et al., 1993b). Results indicated elevated CORT in animals that were deprived, non-fed, and non- stressed vs. those that were deprived, fed, and non-stressed thus linking feeding specifically to the down regulation of deprivation induced CORT increase.

Furthermore, feeding was also able to reduce the larger CORT increase in the non-fed, deprived, stressed animals. ACTH levels were unaffected between fed and non-fed neonates and showed equal elevation after saline injection indicating that feeding did not have regulatory activity over ACTH response to stress.

Taken together, these results are indicative of reduction in sensitivity of the adrenal to increased ACTH via the hidden regulatory function of feeding

(Suchecki et al., 1993a;Suchecki et al., 1993b).

It is important to note that results from the experiment by Sucheki et al.

(Suchecki et al., 1993b) did not show ACTH elevation to the extent observed previously as a result of deprivation. The authors noted that the only difference in the current experiment (vs. those involving deprivation alone) was the action of anogenital stimulation to induce waste excretion in the pups following feeding.

Therefore, an experiment was specifically designed to address the contribution of anogenital stroking relative to ACTH regulation. Examination of both basal and

81 stress levels of ACTH and CORT was performed in P9 or P12 pups following 4

different conditions: control (non-deprived); deprived only, deprived + anogenital

stroking, deprived + handled (no specific stroking) (Suchecki et al., 1993b).

Results indicated that the addition of the stroking behavior suppressed the stress-

induced increase in ACTH to levels not significantly different than those observed

in the non-deprived group. Stroking was thus concluded as a maternal hidden

regulator of ACTH secretion (Suchecki et al., 1993b).

Results from experiments addressing maternal regulation of SHRP

maintenance have several implications in understanding the nature of this period

of hyporesponsivity and development of the HPA axis. Deprivation experiments

with neonatal pups demonstrated that a stress-response can be observed in the

neonate indicating that the biological processes necessary to produce such a response are in fact present during this stage of development. Therefore, the

SHRP is a period of suppression of the stress response having protective properties. The extrinsic factors of maternal anogenital licking and feeding of the neonates regulate specifically inhibition of ACTH release and reduction of adrenal sensitivity to ACTH, respectively (Levine, 1994;Suchecki et al., 1993b).

This enables protection of the developing system from high levels of CORT that have been otherwise shown to have detrimental effects (Bohn, 1980;Bohn et al.,

1984;Erskine et al., 1979).

Data concerning the ontogeny of MRs and GRs also have impact on neonatal HPA axis function. Adult levels of GR concentration are not present until the third postnatal week in the developing animal while adult levels of MRs

82 are achieved by P8 (Levine, 1994;Rosenfeld et al., 1993b); therefore, GC mediated activity during the SHRP is largely mediated by binding to MRs.

Furthermore, this supports lack of the negative feedback system throughout the first two weeks of development as it is regulated by GR binding. Decreased numbers of GRs may also account partially for prolonged period of CORT elevation in the neonate when CORT release is induced. CORT and ACTH are reported to remain elevated for at least 2 hours once activated in the neonate vs.

30 minutes elevation until return to basal levels in adult animals (Levine, 1994).

83 Hypotheses and Aims

Using the rat neonatal model of MA exposure, we have reliably produced

a long-term deficit in spatial learning ability in the Morris Water Maze (MWM)

with MA exposure from P11-20 or P11-15 (Vorhees et al., 1994a;Vorhees et al.,

2000;Williams et al., 2002;Williams et al., 2003c;Williams et al., 2003b;Williams et al., 2004b). The mechanism for this MA-induced long-term learning and memory deficit is unknown. Results also show a significant MA-induced increase in corticosterone (CORT) during the dosing period (Williams et al., 2000) despite this time being during the stress hyporesponsive period (SHRP) of development

(Levine, 1994;Sapolsky and Meaney, 1986). Also, we have recently observed a long-term decrease in adult basal plasma CORT with neonatal MA exposure.

This dissertation addresses the mechanism of the MA-induced long-term learning and memory deficit by two main hypotheses. The first hypothesis states that neonatal MA administration alters neurotrophin levels resulting in impaired spatial learning and memory deficits in the MWM. It is known that neurotrophic factors are in highest concentration during development and are critical for neuronal development processes (Das et al., 2001;Leibrock et al., 1989;Lindsay et al., 1994). Both BDNF and NGF are implicated as necessary components in learning and memory processes (Chen et al., 1997;Figurov et al., 1996;Fischer et al., 1987;Gomez-Pinilla et al., 2001;Kesslak et al., 1998;Lin et al., 1998;Lin et al.,

1999;Linnarsson et al., 1997;Mizuno et al., 2000;Mu et al., 1999;Suen et al.,

1997) as well as being reported to be influenced by levels of CORT (Chao and

84 McEwen, 1994;Kesslak et al., 1998;Mocchetti et al., 1996;Schaaf et al.,

1997;Schaaf et al., 1998;Scully and Otten, 1995).

Due to location in and around the hippocampus, evidence of involvement

in processes of learning and memory, and influence of neurotrophin expression

levels by CORT, brain derived neurotrophic factor (BDNF) and nerve growth

factor (NGF) were examined to determine if they play a role in the mechanism of

MA’s effects on the developing brain. Results from these experiments

demonstrated a clear MA-effect on MWM learning as well as a MA-induced

increase of approximately 8% in BDNF protein on P15, however, the relationship

between these results is unclear. The finding of neonatal MA-induced long-term

reduction of basal plasma CORT (P68) was more promising.

The increase in CORT during the neonatal SHRP with MA dosing

(Williams et al., 2000) and evidence of a long-term basal plasma CORT reduction

in adult animals treated neonatally with MA led to the development of the second

hypothesis addressing the MA-induced learning and memory deficit.

The second hypothesis states that neonatal MA administration alters the

stress response resulting in impaired spatial learning and memory deficits in the

MWM. The hypothalamic-pituitary-adrenal (HPA) axis is the biological system by which the stress response is mediated. Research shows that if development of the (HPA) axis system is disturbed it may result in abnormal adult function and behavior (Schmidt et al., 2003). Human studies examining traumatic events in early childhood years have indicated such events can have lasting behavioral effects and may manifest as anxiety or emotional disorders (Heim and Nemeroff,

85 2001). Also, hyperactive HPA conditions (increased levels of cortisol and abnormal CRF levels) have been reported to be evident in many cases of human clinical depression (Arborelius et al., 1999;Plotsky et al., 1998;Sherman and

Pfohl, 1985) supporting that abnormal HPA axis function is intimately linked to abnormal behavior.

86 Hypotheses 1. Neonatal MA administration alters the neurotrophin levels resulting in impaired spatial learning and memory deficits in the Morris Water Maze (MWM). a. Aim 1: Examine levels of BDNF and NGF following acute (P11) and chronic (P11-15) MA dosing b. Aim 2: Examine levels of BDNF and NGF in adult animals following neonatal dosing (P11-20) and behavioral tasks i. Zero Maze ii. Straight Channel iii. MWM spatial learning and memory 2. Neonatal MA administration alters the stress/CORT response resulting in impaired spatial learning and memory deficits in the Morris Water Maze (MWM). a. Aim 1: Control/block CORT by pharmacological means i. Metyrapone 1. pellets – neonate and adult 2. injection – neonate and adult a. adult behavioral study i. Zero Maze ii. Straight Channel iii. MWM spatial learning and memory ii. Ketoconazole 1. injection – neonate b. Aim 2: Control/block CORT by surgical means i. ADX with CORT replacement via osmotic minipump 1. adult behavioral study a. Zero Maze b. Straight Channel c. MWM spatial learning and memory

87 CHAPTER 2 - BDNF and NGF Protein Levels in the Brains of Rats Neonatally

Treated with Methamphetamine: Implications for Spatial Learning and Memory

Deficits.

Abstract

Methamphetamine (MA) exposure in rats from postnatal day 11-20

(4x/day) results in long-term learning and memory deficits in the Morris Water

Maze (MWM) when the animals are tested as adults. In this study, brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were examined to determine if they are affected by developmental MA exposure. Animals were treated on P11 only, P11-15, or P11-20 and brains assayed for BDNF and NGF.

The P11-20 treated animals were raised to adulthood and tested in the elevated zero maze, straight channel swimming, and MWM prior to assay for BDNF and

NGF on P68. BDNF was elevated (8%) on P15, but not on P11, with a time after learning-dependant trend on P68. There were no changes in NGF following MA, however sex-related differences were noted for both NGF and BDNF. There were no effects of MA treatment on elevated zero maze or straight channel swimming performance. MA-treated offspring had increased latencies, cumulative distance, path length, and first bearing in the MWM. More detailed analyses revealed that MA-treated offspring had deficits in the MWM in spatial mapping and in subordinate functions as evidenced by increased thigmotaxis and fewer direct swims to the platform than saline controls. The findings confirm

88 MA-induced long-term MWM performance deficits but are inconclusive for

association between the drug and changes in BDNF or NGF.

Introduction

Methamphetamine (MA) use increased throughout the 1990’s especially

among adolescents and young adults (Anglin et al., 2000;Anonymous,

2002;Johnston et al., 2004). Approximately half of MA users are women some of

whom become pregnant and continue using the drug, yet there is little information

available on the long-term effects of prenatal exposure on child development. A

few clinical studies demonstrate an association between prenatal MA use and

reductions in infant head circumference, birth weight and length (Little et al.,

1988) and irregular brain ultrasonography (Dixon and Bejar, 1989b). Dixon

(Dixon, 1989) also reported that women who used stimulants, including MA, during pregnancy had increased occurrences of postpartum hemorrhage, and increased incidence of distress (bradycardia, meconium aspiration) among their infants. Others have found increased creatine and glutamate/glutamine peaks in the spectra of MA-exposed children in the striatum with no changes in cortical regions using proton magnetic resonance spectroscopy (Smith et al., 2001).

Consistent with previous birth weight and length findings, it was reported that a higher percentage of the 134 prenatally MA-exposed infants were small for gestational age, especially in a subgroup exposed during all trimesters compared to 160 unexposed infants (Smith et al., 2003). Interestingly, the majority of infants exposed to MA in that study were exposed throughout pregnancy. In a

89 subsequent, more detailed, but smaller study, this group reported that 13 MA-

exposed children had lower scores on visual-motor tests of attention and delayed

verbal and long-term spatial memory compared to 15 matched control children.

Concurrent with the cognitive deficits, the MA-exposed children had reductions

in brain volumes in the putamen, globus pallidus, hippocampus, and caudate

(Chang et al., 2004).

We developed a rat model of human late gestational d-MA exposure, i.e. during the neonatal period because this is when granule cells of the hippocampus are proliferating and we were interested in possible effects of MA on learning and memory. Two effects have been reliably found following P11-20 MA treatment:

(1) long-term spatial learning and memory deficits in the Morris water maze

(MWM) (Vorhees et al., 1994a;Vorhees et al., 2000;Williams et al.,

2002;Williams et al., 2003c;Williams et al., 2004b), and (2) increases in corticosterone (CORT) (Williams et al., 2000). We have also found long-term changes in dopamine (DA), protein kinase A (PKA) and D2-like receptor binding

(Crawford et al., 2000) although the importance of these changes for explaining the cognitive deficits is unknown. Other effects of the drug remain to be identified. The purpose of the present study was to explore the possible effects of

MA on neurotrophic factors, since these factors are critical in brain development.

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are necessary for neuronal development, growth, and survival (de Quervain et al.,

1998;Leibrock et al., 1989;Lindsay et al., 1994;Schaaf et al., 2001). During development, NGF and BDNF mRNA and protein expression peak in the

90 hippocampus on P7 and P14, respectively (Das et al. 2001), i.e., slightly before and during the P11-20 sensitive period for MA-induced MWM deficits. NGF and

BDNF are associated with learning and memory processes in adult rats. For example BDNF mRNA is increased in the hippocampus following 3 or 6 days of spatial learning in the MWM (Kesslak et al., 1998), as is TrkB (Gomez-Pinilla et al., 2001). Impairment of spatial learning following use of antibodies against

BDNF or BDNF antisense oligonucleotides have also been reported (Mizuno et al., 2000;Mu et al., 1999). Furthermore, BDNF mutant mice having only one copy of the gene show learning deficits in the MWM (Linnarsson et al., 1997).

Conversely, increased expression of BDNF and TrkB are associated with enhancement of long-term potentiation (LTP) in the hippocampus (Figurov et al.,

1996) as well as in facilitation of NMDA signaling processes via an increase in phosphorylation of the NMDA NR1 receptor (Lin et al., 1998;Lin et al.,

1999;Suen et al., 1997).

NGF has also been shown to influence learning and memory when administered to adult rats (Fischer et al., 1987). Continuous intracerebral administration of NGF for four weeks produced partial recovery from age- induced atrophy and improved performance in the MWM (Fischer et al., 1987).

Disruption of the NGF gene in heterozygous mutant mice produces deficits in both acquisition learning and retention in the MWM (Chen et al., 1997).

Prolonged infusion of NGF into the lateral ventricle corrected these deficits.

BDNF and NGF have been shown to be altered by increases in circulating

CORT levels, especially in adult animals. Early postnatal treatment with CORT

91 (P1-10 with implanted capsules) has been shown to increase protein levels of

NGF in the hippocampus following 10 days of CORT exposure (i.e., P10, 12, and

18), however no changes were observed in BDNF (Roskoden et al., 2004). The mRNA levels for trkA, trkB, and trkC in the hippocampus were also increased in the CORT exposed animals (Roskoden et al., 2004). In adult rats, a dose- dependant decrease in both BDNF message and protein is observed in the hippocampus following elevated levels of CORT (Chao and McEwen,

1994;Kesslak et al., 1998;Schaaf et al., 1997;Schaaf et al., 1998;Smith et al.,

1995). Increased NGF mRNA in cultured immortalized hippocampal neurons was observed with CORT application and this effect was blocked with a glucocorticoid antagonist (Scully and Otten, 1995). Administration of dexamethasone increases NGF mRNA in the cerebral cortex and hippocampus

(Mocchetti et al., 1996).

Accordingly, the present experiments were intended to explore the effects of neonatal MA treatment on BDNF and NGF concentrations at three different ages: (1) at the beginning of treatment, (2) at the midpoint of treatment when brain BDNF is at its peak (these ages also being when MA-induced CORT release is occurring (Williams et al., 2000)), and (3) in adults following spatial learning in the MWM. The experiment examining neurotrophins at the midpoint was conducted first, since if no effects were seen at this age we did not intend to proceed.

92 Materials and Methods

Subjects and conditions

Nulliparous Sprague-Dawley CD, IGS rats (Charles River, Raleigh, NC) were obtained and bred in-house with males of the same strain and supplier.

Breeding occurred in hanging wire cages following at least two weeks of acclimation to the vivarium. Embryonic day zero (E0) was defined as the day a sperm plug was detected and the females remained with males a period of two weeks. Following this period the females were housed singly in polycarbonate cages. The day of birth was defined as postnatal day 0 (P0). Pups were the subjects for the experiments.

Litters were undisturbed until P1 when litters were culled to eight

(Experiments-1 and 2) or ten (Experiment-3) pups with equal numbers of males and females. Up to two pups per litter were fostered from litters born on the same day if necessary in Experiment-3. In Experiments-1 and 2, twenty litters with eight animals per litter were used for BDNF and NGF protein determinations in the hippocampus and CORT measured in plasma on either P15, Experiment-1 (10 litters) or P11, Experiment-2 (10 litters). Using a within litter design, two males and two females from each litter received MA and the remaining littermates received saline (SAL). In Experiment-3, 24 litters, with ten animals per litter, received either MA (12 litters) or SAL (12 litters) and were used to determine

BDNF and NGF protein levels in the hippocampus and CORT levels in plasma of adult offspring following MWM learning. Pups were ear marked on P11 for identification, weighed daily from P11-20 during drug administration and weekly

93 thereafter. Separation from the dams occurred on P28 with males and females separately caged and then on P42 the animals were separated into same sex pairs.

Animals were housed in a vivarium under a 14/10 hour light/dark cycle with lights on at 0600; food and water were available ad libitum. The vivarium is fully accredited by the Association for the Assessment and Accreditation of

Laboratory and Animal Care (AAALAC). All protocols were approved by the

Cincinnati Children’s Research Foundation Institutional Animal Care and Use

Committee.

Methamphetamine Administration

d-Methamphetamine HCl (National Institute on Drug Abuse, Bethesda,

MD, expressed as the freebase) was administered subcutaneously by injection in the back of the neck at a concentration of 10 mg/kg in a volume of 3 ml/kg. All injections were given at two hour intervals and injection sites were varied.

Animals were weighed prior to each injection.

Experiment-1 and 2

In Experiment-1, animals examined on P15 were administered MA or

SAL 4x/day from P11-14 and 2x on P15 and sacrificed one hour following final injection for tissue and blood collection (P15 group). The time point for tissue collection was based on previous reports that neurotrophin changes could be seen as early as one hour (Smith et al., 1995) with a maximum effect 3-6 hours following a stressor (Schaaf et al., 1998). Therefore, we chose a 3 h time point when the effects were likely to be maximal. In Experiment-2, animals examined on P11 were administered three injections of MA or SAL and sacrificed one hour

94 following the final injection for tissue and blood collection (P11 group). Based

on the results of Experiment-1, the time point for tissue collection in Experiment-

2 was extended to 5 h after the first MA administration.

Experiment-3

In Experiment-3, animals were administered 4 injections of MA or SAL

per day from P11-P20 (Vorhees et al., 1994a;Vorhees et al., 2000;Williams et al.,

2002;Williams et al., 2003c;Williams et al., 2004b) raised to approximately P60,

tested in the elevated zero maze, the straight channel, and the MWM and animals

were sacrificed at 0, 30, 60, or 120 minutes following the last trial of the MWM.

The average day of sacrifice for this group was P68 (P68 group). Eight animals

(4 males, 4 females) per litter were used for behavioral testing. The remaining 2

animals (1 male, 1 female) were not tested, however, they were removed from the

colony room to the testing suites on each of the days testing occurred and therefore served as “untrained,” but disrupted, age-matched controls for P68

BDNF, NGF and CORT analyses.

Corticosterone and Neurotrophin Assessment

Animals were sacrificed by rapid decapitation between 1200 and 1600 h.

Trunk blood was immediately collected in 12 x 70 polyethylene tubes containing

0.05 ml EDTA (2%) and chilled on ice prior to centrifugation. Tubes were centrifuged at 2500 RPM (1399 RCF) for 25 minutes at 4 °C for separation of plasma. Plasma was removed and stored at -80 °C until analysis of CORT.

Plasma CORT concentrations were analyzed via EIA kits obtained from

ALPCO Diagnostics (Windham, NH). Frozen plasma was thawed on ice, and

95 diluted 1:3 for Experiments 1 and 2 and 1:10 for Experiment 3, and then assayed

in duplicate in 96 well plates as described by the manufacturer’s instructions.

The brains of the animals were removed and dissected; the hippocampus

and hypothalamus were collected. These regions were frozen on dry ice

following dissection and then stored at -80°C until BDNF and NGF protein

assessment via ELISA. All tissue was collected between 1200-1600 hours.

BDNF and NGF protein were assessed via ELISA (Promega Corporation,

Madison, WI). Frozen samples were weighed and sonicated in a 1:10 (mg:µl) volume of ice-cold lysis buffer (formula from Promega Corp.). Samples were maintained on ice until assayed. Further dilutions for BDNF (1:2) and NGF

(1:10) were prepared with block and sample buffer supplied by the manufacturer.

Samples were assayed in duplicate and the protocol followed as provided. Total

protein was assessed using the BCA Protein Assay Kit (Pierce Biotechnology,

Rockford, IL). For protein quantification, the samples were further diluted 1:20

in saline before being assayed. The protocol for microplates was followed as

supplied by the manufacturer. Optical densities for all EIAs were measured on a

SpectraMax Plus microtiter plate reader (Molecular Devices, Sunnyvale, CA).

Results for BDNF and NGF were expressed as a percent of total protein in the

region examined.

Elevated Zero Maze

Beginning on the Thursday nearest P60 each animal was administered one

5-minute trial in the elevated zero maze apparatus. The maze consisted of a black

acrylic circular track 10 cm wide and 105 cm in diameter divided into quadrants

96 (San Diego Instruments, CA). Two quadrants opposite each other have 1.3 cm clear acrylic curbs on either side to prevent the animal from falling but otherwise are left open on the sides and are hereafter referred to as the open quadrants. The other two quadrants are referred to as closed and have 28 cm tall black acrylic walls on both sides (Shepherd et al., 1994). Overhead fluorescent lights were on during testing. The maze is elevated 72 cm off the floor. A video cassette recorder attached to an overhead camera recorded each session for later analysis.

At the beginning of each trial the subject was placed in a closed quadrant to start the 5-min trial. For each animal, the number of stretch-attends (defined as an event when the animal has its head and front two paws extending into an open quadrant and the remainder of the body in a closed quadrant), head dips, and open entries as well as the total amount of time spent in the open quadrants of the maze was assessed. The maze was wiped clean of urine and feces with 70% ethyl alcohol between animals.

Straight Channel Swimming

On the day following Zero Maze testing, each animal was administered four timed trials in a straight channel apparatus to test for swimming ability. The straight channel was 15 x 244 cm composed of grey PVC plastic and filled with water (22±1 °C) to a depth of 35 cm. A wire escape ladder was placed at one end.

At the beginning of each trial the animal was placed facing the back wall of the channel at the opposite end from the ladder. A trial began when the animal was released and ended when the escape ladder was grasped.

Morris Water Maze (MWM)

97 Testing in the MWM began three days following straight channel trials.

The MWM is used to test the spatial learning and memory ability of rodents

(Brandeis et al., 1989;D'Hooge and De Deyn, 2001;McNamara and Skelton,

1993;Morris, 1981). The maze used herein consisted of a tank 210 cm in

diameter and painted flat black. The escape platform (10 x 10 cm square) was

made from clear acrylic with a wire mesh attached to the top for traction and was

submerged 2 cm below the water surface; water temperature was 22±1 °C.

Animals were administered four trials per day for 5 days of spatial learning. The

tank was divided into four equal quadrants, with cardinal points designated with

compass headings and north arbitrarily the point furthest from the experimenter.

The escape platform was placed in the center of the SW quadrant and animals

were started from one of four start positions (NW, N, E, and SE) in pseudo-

random order with the restriction that one trial began from each start position per

day. Each trial had a time limit of 120 s with a 15 s inter-trial interval (ITI). If

the animal did not find the platform in the allotted time it was manually placed on

the platform for the ITI. Swim paths and latencies were recorded by an overhead

camera and video-tracking system (Polytrack System, San Diego Instruments, San

Diego, CA).

Principal measures recorded were latency, path length, cumulative distance from the platform (recorded every 55 msec) and angle of deviation (first bearing) from a direct line to the platform (measured during the first 13 cm of recorded path at the beginning of each trial). Supplemental analyses of time spent in specific annuli and number of direct swim events to the target were also

98 analyzed. For annuli determination, a 20 cm wide ring was drawn inside the maze using Polytrack software spanning the inside and outside corners of the platform; this area hereafter is referred to as the target annulus. The ring between the outer wall of the maze and the target annulus was designated the outside annulus and the remainder, the inside annulus. For direct swims (Saucier et al., 1996) a 36 cm corridor was demarcated using Polytrack software from the start position directly to the target with the start position and target being in the center of the corridor

(see Figure 2.1).

On the final day of MWM testing two animals (1 male, 1 female) from each treated and control litter were sacrificed 0, 30, 60, or 120 min immediately following their final trial in the maze. The untrained pair from each litter was sacrificed during the same time period as their littermates.

Varying times of sacrifice were utilized to assess the CORT changes and any BDNF and/or NGF protein changes.

Statistics

Analysis of variance (ANOVA) was used to determine treatment effects.

Neurotrophin levels in the hippocampus and hypothalamus were expressed as a percent of total protein in these regions. Treatment was a within litter factor for experiment-1 and experiment-2 and a between litter factor for experiment-3. All analyses included treatment and sex as within factors and the MWM had additional within factors: day and trial. Direct swim events were calculated as proportions expressed graphically as a percent of total swim events; significance was determined using Fisher’s Test for uncorrelated proportions (Walpole and

99 Myers, 1985). A p<0.05 level of confidence was used to determine significance and trends were noted at p < 0.10.

Results

Corticosterone

Experiment-1: MA-treated animals showed a trend toward decreased plasma CORT on P15 (F (1,9)=4.13, p<0.07; Figure 2.2B).

Experiment-2: There was a main effect of treatment (F (1,8)=48.74, p<0.0001; Figure2. 2A) showing significantly higher levels of CORT in MA- treated animals on P11.

Experiment-3: CORT levels were analyzed separately for the untrained and trained groups. For the untrained group, the values were not normally distributed and therefore were subjected to a log transformation prior to ANOVA.

Among the untrained animals there was a significant difference in CORT levels.

Animals treated neonatally with MA had lower CORT levels (F (1,22)=7.41, p<0.02; Figure 2.3). There was also a main effect of sex (F (1,22)=11.65, p<0.003) with females having higher CORT levels.

For the trained animals, there was no main effect of treatment. As in the untrained animals, females had higher CORT levels in plasma [sex (F

(1,22)=5.57, p<0.03; Figure 2.4A)] NOTE: returning to baseline. A significant main effect of time (F (3,66)=32.81, p<0.0001; Figure 2.4B) was noted showing that animals had higher levels of CORT at 0 and 30 minutes vs. levels at 60 and

120 minutes post training. The levels observed at 120 were comparable to those

100 obtained in the untrained animals. There was also a sex by time interaction

(F(3,66)=4.15, p<0.01; Figure 2.4C). The sex by time interaction demonstrated female CORT levels were increased vs. those in males at 0, 60, and 120 minutes

(see Figure 2.4C).

BDNF

Experiment-1: Significant differences in hippocampal BDNF levels from

MA treatment were observed on P15. BDNF levels were approximately 8% higher in the hippocampus of MA-treated animals compared to SAL controls (F

(1,9)=13.44, p<0.005; Figure 2.5). No sex differences were observed at this age.

No P15 hypothalamic values were obtained due to assay technical problems.

Experiment-2: No MA-induced differences were seen in BDNF at P11; however, there were sex differences in BDNF levels in the hippocampus and hypothalamus at this age. BDNF levels were higher in males for both the hippocampus (F (1,9)=169.18, p<0.0001; Figure 2.6), and hypothalamus (F

(1,9)=7.91, p<0.02; Figure 2.6).

Experiment-3: On P68 following behavioral testing, there was a significant treatment by time interaction (F(3,51)=3.48, p<0.02; Figure 2.7).

Two-group comparisons at each time point failed to show significant group differences; however, MA-treated animals showed a trend toward higher levels of hippocampal BDNF 60 min following their last trial in the MWM (p<0.10). No significant differences were found for BDNF between trained (MWM) and untrained animals inclusive of treatment groups nor for comparison of SAL- trained vs. SAL-untrained animals.

101 NGF

Experiment-1: No treatment effect or treatment x sex interaction was seen for NGF at P15. P15 animals showed a sex difference in the hippocampus with higher NGF levels in females compared to males (F (1,9)=18.39, p<0.002; Figure

2.8A).

Experiment-2: No significant NGF differences were seen at P11.

Experiment-3: Trained P68 animals showed a sex effect in the hypothalamus with lower levels in males compared to females (F(1,18)=11.11, p<0.004) as well as a sex by treatment interaction (F(1,18)=5.18, p<0.04; Figure

2.8B) showing lower levels of NGF in MA-treated females vs. SAL-treated females. Untrained P68 animals showed only a trend toward a main effect of sex in the hypothalamus (F(1,18)=3.04, p<0.098; lower levels in males), but there was a sex by treatment effect (F(1,18)=6.09, p<0.02; lower levels in MA-treated females compared to SAL-treated females; Figure 2.8B). No significant differences were found for NGF between trained (MWM) and untrained animals inclusive of treatment groups nor for comparison of SAL-trained vs. SAL- untrained animals.

Experiment-3: Behavioral Results

Elevated Zero Maze

No differences in any measure were seen between treatment groups or for the treatment by sex interaction. A sex effect was seen for most parameters.

Female rats spent significantly more time in the open (F (1,20)=12.68, p<0.002), showed a trend toward more open entries (F (1,20)=3.83, p<0.06) and performed

102 more stretch attends (F (1,20)=15.72, p<0.001). No sex difference was seen for the number of head dips.

Straight Channel Swimming

No differences in swimming performance were observed between treatment groups or between sexes in this task.

Morris Water Maze (MWM)

There were treatment effects on latency, path length, cumulative distance, and first bearing. ANOVA revealed a significant group effect of treatment on latency (F (1,22)=18.52, p<0.0003; Figure 2.9A); but no significant interactions.

There was also a significant sex effect in which females took significantly longer to reach the goal versus males (F (1,22)=15.16, p<0.0008). The treatment group main effect revealed impairment in reaching the goal in MA-treated animals for path length (F (1,22)=14.13, p<0.001; Figure 2.9B), cumulative distance (F

(1,22)=18.82, p<0.0003; Figure 2.9C), and first bearing (F (1,22)=31.85, p<0.0001; Figure 2.9D). A significant main effect of sex was also present for these measures, path length (F (1,22)=14.89, p<0.0009), cumulative distance (F

(1,22)=16.39, p<0.0005), and first bearing (F (1,22)=6.20, p<0.0208) demonstrating the females did not learn the task as well as their male counterparts in each measure.

Further analyses revealed that MA-treated offspring spent a larger percentage of time in the outer annulus (F (1,22)= 10.30, p<0.004; Figure 2.10A), and a smaller percentage of time in the target annulus vs. controls (F (1,22)= 7.55, p<0.012; Figure 2.10B) as well as a smaller percentage of time in the inside

103 annulus (F (1,22)= 9.11, p<0.006; Figure 2.10C). In addition, an analysis of direct swims to the target revealed MA-treated offspring had significantly fewer direct swim events than SAL-treated animals (Z = -4.606, p<0.01; Figure 2.10D).

Discussion

We hypothesized that animals treated neonatally with MA would have altered levels of neurotrophins that might contribute to long-term spatial learning and memory deficits. The results showed increased levels of BDNF on P15, but not on P11 and not consistently on P68. Hence, the results are not conclusive with regard to what role neurotrophin may play in the mechanism of the MA- induced long-term learning deficit as treatment induced changes in neurotrophin concentrations were not consistent, however only a few time points were assessed thereby limiting the generalization of this interpretation. Neurotrophin levels, specifically NGF and NT-4/5, have been reported to be influenced by CORT in the developing animal (Roskoden et al., 2004). Neurotrophin levels have also been reported to be influenced by CORT in the adult (Kesslak et al., 1998;Schaaf et al., 1997;Schaaf et al., 1998). However, in the developing animal, the CORT- induced changes in neurotrophins appeared only after ten days of CORT administration (Roskoden et al., 2004). This indicates that the developmental response to elevated CORT must be sustained in order to have an effect and this may explain why the shorter period of MA-induced CORT produced by our model may not be sufficient to cause larger neurotrophin changes.

104 It is known that CORT levels are significantly elevated during our MA dosing period, especially on P11 and P15 (Williams et al., 2000) and as shown in

Experiment-2 after P11 MA treatment. Previous work (Schaaf et al., 1997;Schaaf et al., 1998) has demonstrated a dose-dependant response for BDNF message and protein showing decreases with increased levels of CORT administered in adult animals. By contrast, our data show BDNF protein levels are elevated by MA as is CORT release. Whether the change in BDNF is caused by the CORT release is presently unknown. Using continuous infusion of high levels of CORT, it has been shown that P1-10 exposure increase NGF and NT-4/5 (Roskoden et al.,

2004). The BDNF changes seen in P15 MA-treated animals were not seen in P11

MA-treated animals but more than likely this was due to the short drug exposure time in the P11 animals since this exposure does elevate CORT (Williams et al.,

2000). It may be that MA-induced an increase in BDNF at P15 because this is a critical stage of expression for this neurotrophin or because the P15 MA-treated animals had received more doses of MA than the P11 group. We did not include a MA treated group given MA only on P15 so the present experiment cannot determine whether day or number of doses was the determining factor. In addition, it is unknown whether the ~ 8% change seen on P15 is sufficient to induce long-term learning deficits; this will require further studies to resolve.

Neonatal animals did show a MA-induced increase in CORT in

Experiment-2 on P11 similar to the MA-induced neonatal increases shown previously (Williams et al., 2000). A similar MA-induced increase in CORT was not observed on P15 in Experiment-1; these animals were sacrificed 60 minutes

105 following the second MA dose on P15. CORT data for the P15 60 minute time point in the Williams et al. report (Williams et al., 2000) also did not display a significant MA-induced increase in CORT compared to SAL-treated animals but did show significance compared to the handled controls; however, the P15 30 minute time point did display a MA-induced CORT increase compared to SAL animals. As a result, we may have missed the correct time point to show a MA- induced increase in CORT on P15 in the current study. We did not obtain samples 30 minutes post dosing to address this issue.

Previous studies in neonatal animals have not investigated the potential for sex differences in neurotrophin expression. In the present experiments, however, there were sex-dependent differences for both NGF and BDNF. BDNF was lower in females than in males in both the hippocampus and hypothalamus at P11.

Hippocampal NGF, on the other hand, was higher in females at P15 than in males.

Neurotrophin protein levels obtained in the current study are similar in value to those reported in the comprehensive developmental study by Das et al.

(Das et al., 2001). Peak values for BDNF protein occurred near P14 at levels of approximately 90 pg/mg total protein and peak values for NGF protein occurred near P7 at levels of approximately 300 pg/mg total protein (Das et al., 2001).

BDNF protein levels obtained at P15 in the current study averaged 65-70 pg/mg total protein; protein levels on P11 averaged near 60 pg/mg showing that we also saw an increase in BDNF protein from P11 to P15. NGF levels obtained in the current study at P11 averaged near 210 mg/kg, close to the P7 value of 300 pg/mg reported in Das et al. (Das et al., 2001); however, we showed an increase from

106 P11 to P15 in NGF protein with P15 values averaging near 500 pg/mg. Despite

the range between our reported values and those reported by Das et al. (Das et al.,

2001), our values remain comparable in terms of magnitude.

Several studies have suggested that removal or blockade of BDNF

function results in spatial learning impairments (Linnarsson et al., 1997;Mizuno et

al., 2000;Mu et al., 1999). BDNF exposure is also known to enhance LTP

(Figurov et al., 1996) and NMDA signaling (Lin et al., 1998;Lin et al., 1999;Suen

et al., 1997). Our data did not demonstrate a learning-induced increase in BDNF

as might have been expected based on reports of increased BDNF mRNA

following MWM learning (Kesslak et al., 1998) nor did we observe learning-

induced changes in NGF protein. It is possible that we did not see learning-

induced changes in BDNF and NGF because we did not sample at the optimal

time intervals, although this seems unlikely since we see learning deficits after

P11-15 MA treatment (Williams et al., 2003b), or because we did not examine

mRNA levels (Kesslak et al., 1998). It is also possible that the animals did not receive an adequate number of training trials to produce such effects based on the fact that Kesslak et al. (Kesslak et al., 1998) only observed learning-induced increases in BDNF mRNA following 24 trials of acquisition learning administered over the course of three days and also after 48 trials administered over six days of training, the current study contained only 20 trials. Messenger

RNA determination would be necessary to help resolve some of these issues in our experiments.

107 CORT evaluation following MWM testing showed main effects of time, sex, and a sex by time interaction. Overall CORT values in the current study were elevated 0 and 30 minutes following testing and returned to near baseline

(compared to non-tested animals) by 60 minutes. In agreement with previous reports (Beiko et al., 2004) female rats had significantly higher CORT values than males following MWM learning. It is important to note that despite differences in procedure, twelve trials performed in a single day with pretraining (Beiko et al.,

2004;Morris, 1989) compared to our procedure of administering four trials per day for five days, both studies elicited increased levels of CORT in female rats vs. male rats following MWM testing. Furthermore, female rats had poorer behavioral performance than their male counterparts in our study indicating further that there may be a relationship between CORT levels and MWM performance. One of the initial experiments addressing the role of CORT relative to MWM training (Sandi et al., 1997) concluded that CORT serves as a facilitator of information storage during the post-training period. This effect was observed as a result of experimental condition (water temperature of 19° C vs. 25° C) and as a result of exogenously applied CORT but no additive effects of improved behavior were present indicating the level of CORT is a critical factor (Sandi et al., 1997).

Sex differences across factors point to a pattern among the female animals.

Female rats have less BDNF on P11, higher CORT following MWM, and poorer behavioral performance than male rats. Despite not showing a clear correlation

108 between the role of neurotrophins and the mechanism of MA-induced learning deficits, this pattern suggests some relationship may indeed exist.

The results from MWM testing replicated our previous findings that P11-

20 MA treatment results in long-term spatial learning impairments (Vorhees et al.,

1994a;Vorhees et al., 2000;Williams et al., 2002;Williams et al., 2003c). In addition, we analyzed additional parameters in this experiment. Analyses of swimming patterns by annuli showed that MA-treated offspring spent a significantly higher percentage of their swim path in the outer annulus against the sidewall of the tank. Cain and colleagues (Cain et al., 1996) have shown that this pattern is characteristic of animals that have not learned the most fundamental aspects of the task, i.e., that the escape platform can only be found by learning to swim away from the wall. The fact that MA-treated offspring persist in thigmotactic behaviors suggests a fundamental substrategy that is critical for this task is impaired (that is not spatial) in the MA-treated offspring along with spatial mapping difficulties (Williams et al., 2002). Not surprisingly, since the MA- treated offspring swam more in the outer annulus, they swam proportionately less in the target and inner annuli. This experiment included neither a probe trial nor reversal learning trials as was done in previous studies (Vorhees et al.,

1994a;Vorhees et al., 2000;Williams et al., 2002;Williams et al., 2003c). A probe trial was not included because our purpose was to obtain neurotrophin measurements after the last learning trial since previously published data suggested that, at least for BDNF, time since learning is a critical factor in identifying this transient effect (Kesslak et al., 1998;Mizuno et al., 2000;Schaaf et

109 al., 2000;Schaaf et al., 2001). Reversal learning was not included for the same

reason. The effects on BDNF reported previously after MWM testing have been

on acquisition not on reversal learning (Kesslak et al., 1998;Schaaf et al., 2001)

therefore inclusion of reversal trials might have been counterproductive.

Another indication of the MA-induced MWM deficit was seen for direct

swims. This measure assesses the extent to which an animal has learned the

general direction of the goal even if it does not know its precise position. We set a start-to-goal corridor width that was larger than that used by Cain et al. (Cain et al., 1996). However, additional analyses with different corridor widths gave the same result. Controls increased their direct swims across days whereas MA- treated offspring swam outside the direct-path corridor more often. This finding supports the idea that MA-treated animals have a compound deficit in which they acquire both non-spatial and spatial components of the MWM more slowly than controls, yet are not so impaired that they never attain asymptotic performance; rather their rate of learning is significantly lower than controls.

In conclusion, our data do not support a key role for neurotrophins in the mechanism of MA-induced long-term learning and memory deficits. However, the CORT data indicate a possible long-term change in the HPA system as a result of neonatal MA treatment that warrants future exploration.

110

Figure 2.1: MWM Diagrams for Annuli and Direct Swim Corridor Designation Diagrams of MWM with annuli and direct swim corridor designations: target annulus = white area encompassing the target platform (target annulus represents an area 20 cm in width, not drawn to scale); outside annulus = black area; inside annulus = shaded gray; corridor for direct swims having a start position of N and target position of SW Note: alley represents an area 36 cm in width, not drawn to scale.

A. B. P11 P15

35 **** 35

30 30

25 25 ) ml) 20 20 ng/ (ng/ml

T 15 15 CORT ( COR 10 10 †

5 5

0 0 SAL MA SAL MA

****p<0.0001 †p<0.07

Figure 2.2: Neurotrophin Study Neonatal CORT

Mean (± SEM) CORT (ng/ml) in neonatal rats A) P11 SAL vs. MA, † ****p<0. 0001; B) P15 SAL vs. MA, p<0.07

111 120

100 ** 80

(ng/ml) 60

40 CORT

20

0 SAL MA

Figure 2.3: Neurotrophin Study Adult CORT Mean (± SEM) CORT (ng/ml) in untrained adult rats treated P11-20 with MA or SAL (10 mg/kg x 4 per day), **p<0.013 Note: data analyses were based on log transformed data for normalization.

A. Sex; p<0.03 B. Time; p<0.0001 C. Sex x Time; p<0.01

180 250 275 * 160 225 250

200 225 140 200 175 120 ) )

l 175 ml ml

m 150 / / / g g 100 g 150 n (n (n ( 125 T 125 RT R

80 RT 100 CO CO CO 100 60 75 75 40 Male 50 50 Female 20 25 25

0 0 0 Male Female 0 30 60 120 0 30 60 120 Sacrifice time (min) Sacrifice time (min)

Figure 2.4: Neurotrophin Study Adult CORT time course Mean (± SEM) CORT (ng/ml) A) learned adult rats treated P11-20 with MA or SAL (10 mg/kg x 4 per day) in males vs. females, *p<0.03; B) learned adult rats treated P11-20 with MA or SAL (10 mg/kg x 4 per day) main effect of time, p<0.0001; C) trained adult rats treated P11-20 with MA or SAL (10 mg/kg x 4 per day); overall sex x time interaction, p<0.01 Note: data analyses were based on log transformed data for normalization.

112

respec hypothalam Mean (±SE Figure 2.6:Neurotrophin treated animals(10m Mean (±SE Figure 2.5:Neurotrophin

pg/mg protein pg/mg Protein 10 20 30 40 50 60 70 80 0 tive 80 50 55 60 65 70 75 l y u M) BDNF(pg/m M) BDNF(pg/m s inm a Ma le vs.fe Hippocampus l g e /kg x4perdayonP11-14and3P15),**p<0.005 StudyBDNFsexeffect Studytreatmenteffect m Fe g g SAL a m tota totalprot ** le anim a ** le l protein)inthehippocampusand als atP11,****p<0.0001, *p<0.02 ein) inthehippocampusMAvs.SAL 10 20 30 40 50 60 70 80 MA ** 0 Hy Ma pothalamu l e Female * s 113

30 †

28

26 ein ot r 24 /mg p g p 22

20 Saline MA

18 03060 120

Time (min)

Figure 2.7: Neurotrophin Study BDNF time x treatment effect Mean (± SEM) BDNF (pg/mg total protein) in the hippocampus overall time x treatment interaction at P68, *p<0.02 Two way comparison of treatment at time 60, †p<0.1

114 A

P15

600 **

550

500 n ei t o r 450 g p /m

pg 400

350

300 Male Female

B P68 Trained P68 Untrained

110 110

100 100

90 90 n i n ei t ote o r 80 80 /mg pr /mg p g p pg 70 70

60 60

50 50 SAL-Male MA-Male SAL-Male MA-Male SAL-Female MA-Female SAL-Female MA-Female

Figure 2.8: Neurotrophin Study NGF sex effect; sex x treatment effect Mean (± SEM) NGF (pg/mg total protein) A) sex effect in the hippocampus in male vs. female animals at P15, **p<0.002; B) sex x treatment effect in the hypothalamus at P68, trained animals *p<0.04, untrained animals *p<0.03

115

(degrees) *****p<0.0001 (cm Mean (±SE Figure 2.9:Neurotrophin Cumulative Distance (cm) C A

10 11 12 13 14 15 16 Latency (s) ) 00 00 00 00 00 00 00 ***p<0.001;C)Cum 30 35 40 45 50 55 0 0 0 0 0 0 0 M) inMAvs.SALA) SA SAL L u MA MA **** **** Study MWMtreatmenteffect lative D Latency (s)****p<0.0005;B)PathLength i stan ce (cm Path Length (cm) B D First Bearing (degrees) 10 11 12 13 14 15 00 00 00 00 00 00 45 50 55 60 65 40 ) ****p<0.0005;D)FirstBearing SA SAL L MA MA ***** *** 116

A Outside B Target C Inside ** 70 60

) 50 s (

e 40 m i 30 % T * 20 ** 10 0 SAL MA SAL MA SAL MA

D 35 5.5 30 Saline 5.0 MA 25 s 4.5 s m i im

w 20 Sw S 4.0 t

3.5 rec 15 # Di 3.0 10 % Direct ** 2.5 5

2.0 0 SAL MA 012345 Day

Figure 2.10: Neurotrophin Study Annuli and Direct Swim treatment effect Mean (± SE M) Percent A) tim e spent in the outside annulus, **p<0.004; B) time spent in the target annulus, **p<0.012; and, C) time spent in the inside annulus, *p<0.006; D) direct swim events to target, **p<0.01; direct swims, treatment by d

117 CHAPTER 3 - MA-Induced Increased Levels of CORT During the SHRP and

Neonatal MA-Induced Long Term Changes in Basal CORT in the Adult:

Implications for Spatial Learning and Memory Deficits.

Abstract

Long-term spatial learning and memory deficits result from treatment of neonatal rats with methamphetamine (MA) from P11-20 or P11-15 but not from

P1-10 in the Morris Water Maze (MWM). Neonatal MA dosing also causes large increases in plasma corticosterone (CORT) on P11 and P15 despite occurring during the stress hyporesponsive period (SHRP). Increased glucocorticoid concentration during development has been shown to be detrimental to normal development and behavior in rats. Due to evidence of increased CORT during development at a time of hypothalamic-pituitary-adrenal (HPA) axis downregulation we hypothesized that this large CORT increase may permanently alter the development of the HPA axis resulting in altered response to stress in adulthood, and, in our situation, manifest as long-term learning and memory deficits. Experiments were designed to control CORT release. Attempts to block the MA-induced CORT increase during the dosing period in the neonate produced undesirable and inconsistent results. Ability to control CORT release in adult animals during behavioral testing was explored. Animals were administered MA from P11-15 and raised to adulthood. Prior to behavioral testing adult animals were treated with metyrapone (MET) via s.c. injection or were bilaterally adrenalectomized (ADX) with CORT replacement therapy. Tests of anxiety

118 (Zero Maze), swimming (Straight Channel), and spatial learning and memory

(MWM) were performed. Pre-treatment with MET demonstrated a MET attenuation of MA-induced MWM learning deficit in the reversal phase of testing and a MET attenuation of MA-induced MWM memory deficit in probe trials of the acquisition phase. Adult treatment of ADX with CORT replacement (ADX-

C) suggested ADX-C attenuation of MA-induced MWM learning deficit in the double reversal phase of testing and showed an ADX-C attenuation of MA- induced MWM memory deficit in probe trials of the double reversal phase.

Taken together, results suggest a role for CORT in the MA-induced learning and memory deficits observed in the MWM supporting the hypothesis. Other methods of controlling CORT, especially during the period of MA dosing, should be explored.

Introduction

Methamphetamine (MA) use continues to make headlines across the nation. Continued high demand for the drug has enabled spread of drug production in clandestine laboratories from East to West coasts (Brecht et al.,

2004). Reasons expressed in the literature for MA use include drug substitution, cost effectiveness, maintain alertness, enhance sexual performance, and method of weight loss as well as experimentation due to curiosity and ease of acquiring the drug (Brecht et al., 2004;Buffum, 1982;von Mayhauser et al., 2002).

Furthermore, there are reports to suggest that drug use increases the likelihood of sexual contact (Buffum, 1988); this taken together with “enhanced sexual

119 performance” as a motivator of drug use suggests serious implications of potential prenatal exposure to MA. Long-term effects following prenatal human exposure to MA are largely unknown. The limited data that do exist suggest reductions in intrauterine growth, head circumference, and length (Little et al., 1988) and irregularities in untrasonography and fMRI tests (Dixon and Bejar, 1989b;Smith et al., 2001).

We have developed the rat model system of neonatal exposure to MA

(Vorhees et al., 1994a;Vorhees et al., 1994b). Rat brain development through P19 is correlative to human third trimester brain development in respect specifically to continued development of the hippocampus (Bayer et al., 1993;Rice and Barone S

Jr, 2000). MA administration from P11-20 or P11-15 in our experiments has reliably produced long-term deficits in spatial learning and memory in the MWM when animals are tested as adults (P60+) (Vorhees et al., 1994a;Vorhees et al.,

1998;Vorhees et al., 2000;Williams et al., 2002;Williams et al., 2003c;Williams et al., 2003b;Williams et al., 2004b) as well as large increases in corticosterone

(CORT) during the dosing period (Williams et al., 2000). It is important to note that the period of MA dosing occurs during the stress hyporesponsive period

(SHRP) in the rat (Levine, 1994;Sapolsky and Meaney, 1986) a time characterized by low stable circulating CORT levels and largely non-responsive nature to stressors as compared to adult animals. We have also discovered a long- term decrease in basal plasma CORT in animals having received MA from P11-

20.

120 The purpose of these experiments was to explore the relationship of the

MA-induced increase in neonatal CORT and its possible long-term implications on function of the hypothalamic-pituitary-adrenal (HPA) axis and subsequent meaning to MA-induced long-term learning and memory deficits as the behavioral task itself can be considered a stressor (Roozendaal et al., 1996). We hypothesized that neonatal MA administration causes permanent alteration of the stress/CORT response system resulting in impaired spatial learning and memory deficits in the Morris Water Maze (MWM).

Corticosterone (CORT) is a 21 carbon adrenal steroid hormone. It is produced by a series of enzymatic reactions including precursors such as cholesterol and progesterone. The final step of synthesis involves the conversion of 11-deoxycorticosterone to CORT via hydroxylation reaction by the 11β- hydroxylase enzyme. The official chemical name of CORT is 11β,21- dihydroxypregn-4-ene-3,20-dione indicating the type and position of the molecular groups attached to the main carbon chain (Figure 3.1).

CORT is an essential component of normal homeostatic maintenance. A primary function of this steroid hormone is to help regulate the conversion of amino acids into carbohydrates and glycogen by the liver, and to help stimulate glycogen formation in the tissues. CORT is the predominant GC secreted by the adrenal gland in rodents. Glucose production, availability and metabolism via glycolosis are essential to numerous functions in the body through production of

ATP.

121 CORT is a precursor in the synthesis of aldosterone. A chief function of aldosterone is regulation of electrolytic balance in the body through direct interaction with the kidney. Aldosterone causes a decrease in the rate of sodium- ion excretion and an increase in the rate of potassium-ion excretion. Aldosterone secretion is controlled by sodium-ion concentration (aldosterone is released when sodium ions are low) as well as by the renin–angiotensin system. Edema is often a result of pathologic/chronic elevation in aldosterone due to the excessive retention of salt and water by the body. Removal of the adrenal glands through surgery results in a need for sodium supplementation to the animal because of the resulting lack of aldosterone (McEwen et al., 1986).

CORT binds to both mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) but with different affinity. MRs, also known as type-1 receptors, selectively bind aldosterone and are also occupied by CORT at homeostatic levels. These receptors have a high concentration in limbic regions such as the amygdala, hippocampus, and septum. GRs, type-2 receptors, bind CORT with a ten fold lower affinity than MRs. CORT occupies GRs in times of stress; thus,

GRs are widely distributed in high concentrations in areas of the brain associated with the stress system, specifically in the hippocampus, septum, and parvocellular neurons of the PVN (de Kloet et al., 1990). Common antagonists for MC and GC receptors include RU28318 and RU38486 respectively (Korte et al., 1995).

Controlling the stress/CORT response is a way to better understand the system and its interaction with biological and environmental cues.

Adrenalectomy (ADX) involves removal of the adrenal gland (or glands) by

122 surgical means. A “pharmacological adrenalectomy” can be performed with the

use of the drugs to block CORT production. These two means of manipulating

the stress response system have been widely used in research studies.

Metyrapone (MET) was used initially by Jenkins in 1958 for CORT

blockade capacity in the dog (Jenkins et al., 1958). The official chemical name of

MET is 2-methyl-1,2-di(3-pyridl)-1-propanone indicating the type and position of

the molecular groups attached to the main carbon chain (Figure 3.1).

MET specifically blocks the action of the rate limiting enzyme 11β- hydroxylase through inhibition of cytochrome P-450 in the final step of CORT production (Jenkins et al., 1958;Williamson and O'Donnell, 1969a) and reduces plasma CORT levels to that of normal basal levels seen at the beginning of the light phase; MR’s remain occupied, GR’s remain unoccupied (Czyrak et al.,

1997). Due to decreased circulating CORT, the negative feedback loop is less reactive resulting in increased plasma ACTH (Jorgensen and Aakvaag, 1973).

Cytochrome P-450 is the oxygen activating steroid binding component of the enzyme mechanism; this process, and subsequently the blockade by MET, occurs at the level of the mitochondria in adrenal cells (Williamson and O'Donnell,

1967;Williamson and O'Donnell, 1969b). A report by Liddle (Liddle et al., 1958) showed the same MET-induced inhibition of CORT production in humans.

Clinically, MET is used to test adrenal glands for correct cortisol output under perceived normal conditions, or under conditions of stress, or illness; it is also used to treat Cushing’s Syndrome (hypercortisolemia) (Schoneshofer and Claus,

1985).

123 Caution must be taken when using MET to block CORT production as a means to block stress. Some evidence suggests that MET administration is a stressor itself as it has caused increases in plasma ACTH, glucose, and c-fos activity (Herman et al., 1992;Rotllant et al., 2002).

Ketoconazole (KTZ), an imidazole derivative, was found to block adrenal steroid synthesis (Pont et al., 1982;Van den et al., 1980). The official chemical name of KTZ is (±)-cis-1-acetyl-4-(4-[(2-[2,4-dichlorophenyl]-2-[1H-imidazol-1- ylmethyl]-1,3-dioxolan-4-yl)-methoxy]phenyl)piperazine indicating the type and position of the molecular groups attached to the main carbon chain (Figure 3.1).

KTZ was originally approved by the Food and Drug Administration for use as an antifungal agent for the treatment of candidiasis, chronic mucocutaneous candidiasis, oral thrush, candiduria, coccidioidomycosis, histoplasmosis, chromycosis, and paracoccidioidomycosis (Hume and Kerkering, 1983). KTZ works specifically to block GC production through inhibition of cytochrome P450

14α-demethylase during the conversion of lanosterol to cholesterol (Hume and

Kerkering, 1983;Loose et al., 1983;Lyman and Walsh, 1992).

Greater understanding of the biological response to stress and discovery of means to alter this response has lead to a vast area of research. One or more of these principles is often applied in an experiment to further determine the intricate relationship within the system itself and the system’s reaction to outside influence. Devenport and Devenport (Devenport and Devenport, 1983) examined

ADX surgery on P25 age Sprague-Dawley rats. Male rats underwent bilateral

ADX surgery at P25 and were sacrificed on P65 or P145. Significantly increased

124 brain size and decreased body weights were seen in all ADX animals; this effect could be reversed with CORT replacement only and not with aldosterone or deoxycorticosterone replacement. Another study examined the role of CORT in behavior resulting from an inescapable stressor, forced swim (FS) (Baez and

Volosin, 1994). Normal animals facing a FS stress become largely immobile with time; with increasing amounts of MET the animals spent less time immobile indicating CORT’s role in the ability to adapt to the stress (Baez and Volosin,

1994). This report concluded that CORT plays a key role in adaptation when rats are faced with a stressful task; and, that active behavior is relative to lower levels of CORT. An additional report by members of this group stated that an intraperitoneal injection of MET, 150 mg/kg, reduced the inactivity induced by exposure to an inescapable shock (Baez et al., 1996) further validating the role of

CORT in behavioral adaptation. ADX with CORT replacement was performed to examine the HPA response to acute restraint and chronic stress conditions with varying levels of circulating CORT and the subsequent effects on ACTH and CRF

(Akana and Dallman, 1997). Kamphius et al (Kamphuis et al., 2002) reported decreased HPA response (blunted CORT) to stress events in adulthood following neonatal treatment with the GR agonist dexamethasone (DEX).

Effects of both surgical and pharmacological ADX have also been studied relative to processes of learning and memory. A study by Oitzl and de Kloet

(Oitzl and de Kloet, 1992) revealed that ADX impaired spatial learning ability in the MWM in a time dependant manner. ADX animals displayed a learning deficit in locating a hidden platform when tested in the maze three days post-surgery

125 while testing ten days post-surgery resulted in no deficit. ADX animals did not show any deficit in locating a visible platform; bilateral removal of the adrenal medulla (ADMX) had no effect on water maze testing. This study also explored effects of injection of the MR antagonist (A-MR), spironolactone, and the GR antagonist (A-GR), RU38486 on MWM performance. Administration of A-GR

30-45 minutes before the first session of testing (4 trials, day 1) and immediately following the first session increased latency to escape vs. control in session two (4 trials, day 2); this deficit was no longer apparent by session three (4 trials, day 3).

Administration of A-MR 30-45 minutes prior to session 2 resulted in increased latency to escape in session 3. From these results, the authors conclude that GR’s and MR’s serve different roles in the spatial learning process; specifically, GR activation is required for storage and consolidation of spatial information while

MR activation is required for environmental evaluation and response (Oitzl and de Kloet, 1992). Roozendaal and others (Roozendaal et al., 1996) performed a similar experiment examining the effect of 25 or 50 mg/kg MET on CORT levels and performance in the MWM and the elevated plus maze. MWM acquisition performance was impaired by 50 mg/kg MET; retention performance was impaired by both doses of MET. Immediately following exposure to a conditioned stressor, only 25mg/kg MET attenuated anxiety as measured in the elevated plus maze (Roozendaal et al., 1996). A 75 mg/kg intraperitoneal injection of MET reversed anxiogenic behavior caused by restraint in elevated plus maze testing (Calvo et al., 1998). Furthermore, CORT administration without restraint reproduced the anxiogenic behavior.

126 Reasearch has shown that the experience of the water maze task itself induces stress in otherwise untreated (normal) animals as measured by a rise in plasma CORT from approximately 6 µg/dl to 46 µg/dl (Roozendaal et al., 1996).

Learning deficits in the water maze task can be produced by exposure to a footshock stress 30 minutes prior to testing but not by footshock exposure 2 minutes or 4 hours prior to testing (de Quervain et al., 1998) indicating that learning ability is relative to circulating levels of CORT. A subcutaneous injection of MET 50 mg/kg prior to the footshock blocked both the rise in CORT and learning impairment whereas administration of CORT (no footshock) mimicked results obtained with footshock. Experimental results must be examined carefully when CORT levels are a factor; significantly increased or decreased levels of CORT are able to impair the learning and memory process, indicating a biphasic dose-response effect on behavior (de Kloet et al.,

1988;Lupien and McEwen, 1997).

The present set of experiments was intended to explore the effects of controlling CORT on the behavioral deficits resulting from neonatal MA treatment. The first series of experiments explored the capability of blocking the

MA-induced CORT increase in the neonate during the P11-15 MA dosing period.

Neonatal injections of MET and KTZ were employed. Due to the inability to block MA-induced CORT beyond 24 hours or through the P11-15 chronic dosing with the neonatal injection procedures, a second attempt was made to block

CORT in the neonate with MET in a time-release pellet form. MET pellets in concentrations ranging from 200 mg to 35 mg were lethal. MET pellet

127 concentrations 25 mg to 10 mg were not lethal to the animals but did not sufficiently attenuate or block MA-induced CORT during the dosing period. Due to the failure to find a means to block CORT during the dosing period in the neonatal animal, it was decided that the hypothesis be tested at the level of the adult animal and CORT controlled before behavioral testing procedures.

The second set of experiments involving CORT blockade in the adult animal began with MET administration by time-release pellet in 75 mg, 50 mg, and 35 mg concentrations. Although these concentrations of MET were not lethal in the adult animal, none were able to block CORT increase in a preliminary FS stress test. Controlling CORT release in the adult animal was then tested via

MET injection, a procedure supported by the literature (Baez et al., 1996;Baez and Volosin, 1994;de Quervain et al., 1998;Roozendaal et al., 1996). This procedure was successful in blocking CORT increase induced by FS stress and was employed in subsequent behavioral experiments designed to test the hypothesis. A second method of CORT control in the adult, ADX with CORT replacement via osmotic mini pumps, was also successful in blocking FS stress induced CORT increase and was employed in behavioral experiments.

Controlling CORT during behavioral testing for MA and SAL treated animals alike enables equivalent CORT response to the inherent stress of the learning task.

If the hypothesis is proven correct, there should no longer be a deficit in behavioral performance of MA and SAL treated animal groups.

An important caveat of which to be aware when utilizing MET and ADX as methods of controlling CORT is the inherent differences between the two

128 methods. Both methods were used to account for shortcomings of using only one

vs. the other, however, despite ADX compensating for administration of a foreign

substance and daily injections, the ADX procedure has additional factors that should be considered when examining results. Since ADX involved the removal of the entire adrenal gland, factors produced by this gland other than CORT, which was replaced via osmotic mini pumps, were also compromised.

Endogenous maintenance of electrolyte balance is a function of aldosterone which is produced by the adrenal cortex, compensation for this function was provided for by exogenous salt administration via drinking water. Also important to consider are the losses of epinephrine (EPI) and norepinephrine (NEPI), chatecholamines produced and secreted by the adrenal medulla.

EPI and NEPI, a precursor of EPI, are involved in processes including increased heart rate, blood pressure, metabolism, and blood glucose enabling the body to react to and have sufficient energy available to react to a stress event.

NEPI has been implicated to have an important effect on memory; microinfusion of NEPI into the basolateral amygdala after training in the MWM enhanced retention of platform location (Hatfield and McGaugh, 1999). Other reports state the importance of adrenergic signaling in context of spatial and contextual memory specifically (LaLumiere et al., 2003;Murchison et al., 2004). EPI has also been directly implicated in memory as an enhancing factor when administered in rodents improving memory for tasks including inhibitory avoidance, visual discrimination, and appetitive learning (Gold, 2004;Sternberg et al., 1985;Torras-Garcia et al., 1998). Taken together, these data suggest that both

129 EPI and NEPI may impact processes of learning and memory; therefore lack of these catecholamines must be considered a factor in any results obtained demonstrating a change in behavior due to ADX.

130

OH

O CH HO 3

CH3

O

Corticosterone

CH3 O C C

CH3

N N

Metyrapone

N

N O O N N O O H3C H Cl Cl Ketoconazole

Figure 3.1:

Chemical structure of corticosterone and 2 pharmacologic CORT blocking agents, metyrapone and ketoconazole.

131 Materials and Methods

Subjects and conditions

Nulliparous Sprague-Dawley CD, IGS rats (Charles River, Raleigh, NC)

were obtained for in-house breeding with males of the same strain. Breeding

occurred following at least two weeks of environmental acclimation. Embryonic

day zero (E0) was defined as the day a sperm plug was detected. Females

remained with breeder males in hanging wire cages for a period of two weeks to

ensure each had equal time with a male. Following this period the females were

transferred to polycarbonate cages one per cage. The day of birth was defined as

postnatal day 0 (P0). Pups from these matings were the subjects of subsequent

experiments.

Metyrapone neonatal injection

The purpose was to determine if MET treatment would block the MA-

induced CORT increase. Litters for the MET neonatal injection studies were

culled to 10 or 12 pups without regard to sex.

Experiment-1: The first study had 3 treatment groups: VEH/SAL;

VEH/M10; MET/M10, with 4 pups per group (12 pups per litter). Vehicle (VEH)

consisted of 25% dimethylsulfoxide (DMSO) in saline (SAL); metyrapone (MET) was administered at a concentration of 50 mg/kg and 3 ml/kg volume; methamphetamine (MA) was administered at a dose of 10 mg/kg (M10). On P11

MET or VEH was administered followed by M10 or SAL 90 minutes later; four doses of M10 or SAL were given spaced 2 hours apart. One pup from each

132 treatment group was sacrificed 30 minutes after each of the four doses of M10 or

SAL.

Experiment-2: The next study was a replication of the initial experiment using MET at the higher concentration of 100 mg/kg because the 50 mg/kg dose was not effective.

Experiment-3: Additional control groups were added to the design of

Experiment-2 using the MET 100mg/kg dose to determine the effect of MET and

VEH on CORT; these additional groups were: MET/SAL, VEH/SAL, SAL/SAL, weighed only. There were 3 pups per treatment, one from each group sacrificed following the first, third, and fourth M10 or SAL dose.

Experiment-4: This experiment was designed to test effectiveness of MET

100 mg/kg beyond P11. This experiment contained six groups (2 pups each per litter) and followed the same MET (100 mg/kg in 25% DMSO) and MA (10 mg/kg) dosing as Experiments-1 and 2. Groups examined included MET/M10,

VEH/SAL, VEH/M10, SAL/M10, SAL/SAL, weighed only. VEH or MET was administered 90 minutes prior to the first M10 or SAL dose on P11, 4 doses of

M10 or SAL were given on P11 and one dose on P12 (no MET or VEH was administered on P12). One animal from each group was sacrificed 30 minutes and 105 minutes after the first M10 or SAL dose on P12.

Experiment-5: This study used a different VEH, 9.5% ethyl alcohol in

SAL, due to results from Experiments1-4 suggesting DMSO alone caused an increase in CORT. This experiment contained litters composed of 10 animals belonging to 5 different groups (2 animals per group): MET/M10; VEH/M10;

133 VEH/SAL; SAL/M10; SAL/SAL. MET was dissolved in a vehicle consisting of

9.5% ethyl alcohol in SAL. MET (100 mg/kg) was administered 90 minutes prior

to the first M10 or SAL dose; four doses of M10 or SAL were administered. One

animal from each group was sacrificed 30 minutes following the first and fourth

dose of M10 or SAL on P11.

Experiment-6: This experiment examined effects of MET on CORT on

P15 following MA dosing P11-15 (M10 dosing 4x/day 2 hours apart each day

with MET administered as the first dose each day 90 minutes prior to the first

M10 dose). One animal from each group was sacrificed 30 minutes after the 1st

and 4th M10 or SAL dose on P15 to determine if the MET-blockade remained effective after multiple days of MA administration.

All neonatal studies had an N of 6 or more for each individual group.

Metyrapone pellets, neonatal and adult studies

Experiment-7: Although MET blocked MA-induced CORT increase at the higher dose on P11, it was not effective on P15. This could be because of inter-dose rebound effects. Such effects might be suppressed by continuous MET exposure. This experiment sought to test this idea.

Sixteen litters of 8 pups each (preferentially male) were used to determine the effectiveness of metyrapone time release pellets (Innovative Research of

America, Sarasota, FL) in neonatal and adult rats.

For neonatal studies, litters were culled on P1. Pellets were inserted surgically in the nape of the neck under the skin using inhalation anesthesia on

P10 between 1300 and 1400 hours. No ethyl alcohol was used in surgical animal

134 preparation and Betadine solution vs. scrub was used; wounds were closed with

wound clips. Pellets were tested in the following concentrations (all were 21 day

release): 200 mg, 100 mg, 75 mg, 50 mg, 35 mg, 25 mg, 15 mg, 10 mg, 0 mg

(sham). Five litters were used to test the 200 mg pellets; 4 animals per litter received MET and the other 4 received sham pellets. Four litters were used to test the 100 mg pellets; 4 animals per litter received MET and the other 4 received sham pellets. Four litters were used to test the 75 mg, 50 mg, and 35 mg pellets; 2 animals per litter received 75 mg, 2 animals received 50 mg, 2 animals received

35 mg, and 2 animals received sham; any surviving pups received 4 doses of M10

(at 2 hour intervals) on day 1 post surgery and were sacrificed 24 hours after the first dose for trunk blood collection. Three litters were used to test the 25 mg, 15 mg, and 10 mg pellets; 2 animals per litter received 25 mg, 2 animals received 15 mg, 2 animals received 10 mg, and 2 animals received sham; surviving animals were dosed P11-15 with M10 (4x /day at 2 hour intervals) and half of each treatment group was sacrificed 24 hours following the first M10 dose (P12 sacrifice) and the other half was sacrificed 24 hours after the final dose on P15; trunk blood was collected.

Experiment-8: In the adult study, 39 Sprague-Dawley CD, IGS adult male rats (Charles River, Raleigh, NC) were obtained and housed for one week of environmental acclimation before surgical pellet implantation. Animals were prepared for surgery by having their backs shaved, sterile saline and betadine solution used to clean the surgical area, and placed under inhalation anesthesia

(isoflurane). A small incision was made near the nape of the neck and the wound

135 cleaned with cotton swabs. The pellet was placed in a cavity under the skin and the wound closed with surgical wound clips. There were 4 different groups: 75 mg MET, 50 mg MET, 35 mg MET, and 0 mg (sham) with 10 animals in each

MET group and 9 animals in the sham group. Animals were exposed to 10 minutes FS as a means of a stress test 5 days following surgery to evaluate CORT levels; blood was collected immediately following the stress test and again 90 minutes later by cardiac puncture.

Ketoconazole neonatal injection

Since MET was proving to be unable to block the potent MA-induced rise in CORT despite the method of MET administration, the next experiment was designed to test a CORT synthesis inhibitor that works by a different mechanism.

Experiment-9: Eight litters of 12 pups each were culled on P1, male pups were preferentially retained (no sex differences in CORT response have been seen on P11; future behavioral experiments will use male subjects only). There were

4 treatment groups with 3 subjects in each: ketoconazole (KTZ) 25 mg/kg /M10;

KTZ 50 mg/kg/M10; VEH/M10; SAL/M10.

KTZ was dissolved in 100% corn oil and administered in a volume of 6 ml/kg as the first dose of the day on P11. Sixty minutes following the KTZ dose, the first dose of M10 was administered and 3 additional doses followed at 2 hour intervals. An animal from each treatment group was sacrificed by decapitation 1 hour, 7 hours, or 24 hours following the first M10 dose; trunk blood was collected.

136 Experiment-10: Eight litters of 8 pups each were culled on P1, male pups were preferentially retained. There were 5 treatment groups per litter: KTZ 50 mg/kg/SAL; KTZ 50 mg/kg (x1)/M10; KTZ 50 mg/kg (x2)/M10; KTZ 50 mg/kg

(x3)/M10; VEH (x3)/M10; VEH (x3)/SAL. Three pups per litter were placed in the KTZ 50 mg/kg/SAL group; all other groups contained 1 pup per litter. KTZ or VEH was the first dose of the day. Subsequent doses of M10 or SAL occurred

60 minutes later and each additional dose (for a total of 4) at 2 hour intervals.

Groups KTZ 50 mg/kg (x1)/M10; KTZ 50 mg/kg (x2)/M10; KTZ 50 mg/kg

(x3)/M10 received 1, 2, or 3 doses of KTZ throughout the 24 hour period. Group

KTZ 50 mg/kg (x1)/M10 received 1 dose of KTZ 60 minutes prior to SAL dosing on P11. Group KTZ 50 mg/kg (x2)/M10 received 2 doses of KTZ, the first 60 minutes prior to SAL dosing on P11 and the second 12 hours after the first KTZ dose. Group KTZ 50 mg/kg (x3)/M10 received 3 doses of KTZ, the first 60 minutes prior to SAL dosing on P11, the second 12 hours after the first KTZ dose, and the third 24 hours after the first KTZ dose. These additional KTZ doses were administered to determine how often KTZ needed to be given to continue CORT blockade throughout the dosing period. All animals, unless specified otherwise (2 pups in the KTZ 50 mg/kg/SAL group), were sacrificed 24 hours following the first M10 or SAL dose. Trunk blood was collected from all animals for analysis of CORT.

Metyrapone adult injection

Experiment-11: Even though MET was unable to block MA-induced

CORT after multiple doses in neonates, MET has been shown to be effective in

137 blocking swim stress in adults. Since MWM involves swim stress, and since neonatal MA alters basal CORT, we hypothesized that the MWM learning impairment was due to an altered HPA-axis mediated response to swimming.

Such a change, if present, might be blocked by MET treatment prior to each maze trial.

A preliminary experiment was preformed to determine the correct dosage of MET to block swim induced stress and also to determine if the chronic MET dose was toxic during the extended behavioral/adult dosing period. A separate group of 20 Sprague-Dawley CD, IGS adult male rats (Charles River, Raleigh,

NC) was obtained for the preliminary experiment. These animals were allowed one week of environmental acclimation. Half of these animals were administered

MET (50 mg/kg) and half were administered VEH (9.5% ethyl alcohol) prior to stress testing, 10 minutes FS. Post-stress blood samples were obtained via cardiac puncture following anesthetization with isoflurane. A small amount of EDTA was present in the needle (22G) during collection to prevent clotting. Blood was collected in tubes on ice, centrifuged at 2500 RPM for 25 minutes, plasma removed and frozen at -80 °C until assayed for CORT levels. Following FS testing, the animals received MET or VEH (same groups as prior to stress test) once daily for 21 days to mimic the treatment of the animals that go through behavioral testing.

Based on the results from the preliminary experiment, MET injection of

50 mg/kg 90 minutes prior to FS stress in the adult was successful in blocking

138 stress induced increase in CORT. The behavioral experiment proceeded using this method of CORT blockade.

Litters were undisturbed until P1 on which day the animals were culled to seven male pups. Up to two pups per litter were fostered from litters born on the same day if necessary. Sixteen litters were used. Following a within litter design, five pups from each litter were assigned to receive MA treatment, three receiving

MA 15 mg/kg (M15) (an extra was included in case of toxicity) and two receiving

MA 10 mg/kg (M10); the remaining two pups received saline (SAL). At weaning one pup receiving M15 was chosen at random and was sacrificed as only two were needed for behavioral testing. The remaining six pups were raised to adulthood and received a treatment of either metyrapone 50 mg/kg (MET) or vehicle (VEH; 9.5% ethyl alcohol) as adults 90 minutes prior to behavioral testing on a daily basis. One animal from each of the groups, M15, M10, or SAL received MET while the other received VEH. Pups were ear marked on P11 for identification, weighed daily from P11-15, weekly thereafter, and daily throughout behavioral testing. Weaning occurred on P28. Animals were divided evenly into two groups of three animals each. Further separation into pairs occurred on P42. No treatment distinction was made in determining which animals were housed together.

Adrenalectomy with CORT replacement

Experiment-12: Based on the results of the preceding experiment, MET pretreatment via injection appeared to produce some beneficial or corrective effect in the MA-treated groups, but the effects were subtle. However, it could be

139 that a continuous rather than acute CORT blockade would be more effective. The next set of experiments was similar to the preceding experiment, except bilateral

ADX with CORT replacement via osmotic minipumps was used as the method of

CORT control during behavioral testing.

A preliminary experiment was preformed to determine what dose of

CORT after ADX would provide adequate plasma CORT to ensure basal levels, and to determine the time-course of the osmotic minipump CORT output for a duration that would be comparable to that planned for the behavioral experiment.

A separate group of 30 Sprague-Dawley CD, IGS adult male rats (Charles River,

Raleigh, NC) was obtained for a preliminary experiment. These animals were allowed one week of environmental acclimation. The animals were divided into 3 groups following bilateral ADX surgery: Sham (4 animals); ADX-C 10 µg/hr (12 animals); ADX-C 15 µg/hr (11 animals); 3 animals died during surgery. All animals received 0.9% saline to drink following surgery. One week recovery time was given before any further manipulation. Seven days post-surgery a blood sample was obtained to determine a baseline CORT value. Fourteen days post- surgery the animals were put through a 10 minute FS stress and another blood sample was taken immediately following. Two other blood samples were taken at

21 and 28 days post-surgery to determine basal CORT levels and the continued effectiveness of the pump release. All blood samples were obtained via cardiac puncture following anesthetization with isoflurane. A small amount of EDTA was present in the needle (22G) during collection to prevent clotting. Blood was collected in tubes on ice, centrifuged at 2500 RPM for 25 minutes, plasma

140 removed and frozen at -80 °C until assayed for CORT levels. All cages contained an environment enrichment item.

Based on the results from the preliminary experiment, bilateral ADX with

CORT replacement (10 µg/hr) in the adult was successful in restoring CORT to normal basal levels throughout the 28-day life of the pump and in blocking stress induced increase in CORT. The behavioral experiment proceeded using this method of CORT blockade.

Litters were undisturbed until P1 on which day the animals were culled to eight male pups. Up to two pups per litter were fostered from litters born on the same day if necessary. Sixteen litters were used. Following a within litter design, five pups from each litter were assigned to receive MA treatment, three receiving

20 mg/kg (M20) (to try to increase the MA effect) and two receiving 10 mg/kg

(M10); the remaining three pups received saline (SAL). Pups were ear marked on

P11 for identification, weighed daily from P11-15 and weekly thereafter.

Weaning occurred on P28; animals were divided evenly into two groups of four animals each. Further separation into pairs occurred on P42. Pups were raised to adulthood and underwent bilateral ADX or sham surgery between P50-P56.

Animals were divided into 3 surgery groups: ADX with CORT replacement

(ADX-C), ADX only, and SHAM/placebo (sham). Both the ADX-C and

SHAM/placebo groups contained 3 animals, one of each neonatal treatment condition (M20, M10, SAL) per litter. The ADX only group consisted of one animal having received SAL as a neonate per litter. An osmotic minipump

(Alzet, DURECT Co., Cupertino, CA) containing CORT (10 µg/hr release, 28 day

141 duration) was placed under the skin near the nape of the neck of ADX/CORT animals at the time of surgery; sham animals received a control pump containing only propylene glycol. No treatment distinction was made in determining which animals were housed together. All animals received 0.9% saline to drink following surgery. All cages contained environmental enrichment.

For all experiments, animals were housed in a vivarium under a 14/10 hour light/dark cycle with lights on at 0600 with food and water available ad libitum. The vivarium is fully accredited by the Association for the Assessment and Accreditation of Laboratory and Animal Care (AAALAC). All protocols were approved and supervised by the Cincinnati Children’s Hospital Research

Foundation Institutional Animal Care and Use Committee (IACUC).

MA, MET, & CORT Administration & Surgery

MA HCl (expressed as the freebase, Sigma, St. Louis, MO) dissolved in saline was administered subcutaneously by injection near the nape of the neck of the animal at a concentration of 10 mg/kg (M10), 15 mg/kg (M15) or 20 mg/kg

(M20) in a volume of 3 ml/kg. All injections occurred at two hour intervals and injection sites were rotated to minimize irritation. Animals were administered 4 injections of MA or SAL per day from P11-15 unless sacrificed prior to reaching

P15.

KTZ (ICN Biomedicals, Aurora, OH) was administered subcutaneously by injection in the back of the animal. KTZ was dissolved in 100% corn oil in a volume of 6 ml/kg.

142 MET (Sigma, St. Louis, MO) was administered subcutaneously by injection in the back of the animal or by way of a time release pellet inserted surgically under the skin near the nape of the neck (Innovative Research of

America, Sarasota, FL). Surgery for pellet implantation was performed under inhalation anesthesia (isoflurane delivered via a nose cone) and the incision was closed using wound clips. MET pellets were administered in doses of 200 mg,

100 mg, 75 mg, 50 mg, 35 mg, 15 mg, or 10 mg, all in 21 day release form. MET by injection was given at a concentration of 100 mg/kg or 50 mg/kg in a 3 ml/kg volume dissolved in either 25% DMSO in SAL or in 9.5% ethyl alcohol in SAL.

Adult injections of MET were administered 90 minutes prior to behavioral testing each day.

CORT (Sigma, St. Louis, MO) was dissolved in propylene glycol at a concentration of 10 µg/0.26 µl or 15 µg/0.26 µl to obtain a 10 µg/hr release (0.26

µl of solution were released per hour as specified in the pump specific LOT information). The 10-15 µg/hr concentration as well as propylene glycol solvent for use in the CORT replacement experiments was based on previously published data (Fukushima et al., 1985;Will et al., 1980). Solutions were heated to 100 °C and vortexed to enable the CORT to go completely into solution. CORT solution or control (propylene glycol only) was added to the osmotic minipumps (Alzet,

DURECT Co, Cupertino, CA) as described by the manufacturer under sterile conditions. Filled pumps were primed for 48 hours in saline before implantation into the animal.

143 Bilateral ADX surgery took place under inhalation anesthesia using

isoflurane delivered via a nose cone. The dorsal surface of the animal was shaved

and sterilized using a betadine solution and dried before any incision was made.

A single incision approximately 3-4 cm in length was made laterally across the

dorsal surface of the skin just beneath the rib cage. A small hole was created in

the body wall of the animal above the area of the kidney. The adrenal gland was

removed without disturbing the surrounding organs and placed immediately on

dry ice for later analysis. The body wall was then closed using resorbable sutures.

This process was done on each side of the animal to remove both adrenal glands.

The osmotic minipump was then placed beneath the skin toward the nape of the

neck of the animal before the initial incision was closed. Surgical wound clips

were used to close the wound in the skin and tissue glue was added to help ensure

the entire length of the incision was completely closed. Wound clips were

removed six days following surgery.

Corticosterone Assessment

Animals were anesthetized by inhalation of isoflurane in an enclosed container. Approximately 1 ml of blood was collected via cardiac puncture using

3 ml syringes with 22 gauge needles. A small amount of EDTA was present in the syringe to prevent clotting. Blood was dispensed into 12 x 70 polyethylene tubes and chilled on ice. Tubes were centrifuged at 2500 RPM for 25 minutes at

4° C for separation of plasma. Plasma was removed and stored at -80 °C until analysis of CORT via EIA.

144 Plasma CORT concentrations were analyzed via EIA kits obtained from

ALPCO Diagnostics (Windham, NH). Frozen plasma was thawed on ice and added in duplicate to 96 well plates as described by the manufacturer’s instructions.

Behavioral Methods

Elevated Zero Maze

Zero maze was the first behavioral test in which the animals were run.

Animals were 50-62 days of age when this test was run. The maze chamber consists of a black acrylic circular track 10 cm wide and 105 cm in diameter divided into quadrants (San Diego Instruments, CA). Two quadrants opposite each other have 1.3 cm clear acrylic curbs on either side to prevent slipping but otherwise are open on the sides and hereafter are referred to as the open quadrants. The other two quadrants are referred to as closed and have 28 cm high black acrylic walls. The entire maze is elevated 72 cm off the floor. An overhead camera attached to a video cassette recorder recorded the session for later analysis. Behavioral sessions are scored using EventLog tracking software

(Conduit Champaign, IL). Scoring included analysis of the number of stretch- attends (defined as when the animal has its head and front two paws extending into an open quadrant and the remainder of the body in a closed quadrant), head dips, and open entries as well as the total amount of time the animal spent in the open quadrants of the maze. At the beginning of each trial the subject is identified and placed in a closed quadrant at which time the 5 minute trial is begun. When complete, the animal is removed and the maze wiped clean of urine

145 and feces with 70% ethyl alcohol. Overhead lights were off during this task and

only one halogen light was used to illuminate the room. The purpose of this task

is to measure the level of anxiety of the subject.

Straight Channel

On the Friday following Zero Maze testing, each animal was administered four timed trials in the straight channel apparatus. This apparatus consisted of a

15 x 244 cm straight water channel consisting of grey polyvinylchloride plastic.

The channel is filled with water (22±1° C) to a depth of 35 cm with a wire escape ramp at one end. At the beginning of each trial the animal was placed facing the back wall of the channel at the opposite end from the ramp. Timing began when the animal was released and continued until the escape ramp was found using a hand held timer. The purpose of this task is to measure swimming ability and motivation to escape a water task.

Morris Water Maze (MWM)

Testing in the MWM began two days following Straight Channel Trials.

The MWM is an apparatus known to test spatial learning and memory ability

(Brandeis et al., 1989;McNamara and Skelton, 1993). Our tank is 210 cm in diameter and is flat black in color. The escape platform is made from clear acrylic and has wire screening attached to the top for improved grip; the platform is invisible to the eye and sits 2 mm below the water surface; water temperature is

22±1° C. The platform surface used was 10 x 10 cm square for acquisition trials and 5 x 5 cm square for reversal trials for the MET study animals and 10 x 10 cm square for acquisition and reversal trials and 5 x 5 cm square for double reversal

146 trials for the ADX study animals. Animals were administered four trials per day

for 5 days of spatial learning. One probe/memory trial was administered the

following day. The animals then had one day of no testing followed by 5 days of

reversal learning (4 trials per day) and one day of probe/memory trials (one trial per animal). When double reversal was run, a day of no testing was given between the reversal probe trial and the first day of double reversal trials. Double reversal

was run for 5 days with four trials per day for spatial learning followed by one

day with a single probe trial.

The tank was divided into four quadrants along an ordinal axis. The North was defined as the far point exactly opposite the operator and South the near point. West and East were left and right respectively; thus, the quadrants were defined as north-east (NE), south-east (SE), south-west (SW), and north-west

(NW). The escape platform was placed in the center of the SW quadrant for acquisition and double reversal trials and in the center of the NE quadrant for reversal trials. Start positions consisted of four positions distal to (NW, N, E, SE, for a SW target or SE, S, W, NW, for a NE target). Each learning trial had a maximum time limit of 120 seconds with a 15 second inter-trial interval (ITI). If

the animal did not find the platform in the allotted time it was placed on the

platform by the operator for the ITI. Probe trials were run for 30 seconds with the

platform removed from the tank; the animal was placed in either the NE or SW

starting position for acquisition/double reversal or reversal, respectively. Swim

paths were recorded by an overhead camera and video-tracking system (Polytrack

147 System – used in the MET study; SMART System – used in the ADX study, San

Diego Instruments, San Diego, CA).

Statistics

Adult MET injection behavioral experiment

Preliminary experiment CORT results were subjected to the T-test

procedure in SAS® (SAS, 1999b); equality of variances was determined using the

folded F method. For all other data, analysis of variance (ANOVA) was used to

determine group differences using the GLM (general linear model) procedure in

SAS® (SAS, 1999a). Treatment group was a repeated measure (within) factor.

Day and dose were repeated measure (within) factors for statistical analysis of

weight; day and trial were repeated measure (within) factors in analysis of

behavioral testing. Significant main effects were further analyzed by using the

step-down F-test procedures (Kirk, 1995b). A p<0.05 level of confidence was

used to determine significance and p<0.10 for trends.

Adult ADX with CORT replacement behavioral experiment

Analysis of Variance (ANOVA) was used to determine group differences

using the GLM (general linear model) procedure in SAS® (SAS, 1999a).

Treatment group was a between factor. Day, dose, and week were repeated measure factors for analysis of weight; day and trial were repeated measure factors in analysis of behavioral testing. A test was performed for extreme outliers and several animals were removed from the final analysis. Least Squares

Means were generated for all ANOVAs; and, comparisons of interest were further

148 analyzed using the step-down Bonferroni method (Kirk, 1995a). A p<0.05 level

of confidence was used to determine significance.

Results & Discussion

Neonatal MET injection experiment series (Experiment-1 through 6)

Results

Results from Experiment-1 showed some attenuation of the MA-induced

CORT increase with MET pretreatment at 50 mg/kg (Figure 3.2A). Experiment-2 used a higher concentration of MET, 100 mg/kg, to determine if this attenuation could be enhanced; results showed a greater attenuation of the MA-induced

CORT increase (Figure 3.2B). Experiment-3 examined the effects of VEH and

MET on CORT increase without MA present. Results suggested that both the

VEH and MET caused slight increases in CORT levels following the 3rd and 4th

dose on P11 (Figure 3.2C). Experiment-4 results showed no CORT attenuation

on P12 (following four doses of M10 on P11 and one dose on P12) (Figure 3.2D)

as compared to CORT attenuation seen on P11 alone as seen in Experiment-2. In

Experiment-5, use of a different vehicle, 9.5% ethyl alcohol vs. 25% DMSO,

eliminated the VEH-induced CORT increase and attenuated MA-induced CORT

increase as shown on P11 (Figure 3.2E); however, Experiment-6 results showed

MET treatment did not block M10-induced increases in CORT on P15 after P11-

15 dosing (Figure 3.2F).

149 Discussion

We have previously demonstrated that MA administration on P11 has

pronounced effects on the release of CORT (Williams et al., 2000). The purpose

of the present studies was to determine what dose of MET, if any, would be

effective in blocking or attenuating this increase during the neonatal period via

blockade of the enzyme, 11β-hydroxylase, that is required for conversion of 11- deoxycorticosterone to CORT. Behavioral studies using MET to block CORT production have been successful in adult animals (Calvo et al., 1998;Roozendaal et al., 1996).

Results from Experiment-1 using a MET dose concentration of 50 mg/kg showed a moderate attenuation of the MA-induced CORT increase. The results from the VEH/SAL control group also showed an increase in CORT levels from doses 2 through 4. The results suggested the need for a higher MET dose and further examination of any effect of the DMSO vehicle.

Experiment-2 results using MET at a concentration of 100 mg/kg showed a greater attenuation of the MA-induced increase in CORT on P11 after all doses but increase in CORT in the VEH group remained. Results from additional control groups in Experiment-3 indicated that both MET and 25% DMSO alone cause slight increases in plasma CORT consistent with other studies (Herman et al., 1992;Rotllant et al., 2002).

Examination of results from animals sacrificed on P12 following one full day of dosing on P11 (Experiment-4) demonstrated CORT levels from MET/M10 treated animals that were not different than CORT levels from VEH/M10 or

150 SAL/M10 animals when sacrificed 30 minutes following the first dose of SAL or

M10. These data showed that MET was not effective in attenuating the late-phase of MA-induced increase in CORT.

To address the issue of interference by the 25% DMSO VEH, a new VEH,

9.5% ethyl alcohol, was used in subsequent experiments. Results from

Experiments-5 and 6 showed no VEH-induced increase in CORT following 4 doses on P11 or following 5 days of dosing on P15. Results from P11-15 dosing

(Experiment-6) revealed however that MET pretreatment was not effective in blocking the MA-induced late-phase increase in CORT as demonstrated in the data collected 30 minutes following the first M10 dose on P15.

In conclusion, this series of experiments aimed at blocking the MA- induced increases in CORT during the neonatal dosing period with MET did not produce evidence that MET was effective beyond P11. We next turned to a different CORT inhibitor to determine if blocking CORT synthesis at another point in the pathway might prove to be more effective.

151

CORT (ng/ml) E C

CORT (ng/ml) A increase at atten B) MET100m A) MET50m Figure 3.2:NeonatalMETinjection F) MET100m E) MET100m D) MET100m C) MET100m CORT (ng/ml) 10 12 20 40 60 80 10 15 20 25 30 35 10 20 30 40 50 60 70 0 0 0 0 5 0 u W V a e EH ig /S S h/ Do A tion A W L L/ ei V SA gh EH L se 1 /M SA A V Do L/ M E SA ET H/ L /M 30 S A A s V L E e

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= A le t VE o VEHalone * retain l arge CORT

H interfe CORT 152 rence

Neonatal Ketoconazole injection experiment series (Experiment-7 and 8)

Results

Ketoconazole (KTZ) 25 mg/kg was ineffective in blocking MA-induced

CORT release (Figure 3.3). KTZ 50 mg/kg was effective in blocking MA- induced increase in CORT 1 and 7 hours post drug administration on P11, however, results at 24 hours on P12 revealed the need for an additional dose of

KTZ to continue blocking CORT throughout the 24 hour period (Figure 3.3).

Results from the addition of more control groups and additional doses of KTZ 50 mg/kg showed no blocking of the CORT increase with KTZ treatment (Figure

3.3). Evidence of KTZ itself causing increased CORT was shown by the high

CORT values obtained from the groups receiving multiple injections of KTZ as the CORT values from these groups appeared higher than that obtained from the

VEH/M10 group (Figure 3.3).

Discussion

We previously demonstrated that MA administration on P11 has pronounced effects on the release of CORT (Williams et al., 2000). The purpose of these studies was to examine the effect of KTZ on CORT increases during the neonatal dosing period. Specifically, KTZ inhibits the action of 14α-dimethylase

(a cytochrome P-450 enzyme) in the conversion of lanosterol to cholesterol in steroidogenic pathways thereby blocking normal CORT production (Hume and

Kerkering, 1983;Loose et al., 1983). KTZ has been used to treat abnormal corticosteroid secretion associated with Cushing’s syndrome in humans (Sonino,

1986).

153 Results from the first experiment using KTZ at a dose of 25 and 50 mg/kg demonstrated that 25 mg/kg KTZ was not effective in blocking CORT increase but 50 mg/kg KTZ was effective at 1 and 7 hours post drug but not at 24 hours.

The results from the initial experiment revealed the need for an additional dose of KTZ to continue blocking the CORT increase throughout the 24 hour period; only the 50 mg/kg KTZ dose was further examined. Additional control groups were added to complete the design of the initial experiment to ensure there was no KTZ-induced CORT increase. Groups KTZ 50 mg/kg (x2)/M10 and KTZ

50 mg/kg (x3)/M10 with added doses of KTZ throughout the 24 hour period did not show additional attenuation of CORT at 24 hours; furthermore, plasma CORT levels from these groups appeared even higher than that of the VEH/M10 group at

24 hours (Figure 3.3).

In conclusion, this series of experiments to block the MA-induced increase in CORT during the neonatal dosing period with KTZ did not produce evidence of effective CORT blockade or attenuation through a 24 hour dosing period.

Manipulation of the neonatal animal with KTZ administration in addition to the

MA dosing regimen appeared to add further complexity to the examination of the nature of the MA-induced CORT increase.

154

P11 KTZ injectionof25or50m X-axis * * Figure 3.3: following firstMAorS SAL; P11(1or7hoursfollowingfirs CORT (ng/ml) i

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155

MET time-release pellet experiment series (Experiment-9 and 10)

Results

All pups from the 200 mg and 100 mg pellet experiments died within 2 hours of surgery/pellet implantation. Three of four pups having received 75 mg pellets died within 6 hours after surgery; the remaining 75 mg pup died following the first dose with M10. Three of four pups having received 50 mg pellets died within 6 hours after surgery; the remaining pup survived through 4 doses of M10 but was found dead the following morning before a blood sample could be obtained. One of four pups having received the 35 mg pellet died within 6 hours after surgery. Another pup having received a 35 mg pellet died after 2 doses with

M10. A third pup having received a 35 mg pellet survived 4 doses of M10 but was found dead the following morning before blood could be obtained. The remaining pup survived 4 doses of M10 and survived until sacrificed to obtain trunk blood for CORT analysis. All pups having received 25 mg, 15 mg, or 10 mg pellets survived surgery and the dosing period until time of sacrifice (P12 or

P15); trunk blood was collected for CORT analysis.

All adult rats receiving pellet implantation survived surgery and were alive the following morning. One rat having received a 35 mg pellet did not recover from the first exposure to anesthesia/cardiac puncture procedure. One placebo rat died before stress testing.

All CORT levels obtained from neonatal animals having received MET time-release pellets were inconclusive due to inconsistent results that showed no significant MET-induced CORT blockade. CORT levels obtained from adult

156 animals both immediately following forced swim (FS) stress and 90 minutes post

stress showed no treatment effect of MET blocking the stress induced increase in

plasma CORT.

Discussion

We have previously demonstrated that MA administration on P11 has

pronounced effects on the release of CORT (Williams et al., 2000). The purpose

of these studies was to determine what dose of MET was effective in blocking or

attenuating this increase during the neonatal period via blockade of the enzyme,

11β-hydroxylase, that is required for conversion of 11-deoxycorticosterone to

CORT. MET was administered in time-release pellet form to allow delivery of a continuous level of drug so as to produce consistent and prolonged CORT blocking instead of intermittent peak concentrations as occurs after MET injections.

Data from the neonatal experiments showed that the MA-induced CORT increase could not be effectively blocked with the use of time release pellets.

Furthermore, the highest pellet concentrations were fatal to the animals in a very short period of time following implantation indicating that the pups may have received a bolus of drug or an interaction occurred between the drug and surgical recovery.

Failure to effectively block the MA-induced increase in CORT in neonatal animals, attention turned toward controlling CORT levels in adult animals.

Results from the adult MET pellet implantation showed that this method failed to block the CORT response induced by swim stress. Upon physical

157 inspection of an animal having received a MET pellet, the pellet appeared to have changed form from its initial solid white form to a transparent gelatinous mass within 5 days of implantation. This observation made the proper time-release functioning of the pellets suspect as the pellet appeared disintegrated following only 5 days of its 21 day life-span.

This set of experiments designed to block the MA-induced increase in

CORT during the neonatal dosing period or in adult animals following swim stress using MET time-release pellets did not produce CORT blockade or attenuation.

158 Adult MET injection behavioral experiment (Experiment-11)

Preliminary Experiment – CORT assessment

There was a significant treatment effect for levels of CORT following 10 minutes FS stress. The MET treated animals had a significantly lower level of plasma CORT than the VEH treated animals (t(18)=-6.78, p<0.0001, Figure 3.4).

Experiment-11

Weights

Dosing Weights

There was a significant main effect of neonatal treatment on body weight during the dosing period. Each treatment was significantly different from the other two treatments; SAL vs. M10 (F(1,47)=97.36, p<0.0001), SAL vs. M15

(F(1,46)=187.63, p<0.0001), M10 vs. M15 (F(1,46)=10.45, p<0.005). There was a significant interaction of neonatal treatment by day as well as a significant three way interaction of neonatal treatment by day by time of day (4 weights were taken per day during dosing). Specifically, neonatal treatment by day values for SAL vs. M15 groups were significantly different each day; P11 (F(1,46)=6.17, p<0.05), P12 (F(1,46)=46.30, p<0.0001), P13 (F(1,46)=189.10, p<0.0001), P14

(F(1,46)=278.35, p<0.0001), P15 (F(1,46)=337.30, p<0.0001) Figure 3.5.

Neonatal treatment by day by time of day interactions between SAL and M15 were significant for all comparisons (equivalent day and time of day) except the first weight on P11; the P11 second weight comparison was significant

(F(1,46)=5.91, p<0.05). Neonatal treatment by day values for SAL vs. M10 groups were significantly different each day; P11 (F(1,47)=4.22, p<0.05), P12

159 (F(1,47)=29.38, p<0.0001), P13 (F(1,47)=91.72, p<0.0001), P14

(F(1,47)=132.98, p<0.0001), P15 (F(1,47)=149.17, p<0.0001) Figure 3.5.

Neonatal treatment by day by time of day interactions between SAL and M10

were significant for all comparisons (equivalent day and weight) except the first

and second weights on P11; the P11 third weight comparison was significant

(F(1,47)=5.49, p<0.05). Neonatal treatment by day values for M15 vs. M10

groups were significantly on P13-15 only; P13 (F(1,46)=8.92, p<0.005), P14

(F(1,46)=21.27, p<0.0001), P15 (F(1,46)=25.90, p<0.0001) Figure 3.5.

Straight Channel

There were no significant treatment effects on swimming ability.

Elevated Zero Maze

There were no significant treatment effects observed.

Morris Water Maze

Acquisition Phase

There was an effect present for the 3-way interaction of neonatal treatment by adult treatment by trial for latency (F(6,90)=2.15, p=0.05) demonstrating that the M10/VEH group took longer to reach the goal than the M10/MET group on trial 1 (Figure 3.6). There was a trend for the 2-way interaction of adult treatment by trial (F(3,45)=2.65, p=0.06) demonstrating that the VEH treated group took longer to reach the goal than the MET treated group.

There was a significant effect for the 3-way interaction of neonatal treatment by adult treatment by trial for path length demonstrating that the

160 M10/VEH group had a longer path length to reach the goal than the M10/MET

group on trial 1 (F(1,15)=4.67, p<0.05).

There was a significant effect for the 2-way interaction of adult treatment

by day for cumulative distance demonstrating that the MET treated group was

significantly farther from the goal than the VEH treated group (F(1,15)=4.60,

p<0.05). There was a trend for the 3-way interaction of neonatal treatment by

adult treatment by trial (F(6,90)=2.00, p=0.07) demonstrating that the M10/VEH

group was located farther from the goal than the M10/MET group on trial 1.

There was a trend for the 2-way interaction of adult treatment by day for

first bearing (F(4,60)=2.22, p<0.08) demonstrating that the initial direction of the

MET group was at a greater angle from the goal than the first bearing of the VEH group on day 1. There was a trend for the 3-way interaction of neonatal treatment by adult treatment by day (F(8,120)=1.84, p<0.08) demonstrating that the initial direction of the M10/MET and M15/MET groups was at a greater angle from the goal than the first bearing of the SAL/MET group on day 1; the initial direction of the SAL/MET group was at a greater distance from the goal than the first bearing of the SAL/VEH group on day 2; the initial direction of the M15/VEH group was at a greater distance from the goal than the first bearing of the SAL/VEH group on day 2; and, the initial direction of the M10/MET group was at a greater

distance from the goal than the first bearing of the M10/VEH group on day 1.

Reversal Phase

There was a trend for the main effect of neonatal treatment for latency

(F(2,30)=2.67, p<0.09) demonstrating the M10 groups took longer to reach the

161 goal than the SAL groups. There was a trend for the 3-way interaction of neonatal treatment by adult treatment by trial (F(6,90)=1.96, p<0.08; Figure 3.7) demonstrating that the M10/VEH group took longer to reach the goal than the

SAL/VEH group on trials 1 and 2; the M15/VEH group took longer to reach the goal than the SAL/VEH group on trial 1; the M10/MET group took longer to reach the goal than the SAL/MET group on trial 3; and, the M15/VEH group took longer to reach the goal than the M15/MET group on trial 1.

There was a trend for the main effect of neonatal treatment for path length

(F(2,30)=2.56, p<0.09) demonstrating the M10 groups had a longer path length to reach the goal than the SAL groups. There was a trend for the 2-way interaction of neonatal treatment by day (F(8,120)=1.78, p<0.09) demonstrating that the M10 groups had a longer path length to reach the goal than the SAL groups on days 2,

4, and 5; and, that the M15 groups had a longer path length to reach the goal than the SAL groups on day 5.

There was a trend for the 2-way interaction of adult by trial for cumulative distance (F(3,45)=2.29, p<0.09) demonstrating the VEH groups were farther from the goal than the MET groups.

There was a significant main effect of neonatal treatment for first bearing demonstrating that the initial direction of the M15 groups was at a greater angle from the goal than first bearing of either the SAL (F(1,15)=12.21, p<0.005) or

M10 (F(1,15)=4.98, p<0.05) groups. There was a significant main effect of adult treatment for first bearing demonstrating that the initial direction of the VEH

162 groups were at a greater angle from the goal than the first bearing of the MET groups (F(1,15)=10.32, p<0.01).

Probe Trial

Acquisition

There was a significant main effect of adult treatment (F(1,15)=7.58, p<0.05) for average distance from the goal demonstrating that the MET treated groups were significantly closer to the goal than the VEH treated groups. There was as significant interaction of neonatal by adult treatment demonstrating that the M15/MET group was significantly closer to the goal than the M15/VEH group

(F(1,15)=12.09, p<0.005) and that the SAL/VEH group was significantly closer to the goal than the M15/VEH group (F(1,15)=5.28, p<0.05), Figure 3.8.

There was a significant main effect of neonatal treatment for the number of platform crossings demonstrating that the M10 groups crossed the platform significantly more times than the M15 groups (F(1,15)=6.73, p<0.05).

Reversal

There was a significant main effect of adult treatment for first bearing

demonstrating that the initial direction of the VEH groups was at a greater angle

from the goal than the first bearing of the MET groups (F(1,15)=6.84, p<0.05).

Discussion

Neonatal MA treatment produces a reliable deficit in adult spatial learning

and memory as well as a substantial increase in plasma CORT during the dosing

period (Vorhees et al., 1994a;Vorhees et al., 2000;Williams et al., 2000;Williams

et al., 2002;Williams et al., 2003c). The purpose of the current set of experiments

163 was to examine the effects of inhibiting CORT release of the adult animal during behavioral testing to determine if neonatal MA treatment caused long-term abnormal functioning of the HPA axis thereby resulting in learning and memory changes. The approach was via MET injection.

Adult inhibition of CORT synthesis was by MET pretreatment and was effective following a 10 minute FS stress test, Figure 3.4. Plasma CORT levels in animals treated with MET 90 minutes prior to FS stress were significantly lower than levels in VEH treated animals administered stress following the testing procedure. This method of MET administration was then used in the subsequent experiments and administered each day prior to behavioral testing. It is important to note that this group of preliminary animals was subjected to MET injections once daily for 21 days following the FS test; no animals showed any physical signs of sickness or toxicity from chronic MET dosing.

The lack of significant effects in the straight channel swimming task and in the anxiety task indicated that the animals did not have altered anxiety on the zero maze or altered swimming ability. The MWM results did not demonstrate as clear of a MA-induced learning and memory deficit as seen in our previous work.

Trends were present among the groups indicating that the MA exposed animals had some learning deficit as compared to the SAL treated animals (see Figure

3.7) on early trials. Acquisition learning demonstrated several trends between the

M10/VEH and M10/MET groups showing that the M10/VEH animals had deficits in latency, path length and cumulative distance measures vs. their M10/MET treated counterparts. Trends present during acquisition for first bearing toward

164 the goal also indicated the MA effect was present as the M10 and M15 groups performed worse than the SAL animals within both the VEH and MET adult treatment groups on early trials. MWM probe trial data after the acquisition phase demonstrated significant evidence of the MA effect within VEH groups (Figure

3.8) in terms of average distance from goal.

Despite the modest effect size in the MA VEH groups, MET treatment did reverse many of these effects. The 3-way interactions of neonatal treatment by adult treatment by trial seen in the reversal phase were attenuated by MET treatment; trends seen on trials 1 and 2 in VEH groups were no longer apparent in

MET groups. The significant result seen in the acquisition phase probe trial was also blocked by MET treatment. Furthermore, upon inspection of the data

(Figure 3.8) the M10/MET and M15/MET groups appear to have performed better than the SAL/VEH animals in this measure.

In conclusion, most of the evidence supporting the MA-induced deficit was corrected with the adult MET treatment thus supporting the hypothesis that the HPA axis responds differently in the adult animal to swimming when exposed to neonatal MA treatment. We suggest that the added stress of daily injections in the adult animals may have added to the complexity of the experiment thus partially masking the MA effect produced by neonatal MA treatment. Additional experiments were justified to further examine these effects. Alternative methods of CORT inhibition may be more effective than MET injection.

165

**p<0.005, **p<0.0001, and**p<0.0001forP13,14,15respectively M10 vsM1 *p<0.05, *p<0.0001forP11andeachdayP1 SAL vsM10anim *p<0.05, *p<0.0001forP11andeachdayP1 SAL vsM15anim mg/kg, orSAL. Mean (±SE Figure 3.5:METadultinjection,dosingweights P11-15 Figure 3.4:METadultinje Weight (g) to 10m Mean (±SE CORT (ng/ml) 10 20 30 40 50 60 32 34 36 38 40 42 26 28 30 0 0 0 0 0 0 0 i nutes FS;t-test****p<0.0001 5 anim * M) weight(g)ofratpups * P11 M) CORT(ng/m M15 M10 Saline VE al weightsweresi al weightsweresigni al weightsweresigni H P 12 * *

P13 ction, Preliminary[CORT] l ** * * ) instressed MET *** * gnificantly d P by daytreatedwithMA 14 ** * * ficantly differenteachdayofdosing, ficantly d rats treatedwith50m P15 ** * * 2-15 respectively. 2-15 respectively. i i fferent each fferent each

dayP13-P15, dayofdosing, 10m g /kg METprior g /kg, MA15 166

Vehicle Metyrapone

80 80

70 70 †

60 60 ) s (

e 50 50 Tim

40 40

SAL SAL 30 M10 30 M10 M15 M15

20 20 1 2 3 4 1 2 3 4

Trial Trial

Figure 3.6: MET adult injection, MWM AQ latency; neonatal treat x adult treat x trial Mean (± SEM) in Latency (s) treatment by trial; †p<0.06 for M10/VEH vs. M10/MET on trial 1

Vehicle Metyrapone

80 † 80 †

70 70

60 60 ) s (

e 50 † 50 † Tim

40 40

SAL SAL 30 M10 30 M10 M15 M15

20 20 1 2 3 4 1 2 3 4

Trial Trial

Figure 3.7: MET adult injection, MWM RV latency; neonatal treat x adult treat x trial Mean (± SEM) in Latency (s) treatment by trial; †p<0.08 for M10/VEH vs. SAL/VEH on trials 1 and 2, M15/VEH vs. SAL/VEH on trial 1, M10/MET vs. SAL/MET on trial 3, M15/VEH vs. M15/MET on trial 1. Note trends present between VEH groups largely do not appear between MET groups.

167 Vehicle Metyrapone 100 100

90 90 *

80 80 ) m

c 70 70

ance ( 60 60 st Di

50 50

40 40

30 30 SAL/VEH M10/VEH M15/VEH SAL/MET M10/MET M15/MET

Figure 3.8: MET adult injection, MWM AQ probe trials average distance from goal neonatal treat x adult treat Mean (± SEM) in average distance from the goal (cm) neonatal treat by adult treat; M15/VEH vs. M15/ MET p<0.005 and M15/VEH vs. SAL/VEH *p<0.05. Note there is no longer a significant difference between the SAL and M15 treatment groups with MET treatment.

168 Adult ADX with CORT replacement behavioral experiment (Experiment-12)

Corticosterone Assessment

Preliminary Experiment

The 15 µg/hr CORT group did not produce plasma CORT on post-surgery

D7 that were much above ADX alone. We surmised this was due to the CORT concentration being too high causing the CORT to precipitate out of solution, crystallize and not be properly released. The 10 µg/hr CORT replacement values on post-surgery D7 appeared acceptable (not significantly different) as compared to Sham/Placebo animals (Figure 3.9A).

Final group N’s for 28 day analysis were determined by survival of the

animal. Data are from those animals that survived the 28 day period. The

Sham/Placebo group N = 4. The 10 µg/hr CORT group N = 7. The 15 µg/hr

CORT group was not analyzed beyond post-surgery D7 due to ineffective CORT

release.

There was a significant treatment by day interaction (F(3,27)=10.23

p<0.001 Figure 3.9B). For the Sham treatment group, there was a significant

interaction of day on D14 (F(3,12)=57.10 p<0.001).

Behavioral Experiment

Figure 3.10 depicts CORT concentrations from blood samples taken on

post-surgery D29 from ADX animals that underwent behavioral testing. There

are no significant differences between adrenalectomized-CORT replaced (ADX-

C) groups or between Sham groups. There was also no significant difference

169 between the two ADX groups treated neonatally with saline, Sal/ADX-C and

Sal/ADX (no CORT replacement following ADX).

Weights

Dosing Weights

There was a significant main effect of neonatal treatment during the dosing period. Both the M10 group and the M20 group weighed significantly less than the SAL-treated animals during the dosing period (F(2,132)=30.41, p<0.001). There was also a significant neonatal treatment by day interaction during the dosing period (Figure 3.11). There were no significant differences between treatment groups on P11. The M20 group weighed significantly less than the SAL group on P12 (F(8,528)=155.20, p<0.05). Both the M10 and M20 groups weighed significantly less than the SAL group on P13, 14, and15

(F(8,528)=155.20, p<0.005). The M20 group also weighed significantly less than the M10 group on P15 (F(8,528)=155.2, p<0.05).

Post-Dosing Weights

There were no significant interactions of neonatal treatment by week indicating that the weights of the animals were statistically equivalent at this point in time despite neonatal treatment differences (Figure 3.12). There was a significant interaction of adult treatment by week. Specifically, at P56 and P63 the ADX-C animals weighed significantly less than the Sham animals

(F(16,744)=24.64, p<0.05 and p<0.005 respectively); at P70 and P77 the ADX-C and ADX only animals weighed significantly less than the Sham animals

170 (F(16,744)=24.64, p<0.005 and p<0.05 respectively for ADX-C and ADX only on each week); Figure 3.12.

Straight Channel

No differences in swimming performances were seen between sham operated and ADX-C groups in this task. The Sal/ADX group took significantly longer to reach the escape platform than the other groups treated neonatally with saline (F (2,32)=9.74, p<0.005; Figure 3.13).

Elevated Zero Maze

There was a significant neonatal by adult treatment interaction for number of entries into the open; the M20/Sham group made significantly more entries into the open than its ADX-C counterpart (M20/ADX-C) (F(2,81)=4.23, p<0.01) and as compared to the M10/ADX-C group (F(2,81)=4.23, p<0.05); Figure 3.14A.

There was a significant neonatal by adult treatment interaction for time spent in the open; the M20/Sham group spent significantly more time in the open than the

Sal/Sham (F(2,85)=5.85, p<0.05) and M20/ADX-C (F(2,85)=5.85, p<0.01) groups; Figure 3.14B. There were no significant neonatal by adult treatment interactions for head dip or stretch attend events. There were no significant interactions of adult treatment between the animals treated neonatally with saline for any of the parameters examined.

Morris Water Maze

Acquisition Phase

There was a significant main effect of adult treatment for latency

(F(1,82)=7.67, p<0.01) demonstrating the ADX-C group took significantly longer

171 to reach the goal than the Sham group. There was a significant effect of neonatal by adult treatment (F2,82)=4.70, p<0.01; Figure 3.15) demonstrating that the

M20/ADX-C group took significantly longer to reach the goal than the M20 sham operated group (M20/Sham).

There was a significant main effect of adult treatment for path length

(F(1,84)=4.03, p<0.05) demonstrating the ADX-C group had a significantly longer path length to reach the goal than the Sham group. There was a significant effect of neonatal by adult treatment (F(2,84)=4.62, p<0.01) demonstrating that the M20/ADX-C group had a significantly longer path length to reach the goal than their sham operated counterparts (M20/Sham).

There was a significant main effect of adult treatment for cumulative distance (F(1,82)=4.27, p<0.05) demonstrating the ADX-C group was located at a significantly greater distance from the goal than the Sham group. There was a significant effect of neonatal by adult treatment (F2,82)=3.51, p<0.05) demonstrating that the M20/ADX-C group was located at a significantly greater distance from the goal than their sham operated counterparts (M20/Sham).

There were no significant interactions of adult treatment between the animals treated neonatally with saline for any of the parameters examined.

Reversal Phase

There was a significant main effect of neonatal treatment for latency

(F(2,78)=4.83, p<0.01) demonstrating that the M10 groups took significantly longer to reach the goal than the SAL groups. There was as significant main effect of adult treatment between animals treated neonatally with SAL

172 (F(2,30)=4.32, p<0.05) demonstrating that the SAL/ADX group took significantly

longer to reach the goal than either the SAL/ADX-C or SAL/Sham group.

There was a significant main effect of neonatal treatment for path length

(F(2,80)=3.13, p<0.05) demonstrating the M20 groups had a significantly longer

path length to reach the goal than the SAL groups. There was as significant main

effect of adult treatment between animals treated neonatally with SAL

(F(2,29)=3.42, p<0.05) demonstrating that the SAL/ADX group had a

significantly longer path length to reach the goal than the SAL/Sham group.

There was a significant main effect of neonatal treatment for cumulative

distance demonstrating the M10 (F(2,79)=6.06, p<0.005) and M20 (F(2,79)=6.06,

p<0.05) groups were located at a significantly greater distance from the goal than

the Sham groups. There was as significant main effect of adult treatment between

animals treated neonatally with SAL (F(2,30)=4.99, p<0.05) demonstrating

specifically that the SAL/ADX group was located at a significantly greater

distance from the goal than either the SAL/ADX-C or SAL/Sham group.

Double Reversal Phase

There was a significant main effect of neonatal treatment for latency

demonstrating the M10 groups (F(2,80)=5.96, p<0.05) and the M20 groups

(F(2,80)=5.96, p<0.005) took significantly longer to reach the goal than the SAL

groups. There was a significant main effect of adult treatment between animals

treated neonatally with SAL (F(2,28)=4.08, p<0.05) demonstrating specifically

that the SAL/ADX group took significantly longer to reach the goal than the

SAL/Sham group.

173 There were no significant effects found in analyses of path length in the double reversal phase of testing.

There was a significant main effect of neonatal treatment for cumulative distance demonstrating the M10 (F(2,79)=5.52, p<0.05) and M20 (F(2,79)=5.52, p<0.01) groups were located at a significantly greater distance from the goal than the Sham groups. There was a significant main effect of adult treatment for cumulative distance (F(1,79)=4.73, p<0.05) demonstrating the ADX-C group was located at a significantly greater distance from the goal than the Sham group.

There was as significant main effect of adult treatment between animals treated neonatally with SAL (F(2,28)=4.83, p<0.05) demonstrating that the SAL/ADX group was located at a significantly greater distance from the goal than the

SAL/Sham group.

Probe Trials

Acquisition

There was a significant effect of neonatal by adult treatment

(F(2,82)=5.47, p<0.05) for average distance from the goal demonstrating that the

SAL/ADX-C group was significantly closer to the goal than the SAL/Sham group. There was as significant main effect of adult treatment between animals treated neonatally with SAL (F(2,30)=7.19, p<0.005) demonstrating that the

SAL/ADX-C group was significantly closer to the goal than either the SAL/ADX or SAL/Sham group.

There was a significant effect of neonatal by adult treatment

(F(2,84)=5.62, p<0.05) for percent time in the goal quadrant demonstrating that

174 the M20/Sham group spent significantly more time in the goal quadrant than the

SAL/Sham group.

There was as significant main effect of adult treatment between animals treated neonatally with SAL (F(2,29)=4.52, p<0.05) for percent distance traveled in the goal quadrant demonstrating specifically that the SAL/ADX-C group swam a greater percent of total distance in the goal quadrant than the SAL/ADX group.

Reversal

There was a significant main effect of adult treatment (F(1,79)=4.77, p<0.05) for average distance from the goal demonstrating that the ADX-C groups were significantly further from the goal than the Sham groups.

Double Reversal

There was a significant effect of neonatal by adult treatment

(F(2,84)=4.48, p<0.05; Figure 3.17) for average distance from the goal demonstrating that the M20/Sham group was significantly further from the goal than the SAL/Sham group.

There was a significant main effect of adult treatment (F(1,80)=4.56, p<0.05) for percent distance traveled in goal quadrant demonstrating that the

ADX-C group swam a lesser percent of total distance in the goal quadrant than the Sham group.

Discussion

Previously, we have shown that neonatal exposure to MA causes significant increases in plasma CORT levels as well as a long-term learning and memory deficits as evidenced by poorer performance in the MWM when

175 compared to control animals (Vorhees et al., 1994a;Vorhees et al., 2000;Williams et al., 2000;Williams et al., 2002;Williams et al., 2003c). The purpose of these experiments was to evaluate performance in the MWM in animals exposed neonatally to MA and inhibit CORT release via bilateral ADX at the time of testing. This method allowed us to determine more thoroughly the role of the stress response in maze performance.

Results shown in Figure 3.9B revealed CORT replacement via osmotic pumps was effective in releasing a steady level of CORT for 28 days.

Furthermore, the stress test performed on D14 demonstrated the ability of the sham operated animals to show a significant CORT increase following stress while the ADX animals do not show a significant CORT response, as anticipated.

When compared with results shown in Figure 3.9B, the D29 CORT concentrations in animals that underwent behavioral testing shown in Figure 3.10 appear to be abnormally low, raising doubt about the validity of these data. Even though the data in Figure 3.9 and 3.10 are on different groups of animals, Figure

3.10 data suggest that by D29 pump output has sharply declined. Accordingly,

D29 CORT data do not appear useful.

Animal weights during the dosing period displayed an expected pattern of separation with MA treatment. Previous studies using the P11-15 MA dosing protocol (Williams et al., 2003b) show a similar separation of weight gained during the dosing period. The current data revealed a treatment effect with animals having received MA not gaining weight as quickly as their SAL-treated littermates. Furthermore, this effect was dose-dependent; animals having

176 received the highest MA dose gained weight least quickly. It is important to note that there were no differences within any of the 3 neonatal treatment groups indicating that weight and subsequent growth were not factors when examining adult group behavioral performance. Also, animals from all neonatal treatment groups equalized in respect to weight gain shortly following the dosing period.

Adult weights in the weeks following the ADX surgery procedure also showed separation. It can be expected that animals having received ADX surgery will not continue the same rate of growth as their Sham operated counterparts; however,

CORT replacement did not rescue this growth deficit. As stated earlier in the discussion, CORT replacement at 10 µg/hr was sufficient to bring plasma levels back to control levels; however this level of replacement was not sufficient to allow for catch-up growth in these animals.

Data collected from the straight channel swimming task indicate the ADX animals without CORT replacement have added difficulty with the physical task.

Due to the fact that the ADX-C animals did not show a deficit in comparison to the control animals, the complete lack of CORT in the ADX group can be considered a key factor in the performance of the ADX group. This group was thus also expected to perform more poorly in the water maze task as compared to all other groups.

Anxiety testing in the Zero maze task demonstrated a treatment effect in the M20 group. The M20/Sham animals spent more time in the open areas of the maze as well as had a higher number of entries into the open. Taken together, it is difficult to determine if these behavioral changes were due to increased activity or

177 due to decreased anxiety as a result of neonatal MA treatment; additional testing

(i.e. observation of the animals in the home cage, open field testing, or observation in a light/dark box) would need to be performed to determine the true nature of the effect. Also, this effect is not observed in the M10/Sham group indicating a threshold effect of dose. It is also important to note that the MA- induced behavioral change observed in the M20/Sham group was no longer apparent in the M20/ADX-C group. This evidence supports the hypothesis that neonatal MA treatment produces a differential HPA axis response in the adult animal that can be influenced by CORT release.

The three phase design of MWM testing, acquisition, reversal, and double reversal did not produce clear evidence of MA-induced learning and memory deficits. The double reversal phase of learning gave the clearest demonstration that the MA effect was present among Sham treated animals although group differences were not significant. Double reversal probe trials produced the only significant neonatal by adult treatment effect indicative of the MA-induced learning and memory deficit in which the M20/Sham animals were significantly further from the goal location than their SAL/Sham counterparts (Figure 3.17).

Furthermore, this difference was no longer present when animals were subjected to ADX-C indicating some correction of the effect when CORT release was blocked. There were some indications that the MA effect was present when considering only neonatal treatment in the reversal phase as demonstrated by all three of the measured parameters (latency, path length, and cumulative distance; see results section); the same was seen in latency and cumulative distance of the

178 double reversal phase. We expected to see these types of results among Sham animals only; however, as this did not occur perhaps the performance difference was not strong enough among Sham animals alone to reach significance and addition of the ADX-C animals was necessary to make the effect apparent. If this was indeed the case, the results do not collectively support the hypothesis because the ADX-C adult treatment did not have a significant corrective effect on the outcome. Due to the fact that there was no clear demonstration of the MA effect overall, the results of the MWM testing do not support nor negate the original hypothesis. We speculate that the adult treatment paradigm may have interfered with the effect reliably produced by neonatal MA treatment as seen in previous studies using the same or a similar dosing protocol (Vorhees et al.,

1994a;Williams et al., 2002;Williams et al., 2003c;Williams et al., 2003b).

Despite this, some support was provided in the MWM double reversal probe testing phase as well as in the results from the Zero maze task to indicate the hypothesis remains viable.

179 A B

50 225 *** 200 Sham 10 ug/hr replacement 40 175

150 ) ) l l

30 m /

g/m 125 n (ng ( T

R 100 20 CO CORT 75

50 10

25

0 0 Sham CORT 10ug/hr CORT 15ug/hr D7 D14 - Stress D21 D28

Figure 3.9: Adult ADX with CORT replacement, Preliminary [CORT] A) Mean (± SEM) baseline plasma CORT (ng/ml) in adult rats 7 days post bilateral ADX surgery with 0, 10, or 15 µg/hr CORT replacement. The group receiving 10 µg/hr CORT replacement is not significantly different than the Sham operated controls B) Mean (± SEM) plasma CORT (ng/ml) in adult rats 7, 14, 21, and 28 days post bilateral ADX surgery with 0 or 10 µg/hr CORT replacement. Days 7, 21 and 28 are baseline measurements, day 14 is a measurement following 10 minutes FS stress. Sham vs 10 µg/hr was significant only on D14 ***p<0.001

130 130 120 120 110 110 100 100 90 90 ) l 80 80 m 70 70 (ng/ 60 60 RT 50 50 CO 40 40 30 30 20 20 10 10 0 0 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

Figure 3.10: Adult ADX with CORT replacement, [CORT] post-behavior Mean (± SEM) plasma CORT (ng/ml) in adult rats 29 days post bilateral ADX surgery inclusive of 3 weeks behavioral testing with 0 or 10 µg/hr CORT replacement.

180 40

38

36 Saline M10 34 M20

32 * Weight (g) 30 * * 28

26 * * * * **

24 P11 P12 P13 P14 P15

Figure 3.11: Adult ADX with CORT replacement, dosing weights P11-15 Mean (± SEM) weight (g) of rat pups by day treated with MA 10 mg/kg, MA 20 mg/kg, or SAL. SAL vs. M20 animal weights were significantly different each day P12-15, *p<0.05 for P12, *p<0.005 for each day P13-15. SAL vs. M10 animal weights were significantly different each day P13-15, *p<0.005 for each day P13-15. M10 vs. M20 animal weights were significantly different on P15, **p<0.05

181 Post-Dosing Weights 500

450 Sal/ADX-C Sal/Sham 400 Sal/ADX M20/ADX-C 350 M20/Sham M10/ADX-C M10/Sham ) 300

250 ght (g

Wei 200

150 Surgery 100 Separate

50 Wean 0 P21P28P35P42P49P56P63P70P77

Post-Surgery Weights 500

480 Sal/ADX-C Sal/Sham 460 Sal/ADX M20/ADX-C 440 M20/Sham M10/ADX-C 420 M10/Sham

400 ight (g)

We 380

360

340

320

300 P56 P63 P70 P77

Figure 3.12: Adult ADX with CORT replacement, adult weights Mean (± SEM) weight (g) of rat adult treatment by week, ADX, ADX-C, or Sham. ADX-C animals weighed significantly less than Sham animals at P56, P63, P70 and P77, *p<0.05 for P56, **p<0.005 for each P63, P70 and P77. ADX animals weighed significantly less than Sham animals at P70 and P77, *p<0.05.

*Note no significant differences between Sham treated groups.

182 Sham ADX 16 16 ** 15 15

14 14

13 13

12 12 ) s (

e 11 11 m Ti 10 10

9 9

8 8

7 7

6 6 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

Figure 3.13: Adult ADX with CORT replacement, Straight channel Mean (± SEM) Latency to escape (s). Sal/ADX vs Sal/ADX-C and Sal/Sham **p<0.005

183 A) Open Entries Sham ADX * 9 9

8 8

7 7

6 6 )

# 5 ( 5 s e i r t 4 4 En 3 3

2 2

1 1

0 0 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

B) Time in Open Sham ADX 90 * 90 80 80

70 70

60 60 ) s 50 50 ( e

m 40 40 Ti

30 30

20 20

10 10

0 0 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

Figure 3.14: Adult ADX with CORT replacement, Zero Maze A) Mean (± SEM) Entries into the open. M20/Sham vs. M20/ADX-C p<0.01 and vs. M10/ADX-C p<0.05 B) Mean (± SEM) Time in open (s). M20/Sham vs. M20/ADX-C p<0.01 and vs. Sal/Sham *p<0.05

184 A) AQ Sham ** ADX 45 45

40 40

35 35

30 30 (s) e m

Ti 25 25

20 20

15 15

10 10 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

B) RV Sham ADX 45 45

40 40

35 35

30 30 (s) e

Tim 25 25

20 20

15 15

10 10 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

C) DR Sham ADX 45 45

40 40

35 35

) 30

s 30 ( e m 25 25 Ti

20 20

15 15

10 10 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

Figure 3.15: Adult ADX with CORT replacement, MWM latency; neonatal treat x adult treat A) Acquisition, Mean (± SEM) Latency to escape (s) M20/ADX-C vs. M20/Sham **p<0.01 B) Reversal, Mean (± SEM) Latency to escape (s) C) Double Reversal, Mean (± SEM) Latency to escape (s)

185

A) AQ Sham ADX 90 100 SAL/Sham SAL/ADX-C 80 M10/Sham 90 M10/ADX-C M20/Sham M20/ADX-C 70 80 SAL/ADX

70 60 60 50 (s)

e 50 40 Tim 40 30 30 20 20

10 10

0 0 day 1day 2day 3day 4day 5 day 1 day 2 day 3 day 4 day 5

B) RV Sham ADX 60 60 SAL/Sham SAL/ADX-C M10/Sham M10/ADX-C M20/ADX-C 50 M20/Sham 50 SAL/ADX

40 40 ) s (

e 30 30 Tim

20 20

10 10

0 0 day 1 day 2 day 3 day 4 day 5 day 1 day 2 day 3 day 4 day 5

C) DR Sham ADX 60 60 SAL/Sham SAL/ADX-C M10/Sham M10/ADX-C 50 M20/Sham 50 M20/ADX-C SAL/ADX

40 40 ) s (

e 30 30 m Ti

20 20

10 10

0 0 day 1 day 2 day 3 day 4 day 5 day 1 day 2 day 3 day 4 day 5

Figure 3.16: Adult ADX with CORT replacement, MWM Latency by day – Learning curves Mean (± SEM) Latency to escape (s)

186 Sham ADX

150 150 * 140 140

130 130 ) m

c 120 120 e ( anc t 110 110 Dis

100 100

90 90

80 80 Sal/Sham M10/Sham M20/Sham Sal/ADX-C M10/ADX-C M20/ADX-C Sal/ADX

Figure 3.17: Adult ADX with CORT replacement, MWM DR probe average distance to goal; neonatal treat x adult treat Mean (± SEM) Distance (cm) M20/Sham vs Sal/Sham *p<0.05

187 Summary Discussion and Conclusions for CORT experiment series

We hypothesized that neonatal MA administration causes permanent alteration of the stress response system resulting in impaired spatial learning and memory deficits in the Morris Water Maze (MWM) due to the inability to react normally to the inherent stress of the learning task (Roozendaal et al., 1996) following neonatal MA administration. Published reports have clearly indicated that increased levels of CORT (de Quervain et al., 1998) as well as lack of CORT production (due to ADX) (Oitzl and de Kloet, 1992) during behavioral testing results in poorer performance compared to animals with CORT levels that have not been experimentally manipulated. Additionally, evidence showing exposure to repeated stress and/or excess GC’s result in atrophy of hippocampal neurons

(Woolley et al., 1990) gives support to the idea that potential alterations of the stress response system with chronic exposure to MA could have deleterious effects on MWM learning.

The HPA axis stress response system is an essential component of the body’s ability to maintain homeostasis as well as react to periods when homeostasis is disrupted. Proper development of this system has been shown to be essential for normal function and behavior in the adult (Schmidt et al., 2003) or else psychological and behavioral problems can result (Heim and Nemeroff,

2001). Also, a report by Gerra et al. (Gerra et al., 2003) showed differential HPA function under normal and stress conditions in humans following cessation of 3,4- methylenedioxymethamphetamine (MDMA) abuse giving support to the hypothesis that amphetamine derivatives have the potential to alter HPA function.

188 The dosing protocol utilized in these experiments occurs during the SHRP in the rat (Dallman, 2000;Levine, 1994;Sapolsky and Meaney, 1986), a stage of development characterized by a lack of CORT release in response to stress; however, we have seen large increases in plasma CORT as well as increased

ACTH following MA administration during this period (Williams et al., 2000).

Based on these observations as well as the knowledge that neonatal MA administration results in long term learning and memory deficits (Vorhees et al.,

1994a;Vorhees et al., 2000;Williams et al., 2002;Williams et al., 2003b) led us to hypothesize that the MA treatment and resulting CORT increase may disrupt development of the HPA system and thus result in abnormal response to stress events in the adult animal resulting in the observed deficits in learning and memory.

Efforts were taken to determine the proper means by which to control the

CORT increase observed during the dosing period. Neonatal injections of MET

(JENKINS et al., 1958;Williamson and O'Donnell, 1967) and KTZ (Pont et al.,

1982;Van den et al., 1980) were administered in conjunction with MA in initial attempts to block the MA-induced CORT release but neither drug was able to block the CORT release for longer than 24 hours. We suspected that sufficient

CORT blockade was not occurring due to metabolism and clearance of the inhibitor thereby not having the agent consistently present in the system to block

CORT production and release. A time-release method of MET administration was then employed via pellets manufactured by Innovative Research of America

(Sarasota, FL) that were surgically implanted under the skin of the neonate near

189 the nape of the neck. This method also failed to block CORT in the neonatal

animal as it appeared to cause the animals to become sick and at times was fatal.

Despite the failure in neonatal animals with injections and time release pellets, MET was the primary drug candidate to block CORT production due to its extensive use in the literature for CORT control in adult rats (Baez et al.,

1996;Baez and Volosin, 1994;Calvo et al., 1998;de Quervain et al.,

1998;Roozendaal et al., 1996;Schoneshofer and Claus, 1985). By controlling

CORT release during behavioral testing we were able to address the hypothesis and the potential key role of the stress response in relation to the animal’s learning and memory abilities. The time-release method of MET administration via pellets

(Innovative Research of America, Sarasota, FL) was employed in the adult animal with implantation having occurred prior to behavioral testing. This method was again unsuccessful in blocking CORT as tested following a FS stress task apparently because the pellets did not release CORT appropriately. The method of adult MET subcutaneous injection (de Quervain et al., 1998) was chosen to be the means by which CORT release would be inhibited. The preliminary experiment demonstrated this approach was successful with administration of

MET at a concentration of 50 mg/kg each day 90 minutes prior to testing. Results from MWM testing demonstrated a weak effect of neonatal MA treatment among the VEH treated animals. Trends representative of the expected MA–induced deficit were seen in the reversal phase of learning and a significant difference among VEH treated animals was discovered in acquisition probe trial. Despite the weak display of MA-induced effects, adult MET treatment did appear to

190 correct the learning and memory deficits observed. These results supported the

original hypothesis as well as supporting the need for further testing via alternate

means of CORT control.

The final set of experiments designed to test the role of the HPA via

CORT control were designed to mimic the protocol used in the previous MET injection experiments but with the use of ADX as an alternate means of CORT control. The higher MA dose was raised from 15 mg/kg to 20 mg/kg as well as having added an additional week of MWM testing in attempts to make the MA- induced effect on learning and memory more pronounced. The ADX procedure is an effective means of CORT regulation (Devenport and Devenport, 1983;Oitzl and de Kloet, 1992). CORT replacement therapy was also employed via use of osmotic minipumps (Alzet, DURECT Co, Cupertino, CA). Results from preliminary experiments showed that CORT administration of 10 µg/hr

(Fukushima et al., 1985) was a sufficient replacement dose and we showed that these animals showed no CORT in response to FS stress (Figure 3.9). In the

MWM, swimming ability was impaired in ADX animals not having received

CORT replacement therapy. As this was the only group to display swimming difficulty, it was concluded that complete lack of CORT was the cause. Anxiety tests revealed a MA treatment effect not yet reported. The high dose group having received M20 displayed behavior consistent with either increased activity in the maze or decreased anxiety; the experiment as performed did not allow for clear definition of this effect. Furthermore, this MA-induced effect was corrected with ADX-C treatment. Calvo et al. (Calvo and Volosin, 2001) showed a similar

191 decrease in anxiogenic–like behavior in the elevated plus maze with CORT control via MET injection. MWM results again did not display a clear demonstration of the expected MA-induced learning and memory deficit among the control (Sham) group of animals. The clearest indication of a MA-induced deficit in memory was shown in the double reversal phase of testing and, again as was the case with Zero maze results, ADX-C treatment corrected this effect.

Taken together, significant MA-induced effects in the Zero maze and MWM double reversal that were corrected with ADX-C treatment supported the hypothesis indicating a possible role for CORT/HPA axis system in the mechanism of MA-induced learning and memory deficits.

The two sets of experiments exploring CORT control in the adult animal both showed less than the expected MA-induced learning and memory deficit.

We feel this is likely due to increased complexity in experimental design. The

MA effect, although reliable in past reports from our laboratory (Vorhees et al.,

1994a;Vorhees et al., 2000;Williams et al., 2002;Williams et al., 2003b), may be subject to intervening effects that may mask its appearance. Thus, we feel that manipulation of the animal at the adult level may in fact complicate and inhibit the effect caused by chronic neonatal MA dosing. Despite the lack of strong evidence of the MA effect in these experiments, evidence was present to suggest that it was present.

The presence of this issue did impact our ability to interpret our results as it was difficult to determine changes in the MA-effect on learning and memory with the additional adult treatment factors without a strong learning and memory

192 deficit observed in the control groups. Consequentially, it also made it difficult to determine if the lack of catecholamines (NEPI and EPI) in the ADX experiment impacted the results.

Nevertheless, despite these difficulties, results from the MET adult injection and adult ADX-C experiment series suggest a role for CORT and the

HPA axis stress response system in the mechanism of MA-induced long-term spatial learning and memory deficits.

193 CHAPTER 4 - SUMMARY

CONCLUSIONS

Methamphetamine

Methamphetamine use is growing among adolescent and young adults.

Discovered in Japan early in the 20th century, MA remains Japan’s most abused

drug (Matsumoto et al., 2002;Suwaki, 1991). Spread of clandestine laboratories has increased MA availability across the United States, a problem once centered on the west coast and in the Hawaiian Islands. A six hundred percent increase in

MA laboratory seizures occurred from 1981 to 1989 in the US with 3 states encompassing over 75% of these cases: California, Texas, and Oregon (Irvine and

Chin, 1991). Women are using this drug during their peak reproductive years at increasing rates. This pattern will potentially result in increasing numbers of children born to women who used MA during pregnancy. Long-term effects following prenatal exposure to MA are largely unknown at this time.

The evidence reviewed in the Introduction suggests that d-amphetamine and d-MA have low potential for inducing major malformations, except perhaps at very high doses. The limited human data on malformations show no consistent syndrome of effects. On the other hand, these drugs induce fetal growth retardation and increase neurobehavioral symptoms, especially when exposure occurs neonatally in rodents, a period analogous to human brain development during late gestation. Only one long-term study in humans is available from

Sweden on prenatal d-amphetamine. It suggests lower school achievement in the exposed children. Studies in animals are increasingly providing evidence that

194 exposure to MA during later stages of brain development result in long-term

learning and memory impairments. These effects occur at doses that by

allometric scaling are within the range used by humans. Neurochemical changes

are also found in animals exposed during brain development; however, no clear

mechanism of action has been identified. While the current neurochemical

evidence points to changes in dopaminergic systems (changes in PKA, DA, and

dopamine receptors) and in corticosterone, the changes in the developing brain are

unlike those seen in adult animals after similar doses. The available evidence

suggests, however, that there are reasons for concern about the long-term outcome

of infants exposed to MA in utero. Long-term prospective studies following

children throughout school will be required to determine the significance of the

neurobiological effects described in the more recent animal studies.

Brain Derived Neurotrophic Factor

Recent attention has been focused on a family of endogenous proteins

called neurotrophins. These substances are responsible for the growth and

survival of developing neurons and maintenance of neurons throughout

development. Key members of this family include nerve growth factor (NGF),

brain derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3); these proteins are essential for proper brain development and are also necessary for neuronal growth and survival (Leibrock et al., 1989;Lindsay et al., 1994;Schaaf et al., 2001). In addition to the role in maintenance, BDNF and NGF have been shown to be involved in learning and memory processes (Chen et al.,

1997;Fischer et al., 1987;Kesslak et al., 1998;Mizuno et al., 2000;Mu et al.,

195 1999). Additionally, high concentrations of these neurotrophic factors are found in the hippocampus.

Due to high levels of neurotrophins in key areas of the CNS during development (Das et al., 2001) and the suppression of neurotrophins by CORT in adult animals (Chao and McEwen, 1994;Kesslak et al., 1998;Schaaf et al.,

1997;Schaaf et al., 1998;Smith et al., 1995), it was hypothesized that the level of

BDNF and/or NGF would be altered with neonatal MA administration.

Investigation of this possible role of neurotrophins in MA-induced neurotoxicity has provided some useful information. Administration of MA (10 mg/kg 4x daily) to rats from P11-15 showed a small but significant increase in BDNF protein in the hippocampus. Administration of MA (10 mg/kg 3x) on P11 showed no significant changes in neurotrophin protein levels despite elevated CORT levels (Williams et al., 2000). However, a significant sex difference was found for BDNF concentration in the hippocampus and hypothalamus at P11.

Additionally, a significant sex difference in NGF concentration was found in the hippocampus at P15 and in the hypothalamus at P68. Despite these findings, no significant long-term treatment effects were discovered with respect to BDNF or

NGF in the adult animal or following training in the MWM.

The results from neonatal animals are not in agreement with previous reports demonstrating decreases in BDNF following increased CORT in the adult animal (Schaaf et al., 1998). Furthermore, we observed a long-term decrease in

CORT in the adult animal following neonatal MA treatment indicating additional potential for deviation of neurotrophin levels as compared to those normally

196 observed, however, no persuasive evidence of long-term BDNF changes was found. Taken together, our experiments involving MA and neurotrophins indicate that like many biological systems, neurotrophins do not respond to influence in the same manner in the developing animal as in the adult. These data indicate that

BDNF and NGF are not the main source of insult in the mechanism of MA- induced long-term learning and memory deficits.

Corticosterone & the HPA axis

The HPA axis is the body’s natural defense system against stress. It is essential for an organism to be able to recognize and respond to a stressful event.

The stress response includes release of CRF, ACTH, β-endorphin, NE, E, MC’s, and GC’s including CORT. Reaction among these factors allows the organism to be able to speed reaction time, adapt to pain sensation, and focus energy on the body systems necessary to return to homeostasis.

In rats and mice, it has been reported that there is a period of development known as the SHRP (Dallman, 2000;Sapolsky and Meaney, 1986;Schmidt et al.,

2003). This period of development, P4-14 in the rat and mouse is characterized by low stable levels of ACTH and GC’s as well as a lack of CORT release in response to stress as would be seen in the adult animal. It has been hypothesized that this period of low response to stress occurs to allow the development of critical regions of the nervous system as increases in these hormones may be detrimental to proper development and synaptic formation (Cotterrell et al.,

1972;Sapolsky and Meaney, 1986).

CORT regulation is a key factor in the HPA system in times of

197 homeostatic maintenance as well as in response to a stress event. A main function

of CORT is in the formation of carbohydrates and glycogen in the liver as well as

stimulation of glycogen formation in other tissues for use as an energy source.

CORT is also involved in aldosterone production which is responsible for normal

electrolyte balance. In times of stress, plasma CORT is increased and allows for

activation of energy stores as well as providing negative feedback to the HPA axis

when a sufficient reaction has occurred to allow shut off the stress response.

Our laboratory has shown that neonatal treatment with MA on P11-15 or

P11-20 results in long-term learning and memory deficits in the adult animal

(Vorhees et al., 1994a;Vorhees et al., 2000;Williams et al., 2002;Williams et al.,

2003b). The mechanism of this effect is yet unknown. Further experimental

results have shown a large increase in CORT and ACTH during the dosing period

which is also during the SHRP in the rat (Sapolsky and Meaney, 1986;Williams et

al., 2000). This observation provided a potential explanation for how CORT could play a key role in the MA-induced deficit as high levels of CORT are not normally seen during this period of neonatal development.

Efforts to control the CORT increase during the dosing period proved difficult. No effective means of consistent CORT attenuation was discovered.

These results further supported the belief the neonatal rat is very different that the adult in respect to response to MA treatment.

Use of MET treatment and ADX surgery with CORT replacement were successfully employed in the adult rat to help control and equalize the CORT response to the stress of the behavioral tasks among rats treated neonatally with

198 SAL and MA. Behavioral results displayed some correction of the learning and

memory deficit with both MET and ADX-C treatments. However, in both

experiments we had difficulty showing a clear MA-induced learning and memory

deficit among the groups of animals having received MA only. We feel this is

likely due to the increased complexity of the experimental design. Manipulation

of the adult animal may interfere and partially mask the effect of neonatal MA treatment. Despite lack of observation of a clear MA-induced deficit, the results provided some evidence that controlling CORT response during the behavioral tasks helped to correct the MA-induced deficit. This gives support for the idea that CORT may play a role in the mechanism of the MA effect normally observed.

FUTURE DIRECTIONS

Results indicating an attenuation of the MA-induced learning and memory deficit via controlling CORT levels in the adult animal provide a rationale for further experimentation in this area. Data provided herein give support to the hypothesis that neonatal MA treatment does in fact have long-term effects on the functioning of the HPA axis stress response system, effects that may cause an abnormal response to stress events and in our experiments lead to a learning and memory deficit.

Repeating a portion of both the adult MET and ADX studies with additional controls would be helpful to further determine the cause for not having observed a clear deficit in learning and memory tests following neonatal MA

199 administration. Addition of a group of animals, one of each neonatal treatment, having no additional adult treatment would provide the ultimate control group to address this question. This would give information as to whether the added adult treatment of either an injection or surgery adds sufficient complexity to the study design to partially mask the MA effect. Behavioral studies involving MWM testing should use the three week acquisition, reversal, double reversal protocol to help further delineate the effects of MA treatment. Furthermore, an additional experiment designed to mirror the ADX-C experiment could be performed using

CORT pellets of varying replacement value as described by Dallman’s group

(Akana et al., 1992;Akana and Dallman, 1997;Bhatnagar et al., 2000) to provide an alternate means of addressing the hypothesis and account for any shortcomings of using osmotic pumps and give potential for administering a higher level of

CORT replacement.

Prior to any additional ADX experiments, an experiment should be performed to test the effectiveness of the ADX procedure to completely block

CORT production. Some research has provided evidence for extra-adrenal CORT production (Davies and MacKenzie, 2003;Gardiner et al., 1981). The nervous and cardiovascular systems have been identified as potential sites for extra- adrenal CORT production (Davies and MacKenzie, 2003). We have hypothesized that neonatal MA administration results in an abnormal response to stress and thus that varying levels of CORT result in behavior deficits; therefore, any extra- adrenal production of CORT may complicate experimental designs used in ADX studies presented here. Gardiner et al. (Gardiner et al., 1981) have stated that as

200 many as 25% of rats that underwent bilateral ADX surgery in their studies are not adrenocortical insufficient. Their ability to distinguish this group from those that are lacking CORT and aldosterone comes from monitoring weight and salt supplementation. A study replicating the procedures used in Gardiner et al’s report (Gardiner et al., 1981) could be performed; or, additional blood collections for CORT analysis could be taken from all animals following ADX but prior to behavioral testing (and perhaps at several points throughout behavioral testing) to determine if CORT production has been sufficiently depleted. Only animals showing consistently basal levels of CORT should then be used behavioral analyses.

Further exploration of the role of CORT in the mechanism could be determined through examination of CORT receptors. Receptor blocking studies using MR antagonist spironolactone and the GR antagonist RU38486 could be performed in conjunction with the neonatal dosing protocol and adult behavioral testing. Adult or neonatal ADX with receptor agonist administration could also be employed as demonstrated in a study by Calvo and Volosin (Calvo and

Volosin, 2001) indicating involvement of both receptor types in restraint response. These drugs would allow the experimenter to ask if blocking one type of receptor preferentially over the other allows for a more clear effect in attenuating the MA induced deficit, or, if both need to be blocked to account for compensation by one when the other is inactivated.

Additional methods of controlling CORT release during the dosing period in the neonate should also be considered. Successful ADX surgery at this stage of

201 development would allow an alternate means to test the hypothesis. Unilateral

ADX should be explored initially to determine if removal of a single adrenal gland would provide sufficient means to attenuate the CORT response to chronic

MA dosing. If this were successful, the animal could survive and develop normally as the body adjusted to and compensated for having a single adrenal and potentially survive as a normal adult. This would allow the dosing to take place without the CORT increase that may be a key agent in abnormal development of the HPA system and thus abnormal adult behavior. Bilateral ADX can also be explored. In this case, the problem of CORT replacement in the pup would need to be addressed to determine a means by which to continue to allow for proper growth and development in the absence of the adrenal glands. The issue of hormonal replacement following the dosing period would also need to be explored. Transplantation of adrenal glands from a same age pup may be a viable option. Any means by which the lack adrenal glands can be compensated for, short of life-long hormonal replacement therapy via injections or pills, should be determined and strongly considered.

Historically, behavioral studies have been performed in the rat. Studies are currently being performed in our laboratory to determine if the MA-induced learning and memory deficit is also able to be observed in the mouse. Should results from these experiments prove this to be true, there will be multiple avenues by which the current hypothesis could be tested. Technology is such that it is very common to create transgenic mouse models, these models would be helpful in examination of the mechanism of the long-term effect of neonatal MA

202 treatment. Genes involved in CORT production and function could be targeted and knocked-out on a conditional basis. This would allow for CORT production to be effectively turned off during the dosing period and then turned back on to allow for normal growth and development throughout the remainder of the animal’s life. Other areas of the HPA axis could also be targeted for such experimentation. Furthermore, if the gene technology that is currently available in the mouse became as commonplace in the rat, the experiments proposed in the mouse could be extended to the rat model that has been used historically in the field of behavior.

203

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