Neural Processing of Magnetic Intensity Cues by Lesioned Homing Pigeons (Columba Livia) in a Magnetic Conditioning Paradigm

NEURAL PROCESSING OF MAGNETIC INTENSITY CUES BY LESIONED HOMING PIGEONS (COLUMBA LIVIA) IN A MAGNETIC CONDITIONING PARADIGM

Merissa L. Acerbi

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF ARTS

May 2017

Committee:

Verner P. Bingman, Advisor

Anne K. Gordon

William H. O'Brien

ABSTRACT

Dr. Verner P. Bingman, Advisor

The ability to orient in an environment has been shown for centuries to be an important behavior in reproduction and survival. Many scientists agree the avian hippocampal formation

(HF) is a brain structure involved in learning and memory tasks. Specifically, one type of learning and memory task, known as true navigation, defined as an animal’s ability to return home even when navigating in a novel environment. Previous research has shown one way birds are able to successfully complete true navigation is via the earth’s magnetic field. The question of what information is gathered with this geomagnetic sensitivity and where in the brain it is stored has remained unanswered. Currently, scientists theorize there are a number of avian species to use information about magnetic inclination via transduction by way of the Wulst area of the forebrain. However, few scientists have explored the role of magnetic intensity and what brain area(s) may be involved in order to successfully navigate. The current study illustrates the role of the HF as well as the Wulst during a novel magnetic conditioning paradigm where homing pigeons (Columba livia) experienced two different currents in magnetic intensity.

Pigeons (N = 8) were trained, tested preoperatively and underwent one of two previously assigned experimental lesion surgeries (an electrolytic HF lesion, N = 4 or an aspirated Wulst lesion, N = 4). Postoperative data indicates the HF-lesioned pigeons were no longer able to discriminate magnetic intensity while the Wulst-lesioned pigeons continued performing significantly above chance level. Additionally, the Wulst lesions were substantially larger in size versus the HF lesions, suggesting most of the Wulst is not necessary when gathering information iii about magnetic intensity. The results draw attention to a divide in magnetic inclination versus magnetic intensity, and the possible difference in underlying neural mechanisms involved.

There is no one else I could dedicate my thesis to besides the one who showed me what

behavioral neuroscience is all about. v

ACKNOWLEDGMENTS

Above all, I could not have completed the research and writing for this thesis if it weren’t for my advisor, Dr. Vern Bingman. All of his encouragements, strong beliefs in my success, and continually challenging me to reach my goals gave me the courage to become a scientist and pursue research as a career. I would also like to thank Dr. Cordula Mora for being the kindest of mentors. Without any prior experience, she took me in as an undergraduate and walked me through all the steps of applying technique and detail to science.

Throughout the years, I have had a number of truly exceptional research assistants that all helped me stay on top of what it takes to work with pigeons on a daily basis. This includes Mike

Brooks, Lindsey Cunningham, Stephanie Davis, Mary Flaim, Natasha Flesher, Greg Grecco,

Will Gyurgyik, Serena Foor, Stephanie Hylinski, Ashley Meehan, Imani Oliver, Amy

Ruthenburg, Connie Santaigo, Jessica Sharp, Preston Stevenson, Sarah Tolfo, Jazmin Williams and Anna Wittmer. I also am incredibly thankful for my research colleagues, including Jared

Branch, Vincent Coppola, Brittany Halverstadt, Dr. Robert Kirk, Diana Klimas, Mark McCoy,

Lynzee Murray, Timothy Patrick, Josh Ricker and Brittany Sizemore.

Additionally, I am beyond grateful for my committee members, Dr. Anne Gordon and

Dr. Bill O’Brien, for their comments and questions during my proposal and defense meetings. I also received a valuable mentorship from Dr. Linda Rinaman. To add, I would like to thank

Andy Wickiser for building all experimental equipment, Jeni Baranski/University of Animal

Facilities staff for keeping a watchful eye on the pigeons, Dr. Susan Orosz as BGSU’s veterinarian and Russell Mora for writing the virtual map software used throughout the experiments.

Lastly, countless appreciation goes to Christian Jacob Pemberton. Thank you. vi

TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

Avian Navigation ...... 1

Compass Mechanism ...... 2

Map Mechanism...... 3

The Earth’s Magnetic Field ...... 4

Processing Map Information ...... 5

Processing Compass Information ...... 7

Current Study ...... 9

METHODS ...... 10

Subjects ...... 10

Experimental Setup ...... 10

Magnetic Intensity Stimulus ...... 11

Pre-Training Procedure ...... 12

Preoperative Magnetic Conditioned Choice Training: Correction Trials ...... 13

Preoperative Magnetic Conditioned Choice Training without Corrections ...... 13

Postoperative Training ...... 14

Wulst Aspiration Lesions ...... 14

Hippocampal Formation Electrolytic Lesions ...... 15

Wulst Control Series ...... 15

Wulst and Hippocampal Formation Histology and Lesion Damage Reconstruction 16

Statistical Data Analysis ...... 16 vii

RESULTS ...... 18

Lesion Reconstruction ...... 18

Behavior ...... 18

Pre- and Postoperative Learning Curve ...... 18

Sessions to Criteria ...... 20

Session Contrasts ...... 20

Anti-Parallel/Parallel Control Series ...... 21

DISCUSSION ...... 22

Preoperative Magnetic Conditioned Choice Training ...... 24

Preoperative Magnetic Conditioned Choice Training without Correction ...... 24

Postoperative Training ...... 25

Anti-Parallel/Parallel Control Series ...... 25

Interpreting Results ...... 26

Sham Lesion Group ...... 27

Conclusion ...... 28

REFERENCES ...... 29

APPENDIX A. FIGURES ...... 37

APPENDIX B. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL

LETTER ...... 50

INTRODUCTION

Animal navigation is an increasingly popular field of study. Over the past 50 years, researchers have used behavioral measures to answer questions about navigation. Today, many questions require answers at a neurological level: what mechanisms are involved to produce the perception, cognition and behavior exhibited by navigating animals.

Avian Navigation

The ability to navigate to a goal location has been observed across a wide range of animal taxa, including (but not limited to) insects, fish, turtles, birds, and mammals (Able, 1982;

Benhamou et al., 2011; Mouritsen, Atema, Kingsford & Gerlach, 2013; Müller & Wehner, 1988;

Phillips et al., 2013). Navigational ability developed in response to an animal’s need to efficiently move in space and time. Orienting and moving in space can be found in the simplest nervous systems; across the animal kingdom, basic survival requires fulfilling certain needs (e.g., nourishment and reproduction). The rise of mobile animals meant being able to navigate within their environments and successfully finding and remembering locations of food, potential mates and shelter.

The concept of true navigation is the ability to return home even when placed in a completely unknown environment. With time and practice, birds can be trained to become true navigators. The first step involves the ability to determine where the animal is now, such as a remote, distant location, in relation to where the animal wants to go, e.g., a home loft. This requires the use of a map. The second step requires the animal to convert this acquired positional information into a direction (how to get from point A to point B) and makes use of a compass.

The final, third step is achieved once the animal has recognized it is in familiar territory, and thus 2 approached the goal (Kramer, 1953). Past experiments have demonstrated true navigation in a number of different species (Boles & Lohmann, 2003; Rodda & Phillips, 1992).

Compass Mechanism

The compass mechanism has been extensively studied and much is known about how it is used during animal navigation (Alerstam & Pettersson, 1991; Chapman et al., 2008; Quinn,

1980). The magnetic compass is one type of compass mechanism used by animals to navigate and functions with the aid of the earth's magnetic field (Freire, Munro, Rogers, Wiltschko &

Wiltschko, 2005; Gottlieb & Caldwell, 1967; Kiepenheuer, 1984). As an example of an orientation cue, it is based on the physical properties of the earth's magnetic field (Ritz et al.,

2009). Magnetic compass ability is often linked to the inclination parameter of the field

(Lohmann & Lohmann, 1994). Animals use the magnetic compass to be able to define directions in space (Kiepenheuer, 1984). The sand hopper, ant, honeybee, and lobster are all invertebrate species that scientists have consistently shown over a number of years to have some kind of magnetic sensitivity (Banks & Srygley, 2003; Lohmann et al., 1995; Ugolini, 2006; Walker &

Bitterman, 1985). Similarly, migratory birds, mole rats, sting rays, sea turtles, newts, salmon and tuna are all vertebrate animals demonstrated to have magnetic compass capabilities

(Diego‐Rasilla, Luengo & Phillips, 2008; Fransson et al., 2001; Kimchi & Terkel, 2001;

Kalmijn, 1978; Lohmann, Cain, Dodge & Lohmann, 2001; Putman et al., 2013; Walker, 1984).

One intriguing study performed by Thorup et al. looked at the white-crowned sparrows ability to navigate once displaced into a completely unknown environment. When comparing the performance of juveniles versus adults, the researchers found only the adult white-crowned sparrows able to successfully reorient in the right direction, emphasizing a purely compass mechanism resource accessed by the juveniles. 3

Map Mechanism

Map-based navigation is the ability to understand spatially where a goal is without being able to directly sense the goal’s location. Without any direct sensory contact with a goal, map- based navigation has also been termed true navigation (as discussed above) and mirrors Griffin’s

Type III Orientation (Griffin, 1952). Sub-types of map mechanisms include mosaic map navigation and gradient map navigation (Able, 2001).

With regards to the earth's magnetic field, a gradient-like map would be most relevant. A gradient map is also termed a bi-coordinate map, and involves geophysical gradients running in various directions with respect to each other (Wallraff, 1974). A gradient-like map aids an animal in recognizing the physical variables around the goal, for example, the home loft of a pigeon, so the animal can recognize when it has successfully returned home (Wallraff, 1991). In general, the animal making use of a magnetic field gradient map would compare the intensity and/or inclination values of the earth's magnetic field where it currently is to the values of where the goal is in order to successfully navigate.

As introduced above, this type of gradient map is where positional information would be obtained, effectively yielding latitude and longitude measurements. The systematic variation of the earth’s magnetic field results from the earth acting like a large bar magnet (see below).

Consequently, this allows for any point on earth to be defined as a vector that is made up of intensity (length of the vector) and inclination (the dip angle of the vector with respect to the surface of the earth). Past research has only weakly supported the role of the magnetic map in avian map navigation (Wallraff, 2003; Wiltschko & Wiltschko, 2003). Nevertheless, recent work with migratory birds produced a predicted compensatory effect when the birds were virtually displaced with respect to the earth's magnetic field (Kishkinev, Chernetsov, Pakhomov, Heyers 4

& Mouritsen, 2015). Therefore, current research points to the validity of both a geomagnetic map and a geomagnetic compass mechanism during avian navigation.

The Earth’s Magnetic Field

The earth’s magnetic field is spherical in nature and disperses out from the center of the earth’s interior, where the field leaves the earth’s crust at the south geomagnetic Pole (north end), circles out to the earth’s atmosphere, and then enters at the north geomagnetic Pole (south end). The arc that is created carries the magnetic field lines. The placement of the magnetic field within the earth is often compared to a large bar magnet where the field has two ends (a north and a south end) and the field is strongest at either end and weakest in the center, nearest the equator (Macmillan & Rycroft, 2010).

In comparison to the geographical poles, the geomagnetic poles are slightly displaced by an 11.5° tilt. In effect, the rotational axis of the earth is separate from the magnetic axis, and the two axes have separate poles. Although reversals of magnetic field polarity have occurred in the past, this type of natural event has only been recorded to occur about every tens of thousands of years. This type of phenomena forces the large bar magnet in the center of the earth to flip, leading to the north and south ends to reverse positions. However, magnetic field reversals are speculated to not cause a disturbance in animal navigation due to the constant availability of systematic variations (Walker, Dennis & Kirschvink, 2002).

Such systematic variation of the earth’s magnetic field allows any point on the earth’s surface to be represented by a vector. The vector has both a direction and a strength or magnitude

(the length of the vector). When moving towards the magnetic equator, the vector becomes more parallel to the earth’s surface; when moving towards either of the two geomagnetic poles, the 5 vector becomes more perpendicular. Due to the systematic variation of the vector, an animal can use this as a means of locating its position in space (Bloxham & Gubbins, 1985).

The earth’s magnetic field is commonly described with respect to these parameters: inclination and intensity. Inclination is the angle between the magnetic vector and the earth’s surface. It varies from north to south, but varies little when an animal is moving from east to west (or vice versa). Intensity is measured as the strength of the vector at any point within the magnetic field. Intensity decreases when traveling from either geomagnetic pole to the magnetic equator and increases when traveling in the opposite direction (Merrill & McElhinny, 1983;

Figure 1).

Processing Map Information

Understanding how birds process map information was first studied by Kramer in 1953 with his "Map and Compass" theory. In 1957, Kramer discovered differences in first-flight pigeons based on their vanishing bearings. During these studies, pigeons were released one by one from a specific location and data were gathered from the initial direction each took at an unfamiliar location, speculating that this was the bird's first indication of the homeward heading.

Unexpectedly, many pigeons did not initially fly in a homeward direction. Over the course of a number of years, a large amount of data from various lofts and locations found similar results

(Keeton, 1973; Kowalski & Wiltschko, 1987). This phenomenon was subsequently referred to as the release-site bias. It was proposed that this gave clues as to what was speculated to be the

"map" component in the homing pigeon navigational system. From this, scientists now agree that the first step in the three steps of navigation in homing pigeons involves the use of a map. When a bird is able to determine its position in relation to its goal, it is thought to be relying on map information. 6

Navigation near the home loft is thought to rely in part on the avian hippocampal formation (HF). Made up primarily of two prominent sub regions, the HF is comprised of the hippocampus (Hp) and the area para hippocampalis (APH). Recognized as a homologue of the mammalian hippocampus, the avian HF has been argued to be involved primarily in spatial tasks

(Bingman & Mench, 1990). A study by Eichenbaum, Hansen and Singleton (1986), found that rats needed an intact hippocampus in order to perform well on olfactory temporal order discrimination tasks. Additionally, unpublished data from the Bingman lab (2016) found homing pigeons with HF lesions did not decrease in performance during a visual temporal order discrimination task.

Another study whose data added to the idea that the avian HF is programmed primarily for spatial functions involved an open field spatial task. Here, Colombo, Swain and Alsop (1997) asked if the homing pigeon HF played a role in this specific environment. When control pigeons were compared to pre-lesioned pigeons during shaping, there were no between group differences.

However, when the two groups were again compared during the acquisition post-lesion period, the lesioned pigeons took significantly longer to complete this same task.

One experiment performed by Ioalè, Gagliardo and Bingman (2000) found hippocampal- lesioned pigeons to be significantly more error prone than control pigeons when released based on how well the birds’ vanishing bearings were oriented homeward. Nine years later, Gagliardo,

Ioalè, Savini, Dell’Omo and Bingman (2009) used global positioning system (GPS) recorders to study the tracks of homing pigeons. The primary question asked was whether homing pigeons needed an intact HF to successfully navigate back to their home loft from an unfamiliar location.

When the GPS tracks of the control pigeons and lesioned pigeons were compared, it was clear 7 the lesioned pigeons went far out of their way in the wrong direction (and continued flying away from the home loft for substantially longer distances) versus the control pigeons.

Additionally, Keary and Bischof (2012) found that hippocampal activation, evidenced through cFos neuronal activation expression, increased immediately after exposure to either a stationary or rotating magnetic field in zebra finches (Taeniogype guttata). To add, a study conducted in 2006 asked if there were any cells in the homing pigeon HF that responded to changes in the earth’s magnetic field (Vargas, Siegel & Bingman). Based on single unit recordings, the authors found HF cells participate in some aspect of spatially-guided behavior via changes in an ambient magnetic field.

As recent research sheds light on a possible magnetic map used by the homing pigeon, one theory in particular adds to this momentum. In 1998, Walker published the magnetic map hypothesis constructed from changes in the intensity slopes made up in the earth’s magnetic field. As discussed, he presented the idea that a homing pigeon could gather information about latitude from magnetic field intensity and information about longitude from the direction of the slope of magnetic field intensity. To add, the studies discussed above present the avian HF as a prominent brain region in creating a spatial landscape. Could this be referred to in terms of a gradient map, specifically a magnetic gradient map? Together, this information provides some indication that the HF may be used during the processing of map information but it has not yet lead to a theory on geomagnetic sensitivity and the use of map-based cues while navigating through space.

Processing Compass Information

Unlike the neural correlates involved in the processing of magnetic map information, the neural correlates involved in processing compass information have been shown in a number of 8 studies and are widely accepted (Emlen, 1975; Hein et al., 2010; Wiltschko & Wiltschko, 1972).

Specifically, researchers found that birds have nerve receptors in the brain that translate sensory information received through the eye. One study that supports this hypothesis found orientation behavior in European robins to differ when placed in different wavelengths of light (Wiltschko &

Wiltschko, 2001). While the European robins could orient in the correct direction under blue, turquoise and green light, they could no longer discriminate the magnetic field when the experimental arena was flooded with yellow light. Shortly thereafter, the avian magnetic compass was thought to be light-mediated and required visual information about the earth’s magnetic field to navigate correctly.

A subsequent study conducted by Mouritsen, Feenders, Liedvogel, Wada and Jarvis

(2005) further supports the theory behind magnetic compass abilities in birds. These researchers found the visual Wulst (and most notably a sub-region termed “Cluster N”) to be involved in light-mediated magnetoreception in night-migratory songbirds. As assumed by its name, the visual Wulst is a portion of the Wulst and is an avian forebrain structure involved in processing visual information. Located behind the retina, the visual Wulst directly receives inputs from photoreceptors in the eye. While studying migratory restlessness, Mouritsen et al. (2005) found night migratory songbirds to have an increase in neuronal activity in Cluster N. Not only was an increase in ZENK visually apparent when comparing these birds during day time and night time treatments, but this change in activation was also only illustrated when the birds had their eyes opened (versus eyes closed).

Due to recent work with migratory birds, scientists now agree that avian magnetic compass information is thought to be processed via visual cues in Cluster N of the visual Wulst

(within the Wulst). From this, an assumption can be drawn that the magnetic compass is light- 9 mediated and depends on the wavelength of ambient light in space. In effect, Ritz, Adem and

Schulten (2000) introduced the idea that magnetic visual input may overlap normal visual input and produce a specific shading pattern in relation to the direction a bird’s head is facing. Taking this into perspective, if the Wulst provides directional information, could there be a link between magnetic inclination and the direction a bird gathers from the earth’s magnetic field? If so, then magnetic compass information is transmitted via the visual pathway and processed in some area in the visual Wulst area of the homing pigeon Wulst.

Current Study

Inconsistencies can be found regarding the study of magnetoreception in the context of avian navigational research. Although studies have shown the HF to be responsible for avian spatial tasks, the question remains if such information generalizes to homing behavior in

(specifically) navigation via the earth’s magnetic field by the homing pigeon. For a clearer understanding on this topic, further neural and behavioral analyses are needed regarding the avian HF and its role in homing pigeon geomagnetic navigation. Studying the possible role of map-based geomagnetic navigation is an important topic and is best illustrated by comparing the

HF to Wulst (as a type of control lesion).

Therefore, I asked if the Wulst and/or the HF play a role in the ability to discriminate magnetic intensity cues during a spatial conditioning task. Additionally, my main prediction was that if a map-like representation of the earth’s magnetic field is used to interpret intensity cues, then HF-lesioned pigeons will be unable to discriminate intensity cues, whereas the Wulst- lesioned pigeons will continue to discriminate intensity cues.

METHODS

Subjects

Eight adult homing pigeons (Columba livia) with little to no homing experience were obtained from Bowling Green State University’s pigeon colony and were used in the study. The birds were housed at the Bowling Green State University’s Animal Facilities in Bowling Green,

Ohio, USA and lived individually in wire mesh cages in a temperature and humidity controlled room on a 12-12-hour light/dark cycle (lights on at 9:00 AM) with ad libitum access to water.

During the testing period, the pigeons were food deprived to no less than 82% of their free- feeding body weight. Each pigeon’s weight and food consumption were recorded and monitored daily. All experimental procedures and treatment of pigeons were carried out following the recommendations of the Guide of Use of Laboratory Animals of the National Institutes of Health and approved by Bowling Green State University’s Institutional Animal Care and Use

Committee (IACUC).

Experimental Setup

The testing room included a circular arena (dimensions 110 cm in diameter and 38 cm in wall height) and was in the center of a 3-axis magnetic coil system; the same apparatus was used in Mora and Bingman (2013), where a full description can be found (see also Mora, Acerbi &

Bingman, 2014). Located centrally inside the circular arena was a rotatable shaft with a horizontal tracker arm. Each individual pigeon was harnessed to the tracker arm by a clip. The horizontal tracker arm was attached to the circular arena by a vertical shaft. While harnessed, a pigeon could walk the entire 360 degrees of the arena’s periphery freely in either direction

(Figure 2). An angular decoder was placed underneath the circular arena and was used to detect the position of the pigeon to the nearest degree once every 200 milliseconds. Inside the circular 11 arena, there were four automated feeder-response units that lined up with the four cardinal directions (geographic north (N), east (E), south (S), and west (W); Figure 3). Each of the four automated feeder-response units contained a food magazine that held food pellets which were made accessible to a pigeon after a correct response was made by pecking the illuminated key located above the food magazine. Adjacent to the testing room was a control room where the pigeon’s behavior was monitored by a centrally-mounted video camera located above the arena.

Magnetic Intensity Stimulus

A three-axis magnetic Ruben coil system (four 240 x 240 cm square coils per axis, adapted from Merritt, Purcell & Stroink, 1983) was powered by three power supplies, one assigned to each axis of the coil system. This set-up generated a sphere-shaped magnetic field within the center of the coil system and was approximately the size of the diameter of the circular arena. During each testing day, the changing current output through each individual coil axis (see below) only resulted in a change in magnetic intensity (whereas inclination generally remained constant). The field’s magnetic intensity remained at one of two strengths at all times based on a pigeon’s location in the arena. Next to the coil system was a white noise generator that was used to mask any possible humming noise that would have been produced from the coil system and may have been a possible cue for a pigeon to use while in the arena.

A fully-automated, custom software program was written and controlled for the amount of current supplied to each of the three coil axes. The arena was divided into four equal 90 degree quadrants and each of the four cardinal directions were centrally placed in each quadrant.

Each quadrant remained either at 30 nT or 120 nT for the duration the pigeon was located within that quadrant (magnetic intensity changed abruptly when a pigeon transitioned from one quadrant to another). Across trials of a session (see below), the quadrant positions of the two 12 intensities could take two forms. One pattern consisted of N-S trials, where the north and south segments were “rewarded zones” and intensity remained at 120 nT when the pigeon was present in the quadrant. During N-S trials, the east and west segments were “unrewarded zones” and intensity remained at 30 nT. The second pattern consisted of E-W trials, where the east and west segments were “rewarded zones” and the north and south segments were “unrewarded zones”

(Figure 4). The distribution of N-S and E-W rewarded trials for a session was determined pseudo-randomly at the beginning of each testing session. The same rewarded areas were never repeated more than three times in a row.

Pre-Training Procedure

Before experimental training began, each pigeon went through a series of pre-training procedures to acclimate to the conditioning task and shaped to peck at the pecking keys. During the first phase of pre-training, each pigeon was fed in its home cage while harnessed to grow accustomed to wearing the harness. For the second phase of pre-training, each pigeon was placed in a harness in the circular arena, attached to the tracker arm, and was allowed to eat from four small piles of food set up to align with the four cardinal directions thus enabling it to grow accustomed to being in the circular arena. Immediately following, each pigeon received access to food via the food magazine and learned to eat from it. Next, Oreo crème was attached to the pecking key of each feeder and held a pellet of food. Once pecking at the food attached to the pecking key, each pigeon learned to peck at the pecking key first before receiving food from the food magazine. Lastly, the Oreo crème was removed as the pigeon began associating pecking at the pecking key each time a key peck light was illuminated to receive food. Each of the five phases of pre-training listed above took about five days. All levels of training were completed via operant conditioning. 13

Preoperative Magnetic Conditioned Choice Training: Correction Trials

Each pigeon learned the conditioning paradigm associated with the linear, step function change of the magnetic field. For five sessions (one session/day), each of the eight pigeons participated in magnetic conditioned choice training. As noted above, the angular decoder detected the pigeon’s movement through the physical change in location of the tracker arm in real time. Each time a certain position was located, the software would instantaneously generate a magnetic field vector where intensity was adjusted in relation to the position of the tracker arm.

As noted above, the location of the “rewarded 120 nT zones” and the “unrewarded 30 nT zones” was switched in a pseudo-random sequence for the 16 trials of each session of trials with correction.

Each trial consisted of a sample phase and a choice phase. The sample phase began when the trial light was illuminated and the pigeon was then given 15 seconds to move freely around the arena as much or as little as it chose. Once the 15 seconds were up, the choice phase began where all four feeders’ pecking keys were illuminated at the same time. If the pigeon pecked one of the two correct choice key peck lights, it was rewarded with food. A correct choice was one of the two feeders that was located in the “rewarded zones” for that particular trial. If a pigeon made an incorrect choice, it experienced 15 seconds in the dark, and then the same trial was repeated until the pigeon chose correctly.

Preoperative Magnetic Conditioned Choice Training without Correction

Following the five sessions of trials with correction, the pigeons underwent conditioned choice training without correction until the learning criterion was reached. The learning criterion was set at four out of five consecutive sessions in which a pigeon scored a 65.0% or better of correct first choices. Each session consisted of 32 trials and the location of the “rewarded zones” 14 and the “unrewarded zones” were switched in a pseudo-random sequence. Trials with incorrect first choices were now not repeated.

Postoperative Training

Post-Wulst lesion data collection mirrored magnetic conditioned choice testing data collection. Data collection ended once each of the four pigeons reached the same learning criterion as used preoperatively (four out of five consecutive sessions at 65.0% or higher). Post-

HF lesion data collection mirrored magnetic conditioned choice testing data collection. To add, a protocol was created prior to surgery stating that if any of the four pigeons did not once again meet that learning criterion by the 25th session, post-HF lesion data collection would end after session 25.

Wulst Aspiration Lesions

Four of the eight pigeons underwent bilateral, aspiration lesions of the anterior forebrain

Wulst with further intended damage to the deeper mesopallium. On the day of the surgery, each of the four pigeons underwent general anesthesia via the distribution of isoflurane through a vaporizer, overlying skull was removed and brain tissue aspirated. The lesions were targeted to be between anterior coordinates 13.0 and 10.0 taken from the pigeon brain atlas of Karten and

Hodos (1967). The intent of the aspiration lesions was to damage the visual areas of the Wulst.

The vertical depth of the lesions were also intended to cross into the mesopallium, which is included in the Cluster N of nocturnal, migratory songbirds that is thought to support magnetic compass orientation (Zapka et al., 2009). Following surgery, birds were allowed to recover for two weeks while being maintained on ad libitum access to food and water. All pigeons were then put back on food restriction followed by postoperative testing on the magnetic intensity discrimination task. 15

Hippocampal Formation Electrolytic Lesions

In conjunction with the Wulst aspiration lesion surgeries, the other four of eight pigeons underwent bilateral, electrolytic lesions of the HF. On the day of the surgery, each of the four pigeons underwent general anesthesia via the distribution of isoflurane through a vaporizer, overlying skull was removed, and brain tissue electrolytically lesioned. Three bilateral lesion- target coordinates were used to produce the HF lesions: A 3.8, L +/- 0.3, V 12.2; A 3.8, L +/- 0.5,

V 13.3; A 3.5, L +/- 1.0 which were taken from the pigeon brain atlas of Karten and Hodos

(1967). Following surgery, birds were allowed to recover for two weeks while being maintained on ad libitum access to food and water. All pigeons were then put back on food restriction followed by postoperative testing on the magnetic intensity discrimination task.

Wulst Control Series

Immediately following post-operative data collection, all four pigeons in the Wulst lesion group were part of a control series to test their performance when the three-axis Ruben coil system was completely turned off. Once each pigeon reached the postoperative learning criterion

(four out of five consecutive days of 65.6% correct choices), they were placed in the same experimental set-up as previously described.

A device was incorporated into the three-axis coil system where what was typically a parallel current could be turned into an anti-parallel current. During this state, the anti-parallel mode was defined as forcing one half of the total current in the system to move in one direction while the other one half moved in the opposite direction. This created the control state where the total current output remained at 0 nT during the entire 32-trial session.

The entire control series consisted of pairs of sessions where the coils were turned off in the anti-parallel state and where the coils were turned on in the parallel state (normal testing days 16 identical to preoperative and postoperative data collection). The series ran for 12 sessions beginning with two anti-parallel state sessions and ending with two parallel state sessions.

Wulst and Hippocampal Formation Histology and Lesion Damage Reconstruction

Once post-surgery data collection was complete, all pigeons were sacrificed to determine the extent of the according lesion damage. The pigeons received a lethal injection of a pentobarbital-based euthanasia solution (100 mg/kg intramuscularly) and was perfused intracardially with approximately 300 mL of phosphate buffer saline followed by 4% paraformaldehyde. The brains were then harvested and placed in 4.0% paraformaldehyde for 24 hours after which they were transferred to a 30.0% sucrose solution for 48 – 96 hours to ensure cryoprotection. Brains were then sectioned at 40 microns on a freezing microtome platform, with every second section mounted on a subbed slide. Tissue was differentiated with a cresyl violet staining protocol. Lesions were then reconstructed by placing the mounted tissue on a macroprojector.

Statistical Data Analysis

As noted above, each day of data collection consisted of each bird’s performance that was measured through the percentage of correct choices made. One day of data collection included 32 trials, and each trial stood for a choice made by each individual pigeon. Each time a session had been completed by all eight pigeons, a mean discrimination of performance was calculated for that session, analyzed, and graphed together next to each individual pigeon’s percentage of correct choices for that session. Subsequently, the calculation of the mean percentage of correct choices across all pigeons was measured and included as well as the standard error values for each session. 17

Several one-way and two-way repeated measures of analysis of variances (ANOVAs) were conducted to determine the change in performance across the testing sessions. Post-hoc T- tests (independent and paired samples) were used to compare the difference in performance in sessions. Significance was set at p < 0.05. Descriptive statistics were gathered for all figures. All data was entered into SPSS Statistical Package 22.

RESULTS

Lesion Reconstruction

Summarized in Figure 5 is the electrolytic lesion damage sustained by the HF-lesioned pigeons. As typically seen with HF lesions (e.g., Kahn & Bingman, 2009), all pigeons had substantial bilateral damage that was located in the hippocampus proper while damage to the adjacent parahippocampus was less extensive. For some pigeons, lesion damage extended modestly into the hyperpallium apicale, mesopallium, and nidopallium.

Summarized in Figure 6 is the aspiration lesion damage sustained by the Wulst-lesioned pigeons. The intent of the Wulst lesions was to insure that the brain damage extended ventral enough to damage the boundary area between the mesopallium and hyperpallium dorsale, which is the approximate location of the magnetically responsive Field N described in nocturnally migrating songbirds (Zapka et al., 2009). Indeed, this area was damaged in all pigeons, as was a large portion of the entire hyperpallial region (Figure 6a). In fact, one of the most striking aspects of the Wulst lesions was just how large they were. Lesion damage extended not only into the mesopallium, but also the anteromedial portions of the nidopallium and even into the striatum.

One pigeon (Figure 6b) was particularly striking, with substantial portions of the entire anterior forebrain damaged. Given the considerable size and range of the Wulst lesions, it is remarkable that the Wulst lesions did not result in any detectable performance deficit.

Behavior

Pre- and Postoperative Learning Curve. Figure 7 displays the preoperative learning performance of the HF- and Wulst-lesioned pigeons during the five trials of magnetic conditioned choice training (when the correction procedure was employed), the first five trials after the correction procedure had ended and the last five trials prior to lesion surgery (the two 19 latter known as preoperative magnetic conditioned choice testing). The learning curve is presented in this fashion because all the pigeons were trained to a specific learning criterion (see below) and therefore they did not all receive the same number of training sessions.

An ANOVA was carried out on the 15 preoperative sessions (all five sessions of preoperative magnetic conditioned choice training: correction trials, first and last five sessions of preoperative magnetic conditioned choice training: without corrections) and supports what is apparent from the figure. When looking at the difference between sessions, a main effect was illustrated (F(1,14) = 6.546, p = 0.000). When looking at the difference between the two groups, a main effect of treatment was also illustrated (F(1,1) = 5.289, p = 0.024). However, when looking at the difference between the two groups based on session, no interaction was found

(F(1, 14) = 0.699, p = n.s.). Over the course of training, the pigeons from both groups came to reliably perform at about 65.0% - 70.0% of correct choices.

Figure 7 also displays the performance of the HF- and Wulst-lesioned pigeons during the

10 postoperative sessions (first and last five sessions of the postoperative magnetic conditioned choice testing). Again, the learning curve is presented in this fashion because the pigeons were trained to a learning criterion (see below) and therefore they did not all receive the same number of postoperative training sessions. An ANOVA carried out on the 10 postoperative sessions again supports what is apparent from the figure. Unlike previously, when looking at the difference between the two groups, a main effect of treatment was illustrated (F(1,1) = 135.156, p = 0.000). However, when looking at the difference between sessions, no main effect was found

(F(1,9) = 0.799, p = n.s.). Additionally, when looking at the difference between the two groups based on session, no interaction was found (F(1,9) = 0.937, p = n.s.). Whereas pigeons subjected 20 to Wulst lesions discriminated the different magnetic intensities as they did preoperatively, HF- lesioned pigeons brought performance down to chance level (see below).

Sessions to Criteria. The conclusion drawn from the learning curve data are further supported by the analysis of sessions to criterion (Figure 8). Preoperatively, the HF- (M = 19.5,

SE = 2.5) and the Wulst- (M = 12.3, SE = 3.8) lesioned pigeons took a similar number of sessions to reach criterion and no significant difference was found (t(6) = 1.6, p = 0.163).

Postoperatively, the difference between the groups could not be more striking. Whereas the Wulst-lesioned pigeons quickly reached the postoperative learning criterion (sessions: M =

6.5, SE = 1.2); none of the HF-lesioned pigeons approached the learning criterion after 25 postoperative sessions. Assuming a minimum of 29 sessions would be needed by the HF- lesioned pigeons to reach criterion, the postoperative performance of the two groups differed dramatically (t(6) = 18.9, p = 0.000).

Session Contrasts. Additional information on the difference in the performance of the two groups can be gained by comparing performance during some of the sessions. For the last session of preoperative training (Figure 9), no difference was found between the HF- (M = 65.6,

SD = 0.0) and Wulst- (M = 65.6, SD = 0.0) lesioned pigeons with respect to the percentage of correct choices (t(6) = 1.0, p = 0.342). Moreover, the mean percentage of correct choices for both groups was 65.6% (HF: SE = 0.0, W: SE = 0.0).

For the first session during postoperative training (Figure 9), although the Wulst pigeons

(M = 60.9, SE = 3.7) outperformed the HF-lesioned pigeons (M = 50.8, SE = 3.5) no significant difference in the percentage of correct choices was found (t(6) = 2.0, p = 0.093). However, for the last session of postoperative training (Figure 9), a significant difference (t(6) = 4.0, p = 21

0.007) was found between the HF- (M = 53.9, SE = 1.5) and Wulst- (M = 65.6, SE = 2.6) lesioned pigeons with respect to the percentage of correct choices.

Finally, when comparing the HF group’s last preoperative session to the last postoperative session, a significant difference was found (Figure 10, t(3) = 7.9, p = 0.004). By contrast, when comparing the Wulst group’s last preoperative session to the last postoperative session, no difference was found (Figure 10, t(3) = 0.4, p = 0.718).

In summary, examination of various analyses presented above and the figures seen below converges on the conclusion that whereas Wulst lesions do not impact the capacity of the homing pigeons to discriminate differences in magnetic field intensity, HF lesions lead to a profound loss on this capacity.

Anti-Parallel/Parallel Control Series. Wulst lesions had no effect on the ability of the pigeons to discriminate differences in magnetic field intensity. However, to make sure the discrimination displayed by the Wulst-lesioned pigeons was based on differences in the magnetic field, after the postoperative training criterion was reached the Wulst pigeons were further tested with useful (current running parallel in the twin coils) and useless (current running anti-parallel in the coils) magnetic information (see also Mora & Bingman, 2013). Figure 11 illustrates the performance during parallel sessions (M = 67.3, SE = 0.7) and anti-parallel sessions (M = 51.2,

SE = 1.2) and found a significant difference between the mean of each testing condition (t(46) =

11.5, p = 0.000).

DISCUSSION

Past research has shown pigeon HF lesions can cause disruption in the ability to distinguish specific landmark cues while navigating in the environment. However, these studies have emphasized a link between the pigeon HF and a map-like ability. The current study illustrates that homing pigeons can be trained to distinguish magnetic intensity cues in a magnetic conditioning paradigm for the first time in a lab setting. Preoperative figures display similarity in training performance between the HF and Wulst pigeon groups and postoperative figures display a stark contrast in performance. Additionally, the control series further illustrates the Wulst pigeons continued ability to discriminate only when the magnetic field current is parallel (available and useful).

From these results, clues about the ambient field’s magnetic intensity can now be linked to information stored in the homing pigeon HF. Could this implicate a link between gathering information about the magnetic field’s intensity cues and storing such information in the HF? As described above, one key role of the avian HF is to navigate successfully in a spatial environment and provide cognitive skills to spatial obstacles.

These lesion deficits in the HF targeted pigeons are particularly intriguing because they add to the research of previous studies. One particular study found hippocampal lesions did not disturb the homing pigeons’ functional magnetic compass. This was studied through vanishing bearings of intact and HF-lesioned groups. In six different locations, HF-lesioned homing pigeons continually displayed vanishing bearings similar to intact homing pigeons (Ioalè,

Gagliardo & Bingman, 2000b). An additional study from the same group of researchers also found hippocampal ablated pigeons to display nearly identical vanishing bearings to intact 23 pigeons as well as anosmic hippocampal ablated pigeons and anosmic control pigeons (Ioalè,

Gagliardo & Bingman, 2000a).

As a further point of discussion, HF-lesioned Savannah sparrows produced vanishing bearings in a strikingly similar pattern when compared to untreated Savannah sparrows during geomagnetic migratory orientation (Bingman, Able & Siegel, 1999). However, results also indicate a decrease in migratory behavior in the HF-lesioned sparrows when compared to the intact sparrows. All studies discussed above provide data in support of a separation between HF and the ability to make use of a compass mechanism.

One study that possibly provides data in support of the HF being linked to the map ability was found in the research by Mayer, Pecchia, Bingman, Flore, & Vallortigara (2016). Here, domestic chicks were trained to successfully locate a specific rewarded corner using either geometric or feature based rectangular boundaries. After testing, enhanced cFos neuronal activation was found only in the HF and only immediately following the completion of a geometric based task. The authors concluded that HF is “central to processing spatial- geometrical information”.

Additionally, unpublished data from the Bingman lab (2014) studied the role of the homing pigeon HF and Wulst in a magnetic conditioning paradigm where inclination cues were used. The results found the exact opposite effect of what was seen here. Throughout postoperative testing, all Wulst-lesioned pigeons immediately decreased in performance while

HF-lesioned pigeons continued to discriminate inclination cues well above chance level.

Combined with the current work, we now have a double dissociation between HF-lesioned pigeons during an intensity driven task and Wulst-lesioned pigeons during an inclination driven task. 24

Preoperative Magnetic Conditioned Choice Training

Magnetic conditioned choice training occurred prior to any preoperative data collection.

All eight homing pigeons experienced five sessions of 16 trials each where the percentage of correct choices was calculated at the end of each session. It was during this time that a learning curve was established for each pigeon where a majority of the individual pigeons increased in overall performance between the first and fifth sessions of correction trials. Specifically, five out of the eight pigeons improved in session five when compared to session one. Additionally, six out of the eight pigeons had a higher average across the five sessions when compared to session one.

In general, the magnetic conditioned choice training sessions did not produce as clean of a learning curve as originally planned. The Wulst pigeons showed the largest increase in overall average performance but also showed a large amount of inter-individual variability as well. The

HF pigeons displayed the strongest learning curve between sessions two and four. To add, the group’s overall average performance did increase when comparing sessions one and five.

Preoperative Magnetic Conditioned Choice Testing without Correction

Preoperative data collection included two sets of five sessions of 32 trials each where the percentage of correct choices was calculated at the end of each session. The learning curve that formed during the magnetic conditioned choice training was solidified during this period. To emphasize this, six of the eight pigeons improved when the first session of correction trials was compared to the first session of preoperative testing. Furthermore, six of the eight pigeons improved when the first and last sessions of preoperative testing were compared.

Continuing discussion of the learning curve, the Wulst group remained most consistent in performance between sessions during preoperative testing. This learning curve may not have 25 been as steep due to the group’s premature and steeper learning curve during the correction trials.

The HF group showed the most consistent learning curve between the fourth session of the first five preoperative sessions and the second session of the last five preoperative sessions. Across the two groups, performance was most similar in the fourth and fifth of the last five sessions in preoperative testing.

Postoperative Training

As depicted in the learning curve figure (Figure 7), testing following the HF lesion surgeries immediately declined when compared to the last session of preoperative testing

(preoperative testing last session: 65.6% postoperative testing first session: 50.8%). Even though the group’s overall average performance appeared to increase within the first four trials, the entire ten sessions of data collection do not fall within 10.0% of the learning criterion established during preoperative testing (learning criterion: 65.6%, hippocampal group’s highest postoperative session: 55.5%). Not only did the HF group’s performance fall significantly below the performance of the Wulst group’s, it is also the only time for either group to be at or below chance level during any point in time outside of the corrections trials.

As for the Wulst lesions, these pigeons remained steadily and significantly above chance level throughout the course of the postoperative sessions. At two different points in time, the

Wulst group’s overall average peaked at 68.0% (third session in both the postoperative first five and last five). On the other hand, the HF pigeons never leave the 50.0% chance level by +/- 6.0%

(highest postoperative session: 55.5%, lowest postoperative session: 45.3%).

Anti-Parallel/Parallel Control Series

As seen in the Wulst control series image (Figure 11), all pigeons performed as expected when the field was useless (current running anti-parallel in the twin coils) versus when the field 26 was useful (current running parallel in the coils). Inter-individual variability largely varied more during the “Anti-Parallel” sessions versus the “Parallel” sessions. This may be due to the novel state of the static magnetic field for the pigeons. The largest variability was seen during Session

5, where individual data on percentage of correct choices ranged from 43.8% to 59.4%.

Interpreting Results

As discussed previously, the results of this study illustrate a significant difference in the ability to detect magnetic intensity cues after homing pigeons undergo either a bilateral HF electrolytic lesion, a bilateral Wulst aspiration lesion. In addition to overall group performances, the standard error calculations for the HF group varied significantly more throughout the postoperative sessions when compared to the Wulst group performance. This is due to the higher inter-individual variability throughout postoperative testing. The increase in standard error measurements visualizes the inter-individual variability between each pigeon that was not as prominent during preoperative testing.

The results of the lesion reconstructions also provide valuable information about the behavioral data. As noted above, the Wulst lesions were aspirated, or, removal of large portions of brain tissue altogether. Postoperatively, these pigeons were healthy and able to complete the same behavioral task as before surgery while also still excelling in performance. This point is illustrated best in Figure 6b, Bird #896. To emphasize further, the HF lesions were performed electrolytically, or by insertion of a very fine needle and burning only a select portion of tissue.

In effect, the HF lesions produced significantly smaller areas of tissue damage (versus the Wulst lesions) but resulted in far greater deficits within the same behavioral paradigm.

Future research could focus on lesions to specific populations of neurons within the HF.

For instance, one study found homing pigeons with left HF lesions could no longer complete an 27 outdoor navigational task when both the control and right HF-lesioned pigeons still could

(Gagliardo et al., 2001). Select GABA antagonists could also help pinpoint the pigeon HF’s role during various navigational tasks as one study found a high density of GABA receptors in both the ventrolateral and ventromedial cell layers of the HF V-complex (Herold et al., 2014). It may also be worth exploring patterns of activation within the HF, which can be incorporated through immunohistochemistry labeling of cFos (Keary & Bischof, 2012).

Sham Lesion Group

Prior to the start of the study, three additional pigeons were originally included for data collection and testing. After reaching the learning criterion, the three pigeons also went through preparation for one of the two experimental surgeries. This included a period of at least 12 hours of access to water only and occurred directly before the surgery. On the day of the surgery, each of the three pigeons also underwent general anesthesia and the skull was drilled. However, due to unforeseen technical difficulties that arose during surgery, each of the three pigeons were removed as candidates for one of the two experimental lesions and alternatively labeled as part of a sham group. As such, an electrode only touched the brain and did not enter or lesion any part of the brain.

Following surgery, a recovery period of two weeks was maintained while all three pigeons were on ad libitum access to food and water. All pigeons were then put back on food restriction. Post-sham data collection mirrored magnetic conditioned choice testing data collection. The sham lesion group was essentially identical to the HF and the Wulst lesion groups in every aspect of the study preoperatively and significantly similar to the Wulst lesion group in respect to results postoperatively as they continued to discriminate between the two intensity cues during testing. 28

Conclusion

The current study asked the question, do the HF and/or the Wulst play a role in the ability to discriminate magnetic intensity cues during a spatial conditioning task? It was predicted that if a map-like representation of the earth’s magnetic field is used to interpret intensity cues, then

HF-lesioned pigeons will be unable to discriminate intensity cues whereas the Wulst-lesioned pigeons will continue to discriminate intensity cues. The results of our behavioral tests both preoperatively and postoperatively found a statistically significant difference between the HF- lesioned pigeons and the Wulst-lesioned pigeons/sham pigeons. Impairments were immediate and drastic in the birds that underwent bilateral HF electrolytic lesions, a key sign that allows speculation into a link between magnetic map ability and magnetic intensity discrimination.

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APPENDIX A. FIGURES

Figure 1. A visual depiction of the earth’s magnetic field, taken from Magnetic Orientation in Animals by Wiltschko (2012). The bold line illustrates the Earth’s rotational axis and the turning arrow illustrates the axis’ movement in a counterclockwise direction. The vertical line above the earth illustrates where geographic north is located in comparison to geomagnetic north. The vertical line below the earth illustrates where geographic South is located in comparison to geomagnetic South. The dashed line illustrates the geographic equator. The slightly tilted double line illustrates the magnetic equator. The rectangular box illustrates the distribution of magnetic intensity across the earth’s surface. The angled arrows illustrate the changing strengths in magnetic inclination across the earth’s surface. 38

Figure 2. Visual depiction of the arena within the experimental setup. The vertical line illustrates the shaft. The horizontal line illustrates the tracker arm attached to the shaft. The vertical line perpendicular to the tracker arm illustrates the clip used to attach to the harness. The bolded, crossed lines across the body of the pigeon illustrates the harness. The pigeon is fitted into a harness and tethered to the tracker arm by way of the clip and is able to walk freely 360 degrees around the arena.

Figure 3. Visual depiction in bird’s eye view of the arena within the experimental setup. The four dashed lines illustrate the central point of the four quadrants. The four dark squares illustrate the four feeders used to depict one of four cardinal directions (north, south, east and west). The black diagonal line attached to the pigeon illustrates the horizontal tracker arm attached via the harness worn by the pigeon.

Figure 4. A visual depiction of the magnetic intensity stimulus as described in the methods. Black circled cardinal directions illustrate the quadrants where the pigeon was rewarded if it pecked at the feeder located within each quadrant. Dashed lines illustrate the edge of each quadrant.

Figure 5. (above) Schematic coronal sections of the HF lesion reconstructions at 1.0 mm intervals from anterior (A 9.5) to posterior (A 5.5) according to the atlas of Karten and Hodos (1967), labeled according to the revised nomenclature (Reiner et al., 2004). Black areas identify damage in at least two of four pigeons. Abbreviations: APH, area parahippocampalis; E, entopallium; HA, hyperpallium apicale; HD, hyperpallium dorsale; Hp, hippocampus; M, mesopallium; N, nidopallium; V, ventriculus.

Figure 6. (above) Schematic coronal sections of the Wulst lesion reconstructions at 1.0 mm intervals from anterior (A 14.5) to posterior (A 9.5) per the atlas of Karten and Hodos (1967), labeled per the revised nomenclature (Reiner et al., 2004). (A) black areas identify damage in at least two of four pigeons. (B) black areas identify damage in one specific pigeon, Bird #896. This pigeon had the most unusual lesion damage compared to the other three pigeons, specifically due to the lesion size and the pigeon’s ability to continue performing significantly above average postoperatively. Abbreviations: APH, area parahippocampalis; BO, bulbus olfactorius; CPP, cortex prepiriformis; HA, hyperpallium apicale; HD, hyperpallium dorsale; LSt, lateral striatum; M, mesopallium; MSt, medial striatum; N, nidopallium; S, nucleus solitaris; TuO, tuberculum olfactorium; V, ventriculus, Va, vallecula; VO, ventriculus olfactorius.

Figure 7. Percentage of correct choices per session throughout the entirty of the study as depicted in a learning curve. The solid, black line with circular data points indicate the hippocampal formation (HF) group’s average performance and the dashed, black line with the triangular data points indicate the Wulst (W) group’s average performance. The first five data points illustrate the magnetic conditioned choice training sessions, as indicated by “Corrections”. The second five data points illustrate the first five magnetic conditioned choice testing sessions prior to surgery, as indicated by “Pre-op First 5”. The third five data points illustrate the last five magnetic conditioned choice testing sessions prior to surgery, as indicated by “Pre-op Last 5”. The fourth five data points illustrate the first five magnetic conditioned choice testing sessions following surgery, as indicated by “Post-op First 5”. The last five data points illustrate the last five magnetic conditioned choice testing sessions following surgery, as indicated by “Post-op Last 5”. The vertical black bars indicate the separation between various testing phases. The thickest vertical bar indicates the separation between preoperative and postoperative testing. Chance level (50.0%) is indicaed by the horizontal black line.

Figure 8. Number of sessions to criteria when the two experimental groups were compared both preopeartively and postoperatively. Black bars indicate the hippocampal formation (HF) group’s average performance and the white bars indicate the Wulst (W) group’s average performance. Error bars are indicated by the small, vertical black bars on top of each black and white group performance bar. A nonsignificant difference was found when the average number of sessions it took to reach criteria preoperatively was compared between the HF group and the Wulst group. During the postoperative sessions, none of the HF group pigeons reached the predeterminded criteria by session 25. This is indicated by the diagonal lines at the top of the postoperative black bar. When using 29 sessions as the hypothetical average number of sessions it took the HF group to reach criteria, a significant difference was found between the actual average number of sessions it took the Wulst group to reach critera.

Figure 9. Percentage of correct choices when the following sessions were contrasted: the last preoperative session, the first postoperative session, and the last postoperative session. Chance level (50.0%) is indicated by the black horizontal line. Black bars indicate the hippocampal formation (HF) group’s average performance and white bars indicate the Wulst (W) group’s average performance. No significance was found between the two groups during both the last preoperative session as well as the first postoperative session. However, the statistically significant difference between groups during the last postoperative session suggests the group performances (on average) varied due to the type of experimental lesion.

Figure 10. Percentage of correct choices when the last preoperative session was compared to the last postoperative session within groups. Chance level (50.0%) is indicated by the black horizontal line. Black bars indicate the hippocampal formation (HF) group’s average performance and the white bars indicate the Wulst (W) group’s average performance. A statistically significant difference was found when the HF group’s average last preoperative session was compared to the last postoperative session, but no significance was found when the Wulst group’s average last preoperative session was compared to the last postoperative session. This performance difference seems to be tied specifically to the difference in lesioned areas.

Figure 11. Percentage of correct choices during the anti-parallel/parallel control series for the Wulst-lesioned pigeons. The thick, black line indicates the average performance across all four Wulst pigeons. The small, vertical black line that envelopes each data point indicates the error bar for each session. The tall, horizontal black lines indicate the change in session type from the anti-parallel series to the parallel series. “Anti-Parallel” labels the anti-parallel sessions and “Parallel” labels the parallel sessions. Chance level (50.0%) is indicated by the black horizontal line.

APPENDIX B. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL

LETTER

DATE: February 17, 2015

TO: Cordula Mora FROM: Bowling Green State University Institutional Animal Care and Use Committee

PROJECT TITLE: [707209-2] Conditioning of Homing Pigeons (Columba livia) to Magnetic Field Stimuli in Spatial Orientation Tasks IACUC REFERENCE #: SUBMISSION TYPE: Revision

ACTION: APPROVED APPROVAL DATE: February 17, 2015 EXPIRATION DATE: February 16, 2018 REVIEW TYPE: Designated Member Review

Thank you for your submission of Revision materials for the above referenced research project. The Bowling Green State University Institutional Animal Care and Use Committee has APPROVED your submission. All research must be conducted in accordance with this approved submission. Please make sure that all members of your research team read the approved version of the protocol.

Report all NON-COMPLIANCE issues regarding this project to this committee.

Please note that any revision to previously approved materials must be approved by this committee prior to initiation. Please use the Addendum Request form for this procedure.

This project requires Continuing Review via Progress Report on an annual basis. Please use the Annual Renewal form for this procedure.

If you have any questions, please contact the Office of Research Compliance at 419-372-7716 or [email protected]. Please include your project title and reference number in all correspondence with this committee.

This letter has been electronically signed in accordance with all applicable regulations, and a copy is retained within Bowling Green State University Institutional Animal Care and Use Committee's records.

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