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Chapter 1
INTRODUCTION
Purpose of Study
The present study investigated whether auditory cues would exert landmark
control over the brain circuitry that appears to encode the sense of direction in rodents.
In addition, this study sought to determine whether a distal or proximal auditory cue is a
more dominant landmark on this circuitry. The head direction (HD) cell system is a
directionally sensitive neural circuit that is believed to contribute to navigational
processes. These neurons are activated when an animal’s head is pointed in a particular
direction along the horizontal plane, the preferred direction of the recorded cell. Such
activation has been shown to be significantly affected by the location of visual landmarks,
as shifting the location of these landmarks will usually produce a corresponding shift in
the preferred direction of the recorded cell. In addition, visual landmarks which are
located distally in the animal’s surrounding environment have been shown to exert
greater influence on HD cells than do proximal landmarks located in the animal’s
immediate environment. The current investigation seeks to determine if auditory stimuli
can exert similar control over the HD system, and if the same distal and proximal
influences apply.
Background and Significance
Navigation is a multilevel, multifaceted process which occurs when an organism
moves throughout the surrounding environment. The neural processes involved in spatial
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navigation became easier to study with the advancement of electrophysiological
techniques of recording brain cell activity. Measuring neuronal activity is made possible
by implanting an electrode into the brain site of interest. This surgical procedure allows
an experimenter to monitor brain cell activity during normal behavior.
Electophysiologists first discovered place cells in 1971 (O’Keefe & Dostrovsky).
Place cells appear to reflect a sense of orientation by registering the animal’s location
within an environmental enclosure, or arena, where the animal is freely moving. Each
place cell has a particular location in the environment, the place field, where the cell fires
most rapidly when the rat’s head is in that location (Muller, 1996). A typical place field
can be seen in Figure 1 where this particular place cell fires less rapidly the farther the
rat’s head is from its place field. Place cells have been located in the subfields of the
hippocampus, the entorhinal cortex, and the subiculum (Jung & McNaughton, 1993;
O’Keefe & Dostrovsky, 1971).
.
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Figure 1. Place/firing rate plot showing the place field of a place cell. The figure shows
the top down view of the floor of the recording arena. The shade of each pixel indicates
the activity level of the cell when the animal’s head was in that part of the apparatus with
increased firing rates of the place cell indicated by darker shades. A place field for this
place cell can be seen in the southeast location of the arena. The prominent visual
landmark in the arena, a white card affixed to the wall, is indicated as the curved line to
the far right.
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HD cells were discovered in rats in 1984 by James B. Ranck, Jr. (Wiener & Taube,
2005). They were first identified within the rat postubiculum (PoS), also named the
dorsal presubiculum, of the limbic system (Taube, Muller, & Ranck, 1990a, b). HD cells
fire action potentials when the head of the rat is facing a particular direction along the
horizontal (or yaw) plane. Their activity is irrespective of the pitch (tilt) of the head, the
animal’s location, or geomagnetic forces (Mizumori & Williams, 1993). HD cells exhibit
directional preference where each cell has a preferred direction in which it will discharge
maximally. For instance, one HD cell might have a preferred direction of north, while
another HD cell might have a preferred direction of southwest. It is thought that all
possible directions are represented within the HD network and the total pattern of activity
within the HD system could be used as an instantaneous readout of directional orientation
for navigational behavior.
HD cells are typically recorded while the animal is foraging for food inside the
experimental arena (see Figure 2). The directional characteristics of these cells are
evident by constructing a plot of cell firing rate versus head direction for the recording
session (see Figure 3). Plotting the cellular activity in this way produces a Gaussian
tuning curve with the peak firing rate occurring at the preferred direction of the recorded
cell. The preferred direction of the example cell shown in Figure 3 can be seen as 220º.
As seen in the panel on the right, the cell shows maximal activity at this direction and the
firing rate of the cell declines to baseline as the head direction moves away from the
preferred direction of the cell. The range of head directions in which the firing rate is
greater than baseline encompasses the directional firing range for the cell. Typically,
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HD cells demonstrate directional firing ranges of approximately 90˚ (~ 45˚ on either side
of the peak), but ranges from 60˚ to 150˚ have also been observed (Taube & Bassett,
2003). HD cells show persistent activity even when the animal is stationary and do not
appear to exhibit adaptation effects (Taube & Muller, 1998).
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Figure 2. Typical recording arena used when monitoring for HD cells. The rat forages
for food in the recording arena while brain activity is monitored for HD cells. The white
cue card attached to the wall serves as the salient visual landmark in the apparatus.
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90 deg
180 deg
0 deg
270 deg
Firing Rate (Spikes/Sec)
Preferred Direction = 220 deg.
80
60
40
20
0
0
60
120
180
240
300
360
Head Direction (deg.)
Figure 3. Example tuning curve for a recorded HD cell. The left panel shows the top
down view of the apparatus, the position of the cue card, and the preferred direction of
the recorded cell. The right panel shows a plot of the average firing rate of the cell at
various head directions. The preferred head direction of this cell was 220º (southwest),
as indicated by the fact that the cellular firing rate was greatly elevated when the animal’s
head was facing that direction.
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Anatomical Locations of HD Cells
Since their initial discovery in the rat postsubiculum, HD cells have also been
found in the anterior dorsal thalamic nucleus (ADN; Taube, 1995), lateral dorsal thalamic
nucleus (LDN; Mizumori & Williams, 1993), retrosplenial cortex (Cho & Sharp, 2001),
lateral mammilary nucleus (LMN; Stackman & Taube, 1998), and dorsal tegmental
nucleus (DTN; Sharp, Tinkelman, & Cho, 2001) in rats. In addition, HD cells have been
found in the ADN of mice (Yoder & Taube, 2009) and chinchillas (Muir, Brown, Carey,
Hirvonent, Della Santa, Minor, & Taube, 2009), and the presubiculum of rhesus monkeys
(Robertson, Rolls, Georges-Francois, & Panzeri, 1999).
Many of the brain structures containing HD cells have been shown to have direct
or indirect connections with the hippocampal complex, where place cells were first
discovered and a brain region that is strongly linked to both navigation and episodic
memory formation (O’Keefe & Nadel, 1979; Tulving & Markowitsch, 1998; VarghaKhadem, Gadian, Watkins, Connelly, Van Paesschen, & Mishkin, 1997). These regions
form an ascending network called the Papez circuit leading to the hippocampus (Leutgeb,
Ragozzino, & Mizumori, 2000). Both HD and place cells appear to collaborate in
establishing a sense of direction and orientation (Calton, Stackman, Goodridge, Archey,
Dudchenko, & Taube, 2003; Warburton, Baird, Morgan, Muir, & Aggleton, 2001),
sharing many cellular characteristics while remaining independent systems.
Idiothetic and Allocentric Cues
HD cells have been shown to be highly sensitive to the presence of familiar and
salient directional references under most conditions. HD cells appear to be controlled by
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cues that are fixed to the external environment as well as internal cues that are related to
the movement state of the animal (Blair & Sharp, 1996; Goodridge, Dudchenko,
Worboys, Golob, & Taube, 1998; Knierim, Kudrimoti, & McNaughton, 1998; Taube &
Burton, 1995; Zugaro, Arleo, Berthoz, & Wiener, 2003). These fixed external stimuli are
also known as allocentric (or world centered) sources of information. Examples of
allocentric information would be landmarks in the environment composed of visual,
auditory, or olfactory cues. Extramaze allocentric cues are those that are located outside
of the animal’s immediate environment (e.g., a sink in the experimental room visible
from the recording enclosure) and intramaze allocentric cues are those that are within the
animal’s immediate environment (e.g., objects on the floor of the recording enclosure)
(Muir & Taube, 2002). An allocentric reference frame is formed from both of these
sources (Wiener, Berthoz, & Zugaro, 2002), and as the animal moves in the environment
this reference frame changes. New information is then gathered regarding the
environmental stimuli, regardless of the animal’s body position in relation to the stimuli.
In contrast, idiothetic navigational cues are based on internally generated
measures of movement velocity (speed and direction) rather than on fixed landmarks
(Wiener et al., 2002). These sources of movement related signals include vestibular,
proprioceptive, somatosensory, optic flow, and motor efference copy.
Allocentric Cue Control of the HD System
The earliest studies of HD cells discovered that a salient visual landmark could
exert control over the preferred direction of HD cells (e.g., Taube et al., 1990b). The
white cue card attached to the wall of the recording cylinder as shown in Figure 2 is a
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common visual landmark used in studies of HD cells. A typical strategy to assess the
ability of a visual landmark to control HD cell activity is to move the landmark between
recording sessions, after the animal is removed from the arena, in order to determine if
the preferred direction of the cell shifts with the landmark when the animal is returned to
the arena. Experiments using such environmental manipulations have found that the
preferred direction of a cell will usually shift nearly an equal amount and in the same
direction as the cue card during these manipulations, thus illustrating landmark control
from the cue card. Figure 4 illustrates this effect. In this figure, the preferred direction of
the recorded cell is at 136º when the cue card is located on the left side of the arena (west)
for the Standard condition (black line). When the cue is shifted 90º counterclockwise in
the Rotation condition the preferred direction also shifts approximately the same amount
with the new preferred direction of 236º (dotted line). The preferred direction returns to
136º when the cue card is returned to its original location for the Return condition
(dashed line).
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Deviationofof1010
Deviation
deg.
deg.
between
between
cue position
cue position
and preferred
and preferred
head direction.
head direction.
90 deg
Firing
Firing
Rate
Rate(Spikes/Sec)
(Spikes/Sec)
90 deg
180 deg
100
1
00
136
136 deg.
deg.
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180 deg
236 deg.
0 deg
270 deg
80
8
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270 deg
60
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80
240
24
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300
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00
360
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60
Head
HeadDirection
Direction (deg.)
(deg.)
Figure 4. Example tuning curves showing visual landmark control of the preferred
direction of a recorded HD cell. The preferred direction for the Standard recording
session is represented as the solid line at 136°. The preferred direction for the Rotation
session is indicated by the dotted line at 236°. The preferred direction returns to 136° for
the Return to Standard session as shown by the dashed line.
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Further studies of cue control have shown that visual cues which are most reliable
or familiar tend to exert greatest stimulus control over preferred direction (Goodridge et
al., 1998; Goodridge & Taube, 1995; Knierim et al., 1998; Taube & Burton, 1995; Taube,
Goodridge, Golob, Dudchenko, & Stackman, 1996; Taube et al., 1990b). For example,
cue control is strengthened when the animal is not disoriented before being placed in the
arena for all screening sessions (Knierim et al.). In this case it is thought that the
idiothetic cues that are maintained when the animal is placed in an unfamiliar
environment become associated with the new allocentric cues in the environment, thereby
allowing the allocentric cues to control the HD system in future exposures to the
environment.
While nearly all studies of landmark control of HD cells have utilized visual cues,
Goodridge et al. (1998) tested for auditory cue influences over HD cells. In this study,
they presented an auditory cue from one of four speakers mounted to the wall of the
recording cylinder. Similar to the procedures described above to illustrate the influence
of visual landmarks, the experimenters changed the position of the auditory cue by
activating different speakers positioned around the arena to determine if the preferred
direction of the recorded cell would likewise rotate. The room was darkened during
experimental sessions in order to eliminate visual cues while testing. In spite of rotating
the auditory cues by 90º, the HD cell preferred firing directions typically shifted a much
smaller amount (37.8 + 12.9º on average), suggesting only weak control by the auditory
cue compared to what is normally found with visual cue manipulations.
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Despite the apparent lack of control of an auditory cue of the HD network
described above, Rossier, Haeberli, and Schenk (2000) found that auditory cues were
able to exert some behavioral control during a place navigation task when presented
simultaneously with a visual cue. Specifically, rats were able to best discriminate the
location of a platform within a water maze when two auditory cues, which served as
beacons, and one visual cue were present. This was true when comparing sessions where
they either removed the visual cue (leaving only the auditory cues) or removed the
auditory cue (leaving only the visual cue). They concluded that perhaps rats will utilize
different sensory modalities depending on the need for spatial discrimination during a
particular navigational task.
It has been known for many decades that rodents can exhibit accurate localization
of sounds (Barber, 1915; Kelly & Masterton, 1977; Heffner & Heffner, 1985), suggesting
that sounds could in theory serve as directional cues within the appropriate environmental
set-up. There are at least two possible reasons for the lack of influence of auditory cues
over HD cells in the experiments of Goodridge et al. (1998). First, it may be that this
study did not eliminate all available visual cues during screening or testing sessions,
which allowed these visual cues to compete for control over the HD system (Goodridge
et al.; Mizumori & Williams, 1993). Second, the recording arena that was used may have
made sound discrimination difficult (Goodridge, et al.; Rossier, et al., 2000). In their
discussion, Goodridge et al. bring up this latter possibility that the geometric layout of the
circular walled arena that was utilized may not have been conducive for accurate sound
localization. Therefore, the one previous study examining the control of auditory cues on
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the HD system may have been conducted in environmental conditions in which the
auditory cues were perceived as an unreliable indication of directional heading.
Proximal and Distal Cues
Research using multiple visual cues has investigated the effects of landmarks that
are near to the animal (proximal landmarks) and cues that are distant from the animal
(distal landmarks). Proximal landmarks that have been used in such experiments have
been visual, 3-dimensional, and/or tactile objects placed inside the arena, serving as
intramaze allocentric cues, whereas distal landmarks have typically been visual cues that
lie outside the arena, serving as extramaze cues (Cressant, Muller, & Poucet, 1997;
Knierim, 2002; Renaudineau, Poucet, & Save, 2007; Save & Poucet, 2000;
Yoganarasimha, Yu, & Knierim, 2006; Zugaro, Berthoz, & Wiener, 2001). Support for
the role of the hippocampus in the utilization of both proximal and distal visual cues
during navigational tasks was shown in Save and Poucet’s study where rats with
hippocampal lesions were unable to properly utilize either type of cue.
Differences in the characteristics of place cells and HD cells have been discovered
with respect to proximal and distal cue control. When both proximal and distal visual
cues are present, place cells can behave differently either between sessions or between
animals. Unlike typical HD cell recordings, multiple place cells are often recorded
simultaneously bringing up the possibility that not all cells will respond the same to a
manipulation. When proximal and distal cues are rotated in opposite directions, causing
a cue conflict situation, the place fields from all recorded place cells in the same animal
may shift in concert with either the proximal or distal cues, or alternatively some place
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cells may follow the proximal cues while others follow the distal cues. In addition, either
all or some recorded place cells may cease firing, while others may establish entirely new
place fields (Knierim, 2002; Renaudineau et al., 2007; Yoganarasimha et al., 2006). To
summarize, control of place cells by proximal and distal visual cues seems to be
unpredictable from session to session and from animal to animal.
There have been a few studies which have examined proximal and distal visual
cue control over the activity of HD cells. When examined, HD cells behave in a much
different manner than place cells during many of the same environmental manipulations.
When a cue conflict is presented between a set of proximal and a set of distal visual cues,
almost all preferred directions will shift according to the distal cues (Yoganarasimha &
Knierim, 2005; Yoganarasimha et al., 2006; Zugaro et al., 2001). It is unknown, however,
if this same finding can be observed in the case of auditory cues.
As in the case of a distal visual cue, there is reason to believe that a distal auditory
cue may be more effective than a proximal cue at controlling the HD cell network. In
theory, the animal may perceive a proximal auditory cue as having varying intensity
levels depending on the position of the animal within the arena relative to the stable cue.
This may cause the animal to perceive the proximal auditory cue as originating from
slightly different locations as the animal locomotes within the arena. If the auditory cue
appears to resonate from different locations as the animal explores the arena the cue may
then be perceived as unstable and unreliable. In contrast, a distal auditory cue may be
heard at a relatively stable intensity regardless of the animal’s location within the arena.
If the sound is perceived as originating from the same location within the room regardless
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of the position of the animal the cue may be perceived as more stable and reliable.
Therefore, it may be more likely that when a conflict between proximal and distal
auditory cues is presented, the distal auditory cue will exert greater stimulus control over
the preferred direction of the recorded HD cell.
The purpose of the current study was twofold: (1) To determine whether a
complex auditory cue, consisting of simultaneously presented proximal and distal
auditory sounds, would exert control over HD cell behavior and (2) To decipher whether
a proximal or distal auditory cue would exert more salient control over the opposing
auditory cue when the animal is presented with a cue conflict situation.
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Chapter 2
METHOD
Experimental Subjects
Three female Long-Evans rats obtained from Simonsen Labs (Gilroy, CA) served
as subjects for this study. They weighed between 208 and 303 grams at the start of the
study. A food-restricted diet was used to establish the motivation for foraging within the
experimental arena. This entailed maintaining the rats at approximately 85% of free
feeding weight. Water was available ad libitum in the home cage. The rats were housed
individually in clear plastic cages containing wood chips in a room that was maintained
on a 16/8 hour light/dark cycle. The vivarium room temperature remained approximately
70º F. Individual cages were located next to each other in a row to maintain social
contact.
Materials
Electrode Assembly
Recording electrodes were constructed by the experimenter according to the
methods described by Kubie (1984). They consisted of a bundle of ten 25-μm in
diameter nichrome wires (California Fine Wire Co., Grover City, CA). The wires were
insulated except at the tip and were threaded through a stainless steel cannula that was
moveable in the dorsal/ventral direction after being fixed to the skull using dental acrylic.
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Electrophysiological Recordings
To detect cellular activity, electrical signals from the brain were recorded using a
field-effect transistor in a source-follower configuration. Extracellular activity was
amplified by a factor of 10,000-50,000 and bandpass filtered from 500 Hz to 30 KHz.
These single-unit signals were passed through a dual window discriminator (BAK
Electronics, Mount Airy, MD) and oscilloscope for electrical spike discrimination. Spike
information was stored on a desktop computer for offline processing using custom
analysis software (LabView; National Instruments, Austin, TX).
Apparatus
The same arena was used for all cell screening and experimental sessions. The
recording arena was uniquely constructed by the experimenter in order to eliminate all
obvious visual and tactile cues (see Figure 5). The geometric shape of a triangle was
chosen due to potential difficulties of localizing sound through the walls of the traditional
cylindrical arena (Goodridge et al., 1998). Neither a square nor rectangle was appropriate
as rats have not been reported to consistently discriminate between diagonally opposite
corners (Cheng, 1986; Golob, Stackman, Wong, & Taube, 2001; Margules & Gallistel,
1988). The floor of the triangular arena made a solid equilateral triangle with sides
measuring at 94 cm in length. A short wall, measuring at 7 cm in height, bordered this
platform. Both the floor and walls were sanded smooth so as to eliminate any obvious
tactile cues. Black screen was attached to a thin wooden frame over the platform creating
a pyramid-like enclosure in order to prevent the rat from trying to escape. This screen
met 51 cm above the platform at a circular opening (9 cm in diameter) at which the
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recording cable entered the arena. Velcro allowed the experimenter to fold down the top
half of each of the three screen walls of the arena to enable placement of the rat inside.
All wood was spray painted black in order to eliminate any obvious tactile (intramaze)
cues that could be created by paintbrush strokes. The black paint also eliminated any
obvious visual cues while the rat was inside the curtain area because this area was
darkened at all times.
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A
B
C
P
Arena
P
P
D
Curtain
Figure 5. Representations of the triangular recording arena. A, Photograph showing the
triangular recording arena, surrounded by three proximal speakers. It can be seen where
the top of the three screen walls were folded down for placing the rat inside the arena. B,
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Photograph showing the triangular recording arena from above. At the center is the
circular opening where the headstage wire and food pellets entered the arena. C,
Schematic showing the recording arena, surrounded by three proximal speakers (P), a
mobile distal speaker (D), and a black floor-to-ceiling curtain.
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The entire arena was elevated 61 cm above the room floor and rested on a wooden
base, where three removable proximal speakers were fastened to wooden speaker stands
using Velcro. The speakers were black and measured 16.5 cm tall and 9 cm wide. They
stood 9 cm away from the arena and the bases of the speakers were level with the top of
the triangular arena walls. Speakers were positioned facing inward at each point of the
triangular arena so that the sound was directed into the center of the triangular arena. A
fourth speaker (with identical physical measurements as the three proximal speakers)
served as the distal cue. The distal speaker was positioned 91 cm directly behind one
silent proximal speaker, adjacent to the proximal speaker that was active on a given trial
(see Figure 5).
A floor-to-ceiling black curtain surrounded the arena, eliminating all exterior
visual cues. This curtain hung approximately 15 cm outside of the proximal speakers,
with the distal speaker approximately 76 cm outside the curtain. The room that housed
the enclosure had a food pellet dispenser mounted to the ceiling directly above the arena.
The food dispenser released sugar pellets on a variable interval 30 seconds schedule in
order to evoke foraging behaviors. Pellets fell through the circular opening at the top of
the apparatus and bounced to random locations within the arena.
Auditory Cues
All speakers were elevated at equal heights and produced an auditory cue
generated by a computer located within the recording room. The auditory cue was
created by the experimenter using the LabView (National Instruments) computer program,
and consisted of a 2 kHz (Heffner & Heffner, 1985; Kelly & Masterton, 1977; Polley,
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Steinberg & Merzenich, 2006; Rossier et al., 2000) tone emitted from one consistent
proximal and one consistent distal speaker position in an alternating pattern. Specifically,
each tone sounded for one second and was followed by one and one half seconds between
each tone, so that a persistent and alternating beeping was heard throughout all screening
and testing sessions (see Figure 6).
Intensity (dB)
24
100
80
60
40
20
Distal
Proximal
1
2
3
Distal
4
5
Proximal
6
7
Time (Sec)
Figure 6. Pattern of alternating tones emitted by distal and proximal speakers
surrounding the triangular recording arena.
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9
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While the volume of the proximal and distal speakers was set to be equal at the
speakers, the greater physical distance of the distal speaker from the center of the arena
allowed for a difference of 10 decibels (dB) between the proximal and distal speakers
(approximately 66 ½ dB for the proximal auditory cue, with a range of 65-68 dB
throughout screenings, and approximately 56 dB for the distal auditory cue, with range of
54-58 dB throughout screenings), as detected by a sound level meter (Realistic, CAT. No.
33-2050) which was temporarily positioned in the center of the arena facing the sounding
speaker before the screening session began (Bushnell, 1995; Heffner & Heffner, 1985;
Kelly & Masterton, 1977; Polley et al., 2006). Sound level measurements were assessed
six times throughout the 47 week duration of the study.
The efforts made to enhance discriminability and salience of the auditory cues
included: 1) alternating beeps between the speakers (as discussed above) so that there
was a break between beeps (rather than one continuous sound emanating within the
room), 2) allowing for a 10 dB difference to be heard between the proximal and distal
auditory cues from the center of the arena, 3) searching for HD cells and conducting
experimental sessions in a darkened room so that the opportunity to utilize visual cues
was minimized, and 4) using a tone that has been previously found to be within the
hearing range of rats (Bushnell, 1995; Heffner & Heffner, 1985; Kelly & Masterton, 1977;
Polley et al., 2006; Rossier et al., 2000).
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Procedure
Surgical Procedure
Surgical procedures for implantation of the recording electrode were conducted
using an approved protocol from the CSUS Institutional Animal Care and Use
Committee. Rats were anesthetized with an intra-muscular injection of a drug cocktail
containing ketamine (30 mg/kg), xylazene (6 mg/kg), and acepromazine (1 mg/kg). The
head was shaved and disinfected with betadine. The animal was then placed in a
stereotaxic device for support of the head during electrode implantation. The top of the
skull was exposed by a single incision. Six holes were drilled around the edges of the
incision and stainless steel screws were placed in these holes to serve as anchors for the
electrode assembly.
Another hole was drilled in the right parietal plate based on the stereotaxic
coordinates of 1.5 mm posterior to bregma and 1.3 mm lateral to bregma, provided by
Paxinos and Watson (1998), to allow for the electrode to be lowered into the brain tissue.
The electrode was implanted slightly above the ADN of the right hemisphere 3.7 mm
below the dura mater. The electrode was secured to the screws using orthopedic cement.
Once the electrode was secured, a topical antibiotic (Neosporin) was packed around the
incision site and the incision was sutured closed. Analgesics were provided for 1-2 days
following the surgery as needed. Animals were given seven days to recover from the
surgery before the next phase of the experiment.
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Screening Sessions for HD Cells
When searching for HD cells (screening) the activated proximal speaker was
positioned at 120º clockwise (CW) of the activated distal speaker (see Figure 7). The
three identical proximal speakers were positioned around the arena in order to maintain
symmetry and eliminate any potentially obvious visual cues. At the start of a session, the
auditory cues were activated and the rat was brought into the experimental room in its
home cage. The black curtain enclosed the arena so as to hide it from the rat’s view
while the animal was plugged in to the recording equipment. The external area outside of
the curtain was temporarily lighted, whereas the area within the curtain area remained
dark. The experimenter randomly predetermined the rat’s side of entry into the arena by
the roll of a die. After the rat was connected to the recording cable it was then brought
through the curtain and placed into the darkened arena facing the center of the triangle
through one of the three sides of the triangular enclosure. The experimenter secured the
screen wall and exited the curtain from the location of entrance. The exterior lights were
turned off and the door was closed quietly. The screening session began when the food
pellet dispenser was activated from the adjoining room. It is notable that the animal was
not given the disorientation treatment prior to screening sessions that was utilized for
recording sessions (see below). This difference is based on evidence by Knierim et al.,
(1995) that when an animal is consistently disoriented prior to placement into an
environment the HD network fails to be controlled by the relevant cues in the
environment.
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Figure 7. Representation of the triangular recording arena during screening sessions. All
screening sessions were conducted with the proximal speaker at 120º CW, or to the right,
of the distal speaker.
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Each rat underwent screening sessions once per day on an average of three days
per week. Screening for HD cells involved examining the electrical activity on each of
the ten wires in the electrode as the rat foraged in the arena. This process lasted
anywhere from 10-30 minutes per rat. Video tracking hardware (Ebtronics Corp.;
Elmont, NY) monitored the behavior, head position, and directional orientation of the
rat’s head. This tracking system detected the x and y coordinates (256 x 256) of red and
green light emitting diodes (LEDs) secured to the recording headstage six cm apart,
above the head and back of the animal, respectively. Tracking software used the
coordinates of these LEDs to register both head position and head direction.
The presence of directionally specific activity on an electrode wire is an
indication of an isolated HD cell. If an HD cell was not identified during a given
screening session, the electrode was advanced approximately 100 m and the animal was
returned to the vivarium in its home cage. If an HD cell was identified, an experimental
session was conducted either that same day or the following day.
Experimental Sessions for HD Cells
There were four experimental conditions for each HD cell tested. Figure 8
presents the procedure for the study. During testing, the experimenter turned off all
exterior room lights and wore a headlamp when necessary to maneuver within the
darkened room. A session began by placing the rat in a small cardboard box with a
removable lid. There was an opening in the lid that allowed a small space for the
recording cable to remain attached to the rat. The auditory cues were turned off and the
experimenter cleaned the arena floor with household cleaning wipes in order to eliminate
30
any obvious olfactory cues. The speakers were positioned accordingly. The rat was
given disorientation treatment by the experimenter slowly turning the box in full circles
in one direction for approximately six seconds and then in the opposite direction for
about the same duration. This was done in order to disrupt path integration processes and
strengthen the rat’s dependence on experimenter-controlled cues (Biegler and Morris,
1996). After disorientation treatment the auditory cues were turned on. The rat was
taken out of the box and placed into the arena. The experimenter’s headlamp was turned
off, the experimenter left the curtain from a pseudorandom location, and the pellet
dispenser was activated. Cellular activity was recorded for ten minutes while the animal
foraged for food pellets. At the conclusion of each session the rat was removed from the
arena, placed back in the box and the previously mentioned procedures were repeated for
the next session.
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Figure 8. Representations of the triangular recording arena during experimental sessions.
The four conditions were: Standard, Rotate Both, Return to Standard, and Flip, with the
position of the auditory cues manipulated between conditions. For the Standard
condition, the proximal auditory cue was set at 120º CW to the distal auditory cue. For
the Rotate Both condition, both auditory cues were rotated by 120º CW relative to the
standard positions. For the Return to Standard condition, both auditory cues were
returned to the same position as in the Standard condition with a CCW rotation of 120º.
32
For the Flip condition, the proximal auditory cue was rotated CCW by 120º and the distal
auditory cue was rotated CW by 120º, so that they ended up flipping their relative
positions.
33
Within the Standard condition the proximal and distal auditory cues maintained a
similar relationship as within the screening sessions in that if the rat were to face the two
sounds (hence, the side of the triangle between the two speakers) the distal sound could
be heard on the left while the proximal sound could be heard on the right. The Rotate
Both condition maintained this same sound relation while rotating both cues 120º in the
CW direction. Therefore, if the rat was using the auditory cues together to establish head
direction, the preferred head direction would be expected to rotate roughly CW 120º
along with the auditory cues. In the Return to Standard condition, both sound cues were
rotated CCW 120º, returning the cues to the same positions as in the Standard condition.
Here again, if the rat was using the two auditory cues as a single landmark, the preferred
head direction of the cell would be expected to rotate roughly CCW 120º relative to the
previous session. The purpose of the Flip condition was to set up a cue conflict between
the proximal and distal cue to determine if one cue exerts more control over the HD
system than the other. This was accomplished by “flipping” the position of the proximal
and distal cues, so that the proximal auditory cue was now located to the left of the distal
auditory cue, when facing both cues from the center of the arena. If one auditory cue
exerts stronger control than the other then it would be expected that the preferred
direction would shift with the cue that exerts stronger control (Yoganarasimha &
Knierim, 2005; Yoganarasimha et al., 2006).
Data Analysis
Circular statistics (Batschelet, 1981) were used to determine the stability of the
directional signal for all HD cells. The preferred direction shifts between consecutive
34
sessions (i.e., the Standard and Rotate Both conditions, Rotate Both and Return to
Standard conditions, and Return to Standard and Flip conditions) were examined to
determine whether the recorded cells changed their directional specificity with the
position of the auditory cues. These comparisons involved determining directional
deviation scores across the compared sessions using a cross-correlation method (Taube &
Burton, 1995). This analysis involves shifting the firing rate/HD function of the first
condition in 6° increments while correlating this shifted function with the unshifted
function from the comparison condition. The amount of shift required to produce the
maximal Pearson r correlation between the two tuning curves is defined as the directional
deviation score between the conditions.
The directional deviation scores from the entire sample of cells were then
subjected to Rayleigh tests (Batschelet, 1981) to determine if the scores were clustered
randomly, as would expected if the preferred directions were not stable between the
sessions. The critical statistic of the Rayleigh test is the mean vector length, r, which
varies between 0 and 1, with higher values indicating that the distribution of directions is
clustered nonrandomly. The directional deviation scores were also used to calculate the
mean vector angle, m, which estimates the mean angle of the sample. In the case where
the HD cells are controlled by the position of one or both auditory cues, it would be
expected that the mean vector angle would shift 120°along with the position of the
controlling cue(s).
35
Histological Verification of Recording Sites
At the conclusion of recordings, the brains were processed for verification of
recording locations. This involved deeply anesthetizing the animals and a small anodal
current (20 μA, 10 seconds) was passed through the electrode wire(s), in order to conduct
a Prussian blue reaction. The animals were then perfused transcardially with saline,
followed by 10% formalin in saline. The brains were removed and placed in 10%
formalin for at least 48 hours. The brains were then placed in a 10% formalin solution
containing 2% potassium ferrocyanide for 24 hours and then rinsed in 10% formalin for
several days. They were sectioned at 80 to 100 μm in the coronal plane, stained with
cresyl violet, and examined microscopically for localization of the recording sites.
36
Chapter 3
RESULTS
A total of eleven cells were recorded in three animals (range = 1- 6 cells per
animal). Rats JD6 and JD7 had been exposed to the arena and auditory cues for seven
screening sessions before conducting the first cue rotation session. Rat JD5 was exposed
to the arena for 18 screening sessions before this testing occurred. With a typical
screening session lasting ~ 20 minutes, all animals had been exposed to the auditory cues
for at least 140 minutes prior to testing.
Qualitative Analysis of Cellular Activity
Cellular activity was examined subjectively throughout testing conditions to
determine how shifting the position of the auditory cues influenced the preferred
directions of the recorded cells. Representative tuning curves were produced by
averaging firing rate over head directions for each condition. Resulting tuning curves
were subjectively examined to determine directionality and preferred HD cell behavior.
It was observed that all of the cells (11 of 11) exhibited directional tuning, with variations
in strength and isolation for each cell and for each condition. Figure 9 presents example
firing rate versus HD tuning curves for one representative HD cell from rat JD6 during
experimental conditions. For this rat, the preferred direction of approx. 190º remained
consistent throughout all four conditions. Thus, no shift of preferred direction was
observed for this cell across the different conditions despite rotations of the auditory cues.
Other cells showed a similar insensitivity to the position of the auditory cues, some
37
showing preferred directions remaining stationary relative to the cues, while other cells
showed preferred directions that shifted randomly relative to the cues. Figure 10 shows
tuning curves from one representative cell from JD5 where its preferred direction
appeared to shift with the auditory cues during the Rotate Both session, but then the
preferred direction remained the same for the Return to Standard and Flip sessions
despite the moving auditory cues. In another cell (cell JD7-3), the preferred direction
shifted in the opposite direction of the auditory cues during both the Standard to Rotate
Both and Rotate Both to Return to Standard transitions (where the preferred direction
returned back to the original preferred direction for the Return to Standard session), and
appeared to move with the distal cue for the Flip session (Figure 11). Clearly there did
not seem to be consistent findings of auditory cue control from one cell to another across
the sample, or even from one session to another for individual HD cells.
38
Firing Rate (Spikes/Sec) .
cell JD6-1
50
Standard
40
Rotate Both
30
Return to
Standard
Flip
20
10
0
0
60
120
180
240
300
360
Head Direction (Degrees)
Figure 9. Representative firing rate/HD tuning curves for four conditions for cell JD6-1.
The dashed line indicates the tuning curve during the Standard condition. The solid and
bolded line indicates the tuning curve during the Rotate Both condition. The dotted line
indicates the tuning curve during the Return to Standard condition. The shaded line
indicates the tuning curve during the Flip condition.
39
Firing Rate (Spikes/Sec) .
cell JD5-1
60
Standard
40
Rotate Both
Return to Standard
Flip
20
0
0
60
120
180
240
300
360
Head Direction (Degrees)
Figure 10. Representative firing rate/HD tuning curves for four conditions for cell JD51. The dashed line indicates the tuning curve during the Standard condition. The solid
and bolded line indicates the tuning curve during the Rotate Both condition. The dotted
line indicates the tuning curve during the Return to Standard condition. The shaded line
indicates the tuning curve during the Flip condition.
40
Firing Rate (Spikes/Sec) .
cell JD7-3
30
25
20
15
10
5
0
Standard
Rotate Both
Return to Standard
Flip
0
60
120
180
240
300
360
Head Direction (Degrees)
Figure 11. Representative firing rate/HD tuning curves for four conditions for cell JD73. The bold and solid line indicates the tuning curve during the Standard condition. The
dashed line indicates the tuning curve during the Rotate Both condition, representing a
shift ~ 120º in the opposite direction from the auditory cue. The dotted line indicates the
tuning curve during the Return to Standard condition, also showing an opposite of cue
shift. The shaded line indicates the tuning curve during the Flip condition, representing a
shift in the same direction as the distal auditory cue.
41
Quantitative Analysis of Cellular Data
Figure 12 presents the shifts in preferred directions of all recorded HD cells for
the three transitions between conditions that were examined. Rayleigh tests of these
preferred direction shifts between conditions indicated that the shifts were clustered (i.e.,
distributed nonrandomly) for all three transitions [mean vector lengths (r) = 0.500, 0.889,
and 0.840; mean vectors (m) = - 19.0º, - 6.6º, and 1.6º for Standard Vs Rotate Both,
Rotate Both Vs Return to Standard, and Return to Standard Vs Flip conditions,
respectively; ps < 0.05]. Generally a clustering of preferred directions after a landmark
shift could indicate one of two results: either the preferred directions of the population
tended to shift predictably with the shifting landmark (e.g., the shifting auditory cues), or
the preferred directions of the population tended to remain the same as in the previous
session despite the shift of the landmark. An examination of the observed preferred
direction shifts provides the strongest support for the latter outcome. Specifically, for the
Standard to Rotate Both transitions only three cells (27.3%) rotated in the same direction
as the auditory cue (approximately 120º) and one cell rotated approximately 120º in the
opposite direction from the cue. The remaining seven cells (63.6%) rotated
approximately 0º (i.e., showed no change in preferred direction) for this transition. In the
Rotate Both to Return to Standard transitions, one cell rotated approximately 120º in the
opposite direction from the cue while the remaining cells failed to rotate at all. Finally, in
the Return to Standard to Flip transition, once again the majority of the cells failed to
rotate at all, while one cell rotated with the proximal auditory cue and another cell
appeared to rotate with the distal auditory cue.
42
If P
im
rox
If D
ist
al
al
Figure 12. Scatter diagrams illustrating the amount of angular shift between
experimental conditions. The left column shows the amount of preferred direct angular
shift in degrees observed between the Standard and Rotate Both conditions. The middle
column shows the amount of shift observed between the Rotate Both and Return to
Standard conditions. The right column shows the amount of shift observed between the
Return Standard and Flip conditions. The position of each filled circle represents the
amount of angular shift of the preferred direction of a single cell between the first and
second conditions. The curved arrows in each panel indicate the amount and direction of
shift of the auditory cues between the two sessions, with the distal cues shifting
clockwise and the proximal cues shifting counterclockwise in the third transition. The
solid arrow denotes the expected mean vector angle if the angular shift is perfectly
predicted by the auditory cues and the dotted arrow denotes the observed mean vector
angle. The length of the dotted arrow denotes the mean vector length, with a length of
1.0 (no variability in shift scores) represented by a vector spanning the radius of the
circle. Each plot uses Cartesian coordinates with 0º at the 3 o’clock position and
increasing degree values proceeding in a CCW direction.
43
Taken together, poor auditory cue control was shown over the recorded HD cells,
and this is especially apparent when the results of each transition are considered across
individual cells and individual animals. Table 1 shows the results of each condition
transition for each cell from each of the three animals (JD5, JD6, and JD7). For the
purposes of this table, the outcome of each transition was coded as: 1) “Cued Shift” if the
preferred direction shifted within 24º of the new position of the auditory cue(s), 2)
“Opposite Shift” if the preferred direction was within 24º of where the auditory cue(s)
would have been if they had been shifted in the opposite direction, 3) “No Shift” if the
preferred direction shift remained within 18° of its preferred direction for the previous
session, and 4) “Ambiguous Shift” if the preferred direction shift did not meet any of the
above criteria. As described above, the most common outcome of a transition was a
preferred direction that was maintained despite shifting auditory cues (i.e., the “No Shift”
outcome). It is notable that while the preferred direction shifted in the same direction as
the auditory cues in at least five transitions (see Table 1), in each of these instances this
apparent cue control occurred for only one of the three transitions for that particular cell.
Hence, cue control did not seem to be cell specific; rather it seemed to occur randomly on
some sessions but not others, even within the same cell. These results support the
conclusion that anterior thalamic HD cells do not respond to auditory cues, regardless of
whether the cue is located proximal or distal to the rat.
44
Table 1
Shift Outcomes for Session Transitions
Cell
Standard Vs.
Rotate Both Vs. Return
Return Both
Rotate Both
Standard Transition
Vs. Flip
Transition
Transition
JD5-1
Cued Shift
No Shift
No Shift
JD5-2
Cued Shift
No Shift
No Shift
JD6-1
No Shift
No Shift
No Shift
JD6-2
No Shift
No Shift
No Shift
JD6-3
No Shift
No Shift
No Shift
JD6-4
No Shift
Ambiguous Shift
No Shift
JD6-5
No Shift
No Shift
No Shift
JD6-6
No Shift
No Shift
No Shift
JD7-1
Cued Shift
No Shift
No Shift
JD7-2
No Shift
No Shift
Cued Shift
JD7-3
Opposite Shift
Opposite Shift
Cued Shift
Note: See text for definitions of transition outcomes.
45
Point of Entry Analysis
Due to the fact that some cells showed occasional shifts of approximately 120º,
but these shifts appeared largely unrelated to the position of the auditory cues, the
experimenter examined the possibility that the rat’s point of entry (POE) into the arena
may have been controlling the preferred directions of recorded cells. This possibility is
supported by a place cell study that found that the addition of a second visual cue leading
to a visually symmetrical environment (i.e., a cylindrical shaped environment with two
white cards located 180º from each other) resulted in place field locations controlled by
the direction of entry into the environment (Sharp, Kubie, & Muller, 1990). The fact that
POE was randomly altered in 120º increments in this study, and that the cells
occasionally seemed to shift in approximately the same amounts made it important to test
the possibility that POE exerted control over the preferred directions of recorded cells.
This analysis involved determining what the predicted preferred direction shifts between
two consecutive conditions would have been if the POE was the controlling variable.
Based on the geometry of the apparatus, at the beginning of each condition
animals entered the apparatus facing one of three randomly determined directions: 30º,
150º, and 270º. If the POE was truly the variable that determined the preferred direction
of a given condition, then the preferred direction shift between conditions should equal
the POE shift between those conditions. As an example, if an animal entered the
Standard condition at a POE of 30º and entered the Rotate Both condition with the same
POE of 30º, and POE was truly the variable controlling preferred direction, then the
predicted preferred direction shift during the transition between those conditions would
46
be 0º. In contrast, if the POE for the Standard condition was 30º and the Rotate Both
condition POE was 150º, then the shift in preferred direction between the conditions
(again assuming POE is the controlling factor) would be predicted to be +120º.
Figure 13 presents these shifts in preferred directions of HD cells for the three
transitions relative to the POE between the sessions. Rayleigh tests of these preferred
direction shifts between conditions indicated that the shifts were not clustered (i.e., they
were distributed randomly) for all three transitions [mean vector lengths (r) = 0.174,
0.152, and 0.170; mean vectors (m) = - 29.5º, 29.5º, and 129.8º for Standard Vs Rotate
Both, Rotate Both Vs Return to Standard, and Return to Standard Vs Flip conditions,
respectively; ps > 0.05]. Because shifts were distributed randomly relative to the POE
shift between sessions, this is strong evidence that the POE did not appear to have control
over HD cell behavior. In other words, if the rat had used the POE into the arena as the
salient cue, then the shift in preferred directions relative to POE shifts would have been
clustered around zero degrees. This was clearly not the case. At first glance, it may seem
odd that the preferred direction shifts relative to the POE as shown in the figure usually
varied in increments of 120º, however that is explained by the fact that most cells did not
shift between conditions (see Figure 12), while POE did shift randomly between
conditions in multiples of 120º.
47
Figure 13. Scatter diagrams illustrating the amount of angular shift between the observed
preferred directions of recorded cells and the predicted shift of preferred direction if the
cells were using the point of entry (POE) into the recording enclosure. In each transition,
the expected shift would be zero degrees (solid arrow) if the cells were using POE to
maintain a consistent preferred direction. The solid arrow denotes the expected mean
vector angle if the angular shift is perfectly predicted by the POE and the dotted arrow
denotes the observed mean vector angle. The length of the dotted arrow denotes the
mean vector length, with a length of 1.0 (no variability in shift scores) represented by a
vector spanning the radius of the circle. Each plot uses Cartesian coordinates with 0º at
the 3 o’clock position and increasing values proceeding in a CCW direction.
48
Histology
At the completion of the study, histological processing was performed on two of
the three animals (JD6 and JD7). This analysis could not be performed on the third
animal (JD5) due to premature electrode failure. Figure 14 presents a photomicrograph
from the rat in which the majority of cells were recorded (n = 6). For this rat, the
Prussian Blue reaction was performed on the two wires on which the HD cells were
recorded. The dark spots within the ADN and anterior ventral nucleus (AVN) represent
the position of the wires at the time of animal sacrifice. While it is unclear as to the
precise thalamic nucleus containing each recorded HD cell in this animal (ADN or
AVN), it is apparent that the cells were localized in one of those two nuclei of the
anterior thalamus. The second animal showed an electrode localized to the ADN.
49
M2
RSA
M1
S1HL
S1FL
S1DZ
RSGb
cg
S1BF
IG
cc
df
DHC
dhc
LV
fi
sm
st
CA3
DG
D3V
S2
PC
PT
iml
CM
IAM
mt
ic
ns
mfb
VA
Rt
AMV
DI
Re
VRe
SPa
PaV
cst
LGP
VM
IPAC LaDL
PaDC
PaLM
SI
LH
VEn
MeAD
Pir
BMA
Pe
RCh
BLA
IM
SO
sox
AIP
DEn
CeM CeL CeC
AHP
AHC
3V
Cl
ZI
PaMP
opt
GI
CPu
IAD
AM
Sub
Xi
f
AVVL
AVDM
Rh
rf
al
LDVL
MD
PVA
ec
AD
MHb
S1
BAOT
ACo
CxA
LDN
AVN
ADN
Figure 14. Illustration of histology for rat JD6. A diagrammatic representation of a
coronal section of the rat brain at the level of the electrode placement (reprinted from
50
Paxinos and Watson, 1998) is shown at the top of the figure. The bottom of the figure
shows an example brain slice from rat JD6. Two of the ten wires were isolated within the
anterior thalamus as can be seen from the darkened stains from the Prussian blue.
51
Chapter 4
DISCUSSION
The purpose of the current study was twofold: (1) To determine whether a
complex auditory cue, consisting of proximal and distal auditory sounds presented in
synchrony, would exert control over HD cell behavior when significant steps were taken
to minimize the saliency of other landmarks in the environment and (2) To determine
whether either a proximal or distal auditory cue would exert more salient control over the
HD network than the opposing auditory cue when the animal is presented with a cue
conflict situation. The results of the current study showed that under these experimental
conditions, auditory cues do not exert control over anterior thalamic HD cells.
It is not clear why inconsistent patterns for each rat and each cell were found.
The mixed patterns seem to be somewhat animal specific in that none of the HD cells
recorded from JD6 showed a cued shift in their preferred directions for any of the three
rotation transitions but all cells from each of the other two animals showed one (and only
one) transition that was accurately predicted from the cue (see Table 1). It is unusual for
HD cells recorded from the ADN to respond so inconsistently, as they characteristically
tend to either follow landmarks or not, rather than exhibiting landmark control on some
sessions but not others within a single experiment (e.g., Taube, 1995). The least
predictable outcome was seen in cell JD7-3, where the preferred direction shifted for all
transitions, but only one of the shifts was in the same direction as the auditory cue (see
Figure 11 and Table 1).
52
The fact that the HD cells were able to establish a stable preferred direction that usually
did not shift between sessions shows that there was a cue controlling HD cell directional
specificity. However, it is unlikely that this stable salient cue was auditory, visual, or
olfactory because specific steps were taken to eliminate the influence of these extraneous
landmarks. In addition, the observation that preferred directions seemed to shift
randomly in some sessions points to the possibility that if a landmark was being used, it
was not always stable between sessions.
There are various possible explanations for why the current results were obtained.
First, it is possible that even though the rats could hear the proximal and distal sounds,
they could not discriminate between them. The lack of direct evidence that the sounds
used were discriminable is the largest limitation of the current study. The sounds used
were set at a frequency (2 kHz) and intensities (~ 66.5 dB for the proximal auditory cue
and ~ 56 dB for the distal auditory cue) that have been shown to be within rodents’
hearing range: 1-Hz - 80 kHz at 35 - 70 db sound pressure level (SPL) (Bushnell, 1995;
Goodridge et al., 1998; Heffner & Heffner, 1985; Kelly & Masterton, 1977; Polley et al.,
2006; Rossier et al., 2000). Also, rats have exhibited the ability to localize sounds
(Barber, 1915; Heffner & Heffner; Kelly & Masterton) however; perhaps the rats in the
current study were not able to determine the location of the two sounds given the specific
recording environment utilized. This may be true despite efforts to enhance
discriminability, or maximize the likelihood that the HD cells would utilize the
information from the auditory cues, such as alternating the beeps between the speakers so
53
that there was a break in between beeps (rather than one continuous sound emanating
within the room).
Second, even if the locations of the auditory cues were discriminable, it is
possible that the animal (or HD network) failed to properly “attend” to the sounds
consistently. Additional research might examine these two issues by utilizing behavioral
measures to ensure that the animals can discriminate between the positions of the
auditory cues used in the current study. For example, an experiment might attempt to
train an unoperated rat to perform a particular behavior, such as pressing a lever in the
presence of one sound, but not the other. Once it has been determined that the animal can
discriminate the location of a simple sound cue, then further experiments may be
performed to verify the ability to make the more complicated discriminations between
proximal and distal auditory cues.
Another approach would be to examine whether behavioral training to
discriminate sound location leads to HD cells that respond to auditory cues. This would
further address the issue of whether the influence on the HD network by an auditory cue
requires the animal to attend to the cue. In other words, will a rat that has undergone
behavioral training to discriminate the location of an auditory cue possess HD cells that
are more controlled by the auditory cue than an animal that has not had the same
behavioral training? Lastly, an attempt may be made to not only train animals to
discriminate the location of an auditory cue, but to use this cue for directional orientation
in a navigation task.
54
Studies have shown that behavior and head direction can be controlled either by
independent cues or by the same cue depending on the type of task and procedures used
(Dudchenko & Taube, 1997; Golob et al., 2001; Martin, Harley, Smith, Hoyles, & Hynes,
1997; Muir & Taube, 2002; Muir & Taube, 2004). These studies have shown evidence
leading to the possible conclusion that the correlation between HD cell behavior and the
animal’s overt behavior will vary under different settings and for HD cells located in
different brain regions. Dudchenko and Taube (1997) used an eight-arm radial maze to
examine the correlation between spatial behavior and a visual cue for HD cells located
within PoS and ADN. They found that HD cell behavior was not directly related to the
location of a reward for a reference memory task. Therefore, the HD system was shown
to be independent of behavior under those conditions because the directional signal
carried by the HD network was not directly influenced by the reinforcement
contingencies.
Muir and Taube (2002) reviewed all studies which had examined HD cell
behavior during various behavioral tasks utilizing reward contingencies and they
concluded that rats may not consistently rely on their sense of direction to guide behavior
on all spatial tasks. However, the HD network activity may correspond with behavior if
the location of the reward and cues remain consistent over repeated trials (or the animal
learns over the course of training) and if the rat’s point of entry to the environment is
consistent with these cues. In other words, there must be consistency with both the
allocentric cues and the rat’s point of entry into the arena. A future study may mimic the
present experiment by adding a similar behavioral component where a rat performs a
55
reference memory task in which the proximal and distal auditory cues remain in the same
relation to each other and they consistently predict a reward location. In addition, the rat
would not have random point of entries into the environment during screening sessions,
as in the current study, but would instead have the same entrance location into the arena.
The current study varied the point of entry for all testing sessions in order to lessen the
possibility that the HD cell would use the consistent point of entry as the salient cue.
Conversely, given the complex relationship between the HD network and the animal’s
behavior, varying the point of entry during testing may have lessened the HD cell’s
dependency on the auditory cues within the current study.
A third possible explanation for the present findings may be that HD cells, similar
to place cells, may behave differently either between sessions, animals, or brain regions
with respect to proximal and distal auditory cue rotations. Future investigations might
record more than one HD cell within the same brain region during the same session in
order to see if they respond similarly after auditory cue rotations. Although this
phenomenon has not been observed for HD cells, perhaps one HD cell located within the
ADN would respond differently to auditory cue rotations than would a different HD cell
also within the ADN. Likewise, it is possible that HD cells in other brain regions (i.e.,
LDN) may respond differently from HD cells located in the ADN. Goodridge, et al.
(1998) examined auditory landmark control for only one PoS HD cell. This HD cell was
said to have responded similarly to the ADN cells, but perhaps a larger sample size would
have shown different results.
56
A fourth explanation for the current findings is that the HD cells were utilizing the
geometric shape of the environment in order to establish a preferred direction, as was
observed in the study conducted by Golob et al. (2001) where the HD cells could not
distinguish diagonally opposite corners in a square or rectangular arena. This might
explain why shifts of ~ 120º occurred in the wrong direction and for only some
transitions for rats JD5 and JD7. If these HD cells were using a corner as their cue, it
would be reasonable for them to utilize different corners throughout testing.
One notable interpretation of the current findings of poor auditory cue control
over HD cell behavior is that the typical procedure for turning on white noise to mask
auditory cues during experimental sessions is apparently unnecessary. This has been
standard procedure for decades as it was assumed that HD cell behavior might be
controlled by an auditory cue perceptible from outside the recording arena. Eliminating
this aspect to the typical procedures may be acceptable since the present findings suggest
that it is unlikely that latent/ambient auditory cues, such as the sound of the door closing
after the experimenter, would exert HD cell control. If a latent auditory cue were strong
enough to establish cue control it would be expected that the prominent and consistent
beeping in the current experiment would have exerted cue control for all transitions for at
least one HD cell.
In summary, the current study has shown that unlike salient visual cues, salient
auditory cues do not exert stimulus control over HD cells located within the ADN when
rats are given a complex cue consisting of both proximal and distal sounds. Despite
many efforts made throughout experimentation to enhance both the saliency and
57
discriminability of the auditory cue(s), the persistent lack of auditory cue control might
suggest that the brain circuitry directly connected to the ADN is not tight coupled with
auditory centers. Perhaps these HD cells are more strongly linked to the neural pathways
that process visual, tactile, olfactory, and vestibular electrical signals. The results from
this experiment may aide future researchers in determining the neighboring neural
pathways in order to better understand the behavioral characteristics and purpose of HD
cells located within the anterior thalamus. Uncovering these additional fragments will
contribute to the body of knowledge that seeks to understand how HD cells aid an animal
during navigation.
58
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