Stackman, Robert W.

Considerable research has been carried out to establish a rodent model for the
study of human memory, yet functional similarities between the species remain up for
debate. The hippocampus, a region deep within the medial temporal lobe of the
mammalian CNS, is critical for long-term episodic memory. Projections from the medial
entorhinal cortex convey spatial/contextual information, while projections from the
lateral entorhinal cortex convey item/object information to the hippocampus. The
functional significance of these parallel projections to the rodent hippocampus has been
suggested to support spatial processing, while the same projections to the human
hippocampus support spatial and non-spatial memory. Discharging in a location-specific
manner, hippocampal place cells contribute to spatial memory; however, evidence for
neuronal correlates of non-spatial object memory has not been fully defined. The current
experiments were designed to address the following questions, while utilizing
electrophysiology, functional inactivation during a novel behavioral task, and immunohistochemistry. Is the memory for objects hippocampal-dependent, solely due to
the location of the object, or are objects represented within hippocampal activity
independent of location? To tease apart spatial and non-spatial processing by the
hippocampus, the spatial aspects of 3D objects were enhanced by utilizing movement. A
novel discriminatory avoidance task, Knowing Your Enemy, was adapted from an Enemy
Avoidance task to test true object memory in mice. Current findings support the notion
that object-associations acquisition depends upon a specific context. Retrieval of such
object-associations is not context-dependent, yet remains sensitive to temporary
inactivation of the CA1 region of the dorsal hippocampus. The avoidance impairments
observed following hippocampal inactivation were shown to not be a result of reduced
anxiety. Immunohistochemical marker expression suggests that the CA1 region was
highly active during object exposures, yet the hippocampal system responded
differentially to moving and to stationary objects. Recordings of CA1 neurons yielded
non-bursting object-related activity during object exploration, and place cell activity
remained unaffected in the presence of moving objects; supporting independent, yet
simultaneous processing of spatial and non-spatial information within the hippocampus.
Together, the current findings support the notion that the CA1 region of the rodent
hippocampus processes object-related information, independent of spatial information.

The rodent hippocampus is an essential neural substrate for spatial memory. This
functional capacity is considered to rely upon a cognitive map that represents the location where
relevant non-spatial items or objects are encountered and where specific events occur within a
contextual or spatial reference frame. Place cell activity recorded from CA1 pyramidal neurons
of the dorsal hippocampus of freely moving rodents is influenced by distal and proximal cues or
items within an environment, and increases when objects are placed into a familiar arena.
Recently, the CA1 region of the rodent dorsal hippocampus was shown to play a vital role in
object-in-context memory, and object memory independent of context; findings consistent with
the cognitive map view. Here, we tested the influence of 3D objects on the spatial firing
properties of CA1 neurons, since object-specific neuronal activity has not yet been fully
established in mouse hippocampus. In vivo extracellular recordings from intermediate dorsal
CA1 yielded simultaneous recordings of place cells and a pyramidal neuron demonstrating
object-specific activity over two consecutive sessions with objects present. Higher frequency
object-specific activity was recorded from the same mouse again 3 weeks later during a
comparable task. Object-specific activity was observed only when the mouse explored objects in
the arena, and was independent of spatial location or object identity. Recordings from more distal
region of dorsal CA1, which receives input from proximal CA3, yielded two additional neurons
that demonstrated comparable object-related activity. These results further support the
involvement of the rodent hippocampus in non-spatial object memory.

Small conductance Ca2+ -activated K+ (SK) channels have physiological roles in
learning and memory, intrinsic excitability, synaptic transmission and plasticity, and addiction.
While SK2 and SK3 channels have been studied, the role of SK1 has not yet been determined
due to the prior lack of gene-specific antibodies and agonists. SK1 are robustly expressed in the
CA1 pyramidal neurons of the hippocampus and modulate their excitability by affecting
afterhyperpolarization. SK1 subunits are only sited in the plasma membrane when co-expressed
with SK2 or SK3. Co-expressed and co-assembled SK1, SK2, and SK3 subunits form functional
apamin-sensitive channels. SK1 are not apamin selective, suggesting the overriding hypothesis
that SK1 is a subunit of heteromeric SK channels that bind specific interacting proteins. We
examined the effect of a new SK1 selective activator, GW542573X, on hippocampal dependent
object memory in male C57BL/6J mice. The results showed that activating SK1 channels by
systemic injection of the SK1 agonist GW542573X before the sample session, led to impaired
object memory in mice 24 h later. Mice treated with GW542573X acquired the sample object
exploration criterion in a similar latency as the vehicle-treated mice.GW542573X treated mice
exhibited significantly less preference for exploring the novel object during the test session
compared to the vehicle-treated mice. These results suggest that the SK1 activator disrupted the
encoding of object memory without affecting the motivation to explore objects. This supports a
role for SK1 in the modulation of hippocampal synaptic plasticity and hippocampal-dependent
memory.

We recently reported that male C57BL/6J mice navigate in spatial tasks, such as the
Morris water maze MWM, by swimming in a particular direction to a location relative to poolbased
cues, rather than to an absolute location defined by room-based cues. Neural mechanisms
supporting this bias in rodents for relative responding rather than absolute responding in spatial
tasks are not yet understood. Anterior thalamic neurons discharge according to the current
directional heading of the animal. The contribution of head direction HD cell activity to
navigation has been difficult to elucidate. Selective inactivation of anterior thalamic nuclei ATN
by microinfusion of muscimol or fluorophore-conjugated muscimol caused a near complete shift
in preference from relative to absolute responding. Interestingly, inactivation of the dorsal CA1
region of the hippocampus did not affect relative responding. A land based version of the MWM
has been developed to permit the recording of anterior thalamic HD cells during spatial search
behavior. These experiments have been conducted to further examine the contribution of the HD
cell activity to relative responding during spatial navigation.

Chronic activation of the amygdala through repetitive stressful events can lead to
permanent hyper-excitability of its circuitry, which is known to be the root of a number of mood
and anxiety disorders. Small conductance Ca2+-activated K+ (SK) channels expressed on lateral
amygdala (LA) pyramidal neurons shape glutamatergic postsynaptic potentials and module
NMDA receptor-dependent synaptic plasticity. When activated, SK channels reduce neuronal
excitability and LTP. Induction of synaptic plasticity in LA pyramidal neurons causes PKAmediated
internalization of SK channels from the postsynaptic density. The current study
examined whether fear conditioning would affect the subsequent sensitivity of mice to novel fear
memory encoding through SK channel blockade by the bee venom peptide, apamin. Naïve male
C57BL/6J mice received a systemic injection of apamin or saline prior to exposure to a 1 tone
(CS) - foot shock (US) conditioning protocol. Tone fear memory strength was examined 24
hours later. The next day, mice received the same or reversed treatments of saline or apamin and
were conditioned to a novel CS and context. The influence of apamin on anxiety was also
examined in the elevated plus maze to determine whether the drug was able to alter anxiety
independent of conditioning. The fear conditioning results suggest that prior fear conditioning
altered the sensitivity of mice to apamin-induced fear memory encoding during the second
conditioning session. The plus maze results indicate that solely apamin does not alter anxiety,
thus fear conditioning impairment in apamin-treated mice is not a reflection of drug effects
alone.

From locating a secure home, foraging for food, running away from predators,
spatial navigation is an integral part of everyday life. Multiple brain regions work
together to form a three-dimensional representation of our environment; specifically,
place cells, grid cells, border cells & head direction cells are thought to interact and
influence one another to form this cognitive map. Head direction (HD) cells fire as the
animal moves through space, according to directional orientation of the animal’s head
with respect to the laboratory reference frame, and are therefore considered to represent
the directional sense. Interestingly, inactivation of head direction cell-containing brain
regions has mixed consequences on spatial behavior. Current methods of identifying HD
cells are limited to in vivo electrophysiological recordings in a dry-land environment. We
first developed a dry-land version of the MWM in order to carry out behavioral-recording
paired studies. Additionally, to learn about HD cells function we quantified expression of neuronal activation marker (c-Fos), and L-amino acid transporter 4 (Lat4) in neurons
found within the HD cell dense anterodorsal thalamic nucleus (ADN) in mice after
exploratory behavior in an open field, or forward unidirectional movement on a treadmill.
We hypothesize that the degree to which ADN neurons are activated during exploratory
behavior is influenced by the range of heading directions sampled. Additionally, we
hypothesize that c-Fos and Lat4 are colocalized within ADN neurons following varying
amounts of head direction exposure. Results indicate that following free locomotion of
mice in an open field arena, which permitted access to 360° of heading, a greater number
of ADN neurons express c-Fos protein compared to those exposed to a limited range of
head directions during locomotion in a treadmill. These findings suggest that the degree
of ADN neuronal activation was dependent upon the range of head directions sampled.
We observed a high degree of colocalization of c-Fos and Lat4 within ADN suggesting
that Lat4 may be a useful tool to manipulate neuronal activity of HD cells. Identifying
genetic markers specific to ADN helps provide an essential understanding of the spatial
navigation system, and supports development of therapies for cognitive disorders
affecting navigation.

Excessive fear is a hallmark of various anxiety and stress disorders, such as post-traumatic stress disorder (PTSD). Current pre-clinical research has focused on identifying behavioral and neuropharmacological methods of facilitating fear extinction in order to improve well-being of PTSD patients. The present experiment investigated the effects of voluntary exercise on fear memory and object recognition memory in male C57BL/6J mice. Results suggested that mice that exercised voluntarily exhibited significantly less fear-elicited freezing behavior during cued fear extinction trials compared to that of non-exercise control mice. Results from assessment of object memory revealed no difference in object memory retention between voluntary exercise mice and control mice. These results suggest that the beneficial effects of exercise in mice appear to be specific to the task and to the specific memory process. These results suggest that voluntary exercise may hold utility for remediation of PTSD and anxiety symptoms in humans.

Establishing appropriate animal models for the study of human memory is
paramount to the development of memory disorder treatments. Damage to the
hippocampus, a medial temporal lobe brain structure, has been implicated in the memory
loss associated with Alzheimer’s disease and other dementias. In humans, the role of the
hippocampus is largely defined; yet, its role in rodents is much less clear due to
conflicting findings. To investigate these discrepancies, an extensive review of the rodent
literature was conducted, with a focus on studies that used the Novel Object Recognition
(NOR) paradigm for testing. The total amount of time the objects were explored during
training and the delay imposed between training and testing seemed to determine
hippocampal recruitment in rodents. Male C57BL/6J mice were implanted with bilateral
dorsal CA1 guide cannulae to allow for the inactivation of the hippocampus at discrete
time points in the task. The results suggest that the rodent hippocampus is crucial to the
encoding, consolidation and retrieval of object memory. Next, it was determined that there is a delay-dependent involvement of the hippocampus in object memory, implying
that other structures may be supporting the memory prior to the recruitment of
hippocampus. In addition, when the context memory and object memory could be further
dissociated, by altering the task design, the results imply a necessary role for the
hippocampus in the object memory, irrespective of context. Also, making the task more
perceptually demanding, by requiring the mice to perform a two-dimensional to three-dimensional
association between stimuli, engaged the hippocampus. Then, in the
traditional NOR task, long and short training exploration times were imposed to
determine brain region activity for weak and strong object memory. The inactivation and
immunohistochemistry findings imply weak object memory is perirhinal cortex
dependent, while strong object memory is hippocampal-dependent. Taken together, the
findings suggest that mice, like humans, process object memory on a continuum from
weak to strong, recruiting the hippocampus conditionally for strong familiarity.
Confirming this functional similarity between the rodent and human object memory
systems could be beneficial for future studies investigating memory disorders.

SK channels are small conductance Ca2+-activated K+ channels expressed throughout the CNS. SK channels modulate the excitability of hippocampal CA1 neurons by affecting afterhyperpolarization and shaping excitatory postsynaptic responses. Such SK-mediated effects on activity-dependent neuronal excitability and synaptic strength are thought to underlie the modulatory influence of SK channels on memory encoding. Here,the effect of a new SK1 selective activator, GW542573X, on hippocampal-dependent object memory, contextual and cued conditioning, and trace fear conditioning was examined. The results demonstrated that pre- but not post-training systemic administration of GW542573X impaired object memory and trace fear memory in mice 24 h after training. Contextual and cued fear memory were not disrupted. These current data suggest that activation of SK1 subtype-containing SK channels impairs long-term memory. These results are consistent with converging evidence that SK channel activation suppressed behaviorally triggered synaptic plasticity necessary for encoding hippocampal-dependent memory.

Small conductance Ca2+-activated K+ (SK) channels have been shown to alter the encoding of spatial and non-spatial memory in the hippocampus by shaping glutamatergic postsynaptic potentials and modulating NMDA receptor-dependent synaptic plasticity. When activated, dendritic SK channels reduce hippocampal neuronal excitability and LTP. Similar SK channel properties have been demonstrated in lateral amygdala (LA) pyramidal neurons. Additionally, induction of synaptic plasticity and beta-adrenoreceptor activation in LA pyramidal neurons causes PKA-mediated internalization of SK channels from the postsynaptic density. Chronic activation of the amygdala through repetitive stressful stimuli can lead to excitatory synaptic strengthening that may create permanent hyper-excitability in its circuitry. This mechanism may contribute to a number of mood and anxiety disorders. The selective influence of SK channels in the LA on anxiety and fear conditioning are not known. The thesis project outlined herein examined whether SK channel blockade by bee venom peptide, apamin, during a repetitive acute fear conditioning paradigm was sufficient to alter fear memory encoding and the resulting behavioral outcome. Following the final fear memory test session, mice were tested in the open field immediately after the second fear conditioning test session. The findings indicate that intracranial LA microinfusions of apamin did not affect memory encoding or subsequent anxiety.