1. The Effects of Post-training Timolol on Hippocampal Excitability
Priscilla D. Saenz, Eric S. Lovitz, Lucien T. Thompson
Aging and Memory Lab, School of Behavioral and Brain Sciences, The University of Texas at Dallas, Richardson, TX
Methods
Subjects: Experiments were performed using male Long-Evans rats (2-3 mo). Rats were locally bred, and
maintained under conditions approved by the UT Dallas IACUC on a 12/12 hr light/dark schedule prior to
testing. Rats were handled daily for 5 min for 5 days prior to all experimental use.
Cannula Implantation: Rats were anesthetized with isoflurane and implanted bilaterally with cannula (15
mm, 23 ga) stereotaxically into the amygdala (-2.7 mm AP, ±5.1 mm ML, -6.8 mm DV) 5 days prior to
training and subsequent in vitro recordings. 1 mg/kg of antibiotic (enrofloxacin) was injected I.M. post-
surgery.
IA Behavioral Training: In a single trial, each rat was placed into the brightly lit compartment of a
rectangular Plexiglas shuttle box. The rat was allowed to cross to the dark compartment, and a guillotine
door was shut to lock the animal in the dark compartment. When the rat reached the end of the dark
compartment and turned around, a single high intensity (0.5 mA, 1 s) footshock was given. The rat
remained in the dark compartment for an additional 15 s following the footshock. Immediately following
the single IA behavioral training trial or control procedures, rats were infused with timolol (0.75 μg/0.5
μl, made in 0.1 M PBS) in one hemisphere and vehicle (0.1 M PBS) in the control hemisphere
contralateral to the drug infusion. Infusion rates of 0.5 μl over 60 s were used.
Slice Preparation: 24 hr after infusion, rats were anesthetized with isoflurane and decapitated. The
brain was hemisected and immersed in cooled (1°C) oxygenated (95% O2: 5% CO2) s-aCSF [in mM: 124
sucrose; 3 KCl; 1.3 MgSO4; 1.24 NaH2PO4; 2.4 CaCl2; 26 NaHCO3; 10 d-glucose; pH 7.4]. After chilling for
3-4 min, the brain was blocked and 400 µm ventral brain slices were cut using vibratomes. Slices were
placed in room temperature (25°C) aCSF [in mM: 124 NaCl; 3 KCl; 1.3 MgSO4; 1.24 NaH2PO4; 2.4 CaCl2;
26 NaHCO3; 10 d-glucose; pH 7.4]. Slices were continuously oxygenated (95% O2: 5% CO2).
Neurophysiological recording: Neurons were recorded from both drug-treated and control hemisphere
slices. Sharp electrodes were prepared from borosilicate glass (filled with 3 M KCl; 30-80 MΩ), and
intracellular recordings were made (using AxoClamp 2b amplifiers and National Instruments LabView
interfaces) from submerged slices (31°C). Electrophysiological data was analyzed using ANOVA. Post-hoc
Scheffe’s tests were performed to identify individual differences between groups.
Future Directions
In a previous study, post-trial infusion of the β-adrenergic antagonist propranolol into the amygdala
was shown to cause impairment in memory retention for the IA task. Propranolol is shown to have
non-specific effects on voltage-gated K+ channels, and affecting Ca2+ channels. Propranolol could
be tested to see if an optimal dose could block AHP plasticity after a fearful event. Adrenergic
systems are importantly involved in memory storage processes.
Acknowledgements
We would like to thank others who helped on this project: Michelle Chavez, Devin Proch, David Beddow, & Hallie
Cox. We would also like to thank the Undergraduate Research Scholar Award for helping to fund this project.
Neurophysiological Recordings
24 hr
IA Training and Drug Infusion
Handling (5 d)
Recovery (3 d)
Cannulation of the Amygdala
Handling (5 d)
Fig. 3. Infographic on the timeline of
behavioral training.
Fig. 1. The inhibitory avoidance (IA) shuttlebox. Animals are placed into the light-side and once
they entered the dark-side, they received a mild foot-shock.
Fig. 4. Measures of AHPs following inhibitory avoidance task. (A) AHP waveforms from untrained and IA trained rats 24 h after
post-trial unilateral infusions of timolol or vehicle (0 µg; IA trained neurons n = 7, untrained controls: n = 7; 0.75 µg; IA
trained neurons n = 11, untrained controls: n = 10). AHPs were reduced in IA trained vehicle and timolol infusion. (B) Medium
and slow components of AHPs. The amplitudes of mAHPs, measured 250 ms post-burst, and of sAHPs, measured 500 ms, 750 ms,
and 1 s and 2 s post-burst, showed reductions in AHP. Neurons from IA trained timolol-infused hemispheres showed
significant reductions in the sAHP at 2 s compared to neurons from untrained timolol-infused hemispheres (⁺=p<0.04). (C)
Peak AHP amplitudes. Neurons from IA trained vehicle hemispheres showed reduced peak AHP amplitudes compared to
neurons from untrained vehicle-infused hemispheres (ᵃ=p<0.02). Timolol infusion did not cause significant changes.
Fig. 5: Intrinsic excitability as measured by accommodation. (A) Waveforms of accommodation was reduced in neurons
from IA trained vehicle- and timolol-infused hemispheres, and also in neurons from untrained timolol-infused
hemispheres. (B) Action potential firing during an 800 ms sustained depolarization was significantly increased in neurons
from vehicle- and timolol-infused IA trained hemispheres, and from timolol untrained hemispheres. Action potential
firing was decreased (i.e. accommodation was increased) in neurons from timolol-infused IA trained hemispheres
compared to vehicle-infused IA trained hemispheres (*=p<0.001).
0.5 mA, 1 s
Introduction
Stress from fear plays a key role in the formation of memories. This fear
releases a surge of Norepinephrine (NE) which activates the amygdala
through β-adrenergic receptors, in turn enhancing memory formation
mediated by the hippocampus.
In a previous study, timolol (an inverse β-agonist, or functional antagonist) was
shown to dose-dependently impair memory by interfering with consolidation after
a stressful event in a single-trial inhibitory avoidance (IA) learning task, a measure
of retention latency was decreased with bilateral infusion of timolol (0µg, 0.25µg,
0.75µg, 1.25µg) 24 hr after IA training. A dose of 0.75µg was shown to have the
most effective reduction in retention.
In this study, the mechanism behind the effect of timolol (0.75 µg) infused into the
amygdala of male rats immediately after aversive (IA) training was examined.
Neurophysiological recordings were taken from CA1 hippocampal neurons to
determine the effects of unilaterally infused timolol on intrinsic excitability (AHPs,
accommodation). This study determined intrinsic excitability decreased in neurons
from IA-trained timolol infused hemispheres, while IA training combined with
vehicle will enhance intrinsic excitability in hippocampal CA1 neurons when
compared to neurons from untrained vehicle-infused hemispheres.
Results
Afterhyperpolarizations (AHPs) and accommodation were
measured from CA1 neurons in timolol-infused (0.75 μg/0.5 μl) hemispheres,
contralateral vehicle-infused IA trained hemispheres as well as to neurons from timolol-
or vehicle-infused untrained control hemispheres.
Following IA training, recordings showed reductions in both AHP and accommodation, yet
showed intrinsic excitability was enhanced in those that were trained and vehicle-infused
(evaluated 24 h post-training). (Figure 4) AHP plasticity effects were shown in analyses of
both the medium AHP (mAHPs) and slow AHP (sAHPs) (Fig. 4B). CA1 Pyramidal neurons
showed significantly reduced AHP peak amplitudes from IA trained, vehicle-infused
hemispheres compared to neurons from untrained vehicle-infused hemispheres at various
time intervals. (Figure 4C)(ANOVA: F(3) = 3.461, p =0.0181; Scheffe’s test: IA trained
vehicle vs. untrained vehicle: p = 0.02; untrained timolol vs. untrained vehicle: p >0.26; IA
trained timolol vs. IA trained vehicle: p >0.84; Fig. 4C). Effects of AHP were also measured
as the spikes elicited in spike-frequency accommodation. Infusion of timolol blocked the
effect of training in hippocampal excitability (Fig. 5).
Are You Afraid
of the Dark?
Fig. 2. Timolol affects retention latency in the IA task. All rats
showed increased latency to enter the dark compartment during
retention compared to initial latency (*=p<0.007). The 0.75 μg
and 1.25 μg doses significantly decreased latency (a=p<0.0150
µg: n=13, 0.25 µg: n=11, 0.75 µg: n=10, 1.25 µg: n=12). The 0.75
μg dose produced the most significant impairment in memory.