Awake-behaving recordings in mice during fear conditioning. Eric H. Chang, Ph.D. Goldsmith Postdoctoral Fellow
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1 Awake-behaving recordings in mice during fear conditioning Eric H. Chang, Ph.D. Goldsmith Postdoctoral Fellow Weill Cornell Medical College Burke Cornell Medical Research Institute White Plains, NY Noldus Symposium, November 16 th, 1
2 BALB/c and C57Bl/6 mice: What s the difference? BALB/c C57Bl/6 More popular for usage in cancer and immunology research, good for producing monoclonal antibodies Resistant to diet-induced cardiovascular ailments Generally high anxiety and males will fight one another if group housed Originated from Memorial Hospital in New York, NY in 19 First strain to have its genome fully sequenced Commonly used as the background for genetic modifications Resistant to tumors and permissive for maximal expression of most mutations Tendency to be aggressive and irritable towards humans and littermates. Originated from Harvard (Cambridge, MA) in 1921
3 Study design: strain differences in fear memory Context fear memory 24 hrs Habituation 1 min Conditioning - min 5 tone-shock pairings 15 sec tone 1 sec shock,.2 1. ma intensity 24 hr memory 1 min Tone fear memory: Five 15-sec tone presentations Freezing is defined as the absence of all movement, other than that required for respiration (Blanchard and Blanchard, 1972) Range of shock intensities:.2,.4,.6,.8, 1. ma for 1 second n = 1 mice / group, 5 BALB/c and 5 C57Bl/6 mice
4 Actimetrics FreezeFrame with Coulbourn box camera speaker Shock grid Actimetrics FreezeFrame program performs automated freezing analysis by taking the differential between consecutive frames (sampled ~2-5 Hz with a maximum average of 4 Hz). It uses a proprietary motion detection algorithm that can detect movements as small as 1 mm, which produces much more objective and reproducible sampling than traditional methods such as human scoring (one sample per 4 seconds).
5 Total freezing (%) Total freezing (%) Total freezing (%) Total Freezing (%) Fear memory across BALB/c and C57Bl/6 mice C57BL/6 - Habituation C57BL/6 24 hr Context Conditioning 5 tone-shocks Shock intensity (ma) Shock Intensity (ma) BALB/cJ Habituation BALB/c 24hr Context Conditioning 5 tone-shocks Shock Intensity (ma) Shock Intensity (ma)
6 Freezing (%) Total freezing (%) Total Freezing (%) Freezing (%) Total Freezing (%) Fear memory across BALB/c and C57Bl/6 mice 1.6 ma conditioning - 5 tone-shock pairings C57BL/6 Conditioning 24 hr Context Fear: BALB, C57 () BALB/c () C57Bl/ Shock Intensity (ma) 1 1 Time (Sec.) 1.6mA Conditioning - 5 tone-shock pairings BALB/cJ Conditioning Shock Intensity (ma) Shock Intensity (ma) 1 1 Time (sec)
7 Awake-behaving recordings during fear learning In vivo recordings allow for the simultaneous continuous recordings of 1s and 1s of neurons with high fidelity. By filtering the signal for lower frequencies, local field potentials (EEGs) can also be recorded simultaneously to study the behavior of a large population of neurons (thousands and tens of thousands). Also allows for the correlation of neurophysiology with ongoing animal behavior and cognition mm The lateral amygdala (LA) is responsible for triggering a fear response but LA neuron firing alone is insufficient to sustain the freezing. The prelimbic (PL) area of the prefrontal cortex is a candidate structure for this freezing maintenance because it receives convergent input from LA, hippocampus, and auditory cortex, and then projects back to and drives basal amygdala neurons. We implanted three tetrodes and one 5 micron wire into the PL of BALB/c and C57Bl/6 mice to determine whether the differences in fear expression can be detected within PL neurons.
8 Setup for synced electrophysiological-behavioral recordings TTLs signaling trial start and stimulus events Coulbourn FreezeFrame EthoVision experimental control set to Start Trial on TTL signal (Low High) Noldus I/O Box Neuralynx Cheetah 3 computers 4 monitors ~$, 1 mouse Noldus EthoVision
9 Using automated video tracking with implanted mice
10 Using automated video tracking with implanted mice
11 Using automated video tracking with implanted mice: FreezeFrame 24 hr context memory test 24 hr tone memory test
12 Using automated video tracking with implanted mice: EthoVision XT
13 Using the EthoVision immobility measure for freezing EthoVision uses a 3 Hz sampling rate compared to FreezeFrame at maximum average of 4 Hz. We can use the immobility measure to quantify freezing within EthoVision. Averaging Interval (Range 1-1) This is the number of samples across which changes in the subject's area are calculated to determine whether the subject is Mobile, Immobile or Highly mobile. In order to reduce the sensitivity of the Mobility variable to brief changes in area, the area data can be smoothed by taking the average change in area in the last n samples. Enter the averaging interval n or leave 1 if you do not want to smooth area changes.
14 Using the EthoVision immobility measure for freezing EthoVision uses a 3 Hz sampling rate compared to FreezeFrame at maximum average of 4 Hz. We can use the immobility measure to quantify freezing within EthoVision. Averaging Interval Immobile threshold: the percentage change in the subject s area to be considered mobile *immobility threshold set at.5%
15 Using automated video tracking with implanted mice: EthoVision XT Distance moved Velocity immobile mobile
16 Comparison of automated freezing measurements EthoVision uses a 3 Hz sampling rate compared to FreezeFrame at maximum average of 4 Hz. We can use the immobility measure to quantify freezing within EthoVision. *immobility threshold set at.5% Sampling average immobile time (sec) frequency % freezing Time % freeze FreezeFrame measurement for the same sec sample trial = 36.2% freezing *with 4 Hz sampling but only one value per second exportable
17 Frequency (imp/sec) spikes/sec % Frezeframe Frequency (imp/sec) spikes/sec D Freezing % Frezeframe Percent freezing (%) Comparison of automated freezing measurements FreezeFrame Time (freeze frame) Perievent Histograms, referenc TT2d_1 EthoVision Neuralynx: single-unit activity from PL T im e ( s e.8 c ) a _ 1 a _ 1 a _ 1 1 a _ 1 b _ 1 c _ 1 d _ 1 e _ 1 a _ 1 _ 1 1 b _ 1 _ Perievent Histograms, referenc Time (Ethovision) 2 4 h Time (freeze frame) Peri-event Histogram -5 5 TT2e_ /- 5 sec TT1_tt_ time TT3a_1 (sec) TT2_tt_ -5 5 Time (sec) TT3_tt_ +/- 1 sec time (sec)
18 Total Freezing (%) Conclusions and future work 24 hr Context Fear: BALB, C57 () BALB/c () C57Bl/6 There are significant differences in freezing between BALB/c and C57Cl/6 mouse strains. These differences remain across a range of shock intensities Shock Intensity (ma) The lower BALB/c freezing was apparent on fear acquisition (conditioning) and remained through various stages of fear memory testing. Both FreezeFrame and EthoVision are capable of measuring freezing in implanted, tethered mice. The precise (sub-second) syncing of fear conditioning events with neurophysiological events is critical for the examination of fear-related neuronal firing and network-level activity. Higher behavioral sampling rates allow for tighter correlations between ongoing fear behavior and its underlying neural activity.
19 Many thanks to: Patricio Huerta Sergio Robbiati Tom Faust RoseAnn Berlin Support for our work: The Burke Foundation Goldsmith Fellowship NIH RO
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