Mutual Excitation Among Olfactory Bulb Mitral Cells Revealed by
Recurrence Time History Matching (RTHM)
Alexandra Radu, Maame Boateng, Henaa Razzak, Barry K. Rhoades.
Department of Biology, Wesleyan College, Macon, GA 31210.
BACKGROUND:
Olfactory Bulb Function
In the mammalian
olfactory system, the
primary cortical
sensory area is the
olfactory bulb (OB).
BACKGROUND:
Correlational Analysis
METHODS
Surgery and Monitoring
An interspike interval histogram (ISIH) shows
the cumulative distribution of intervals between
action potentials in a unit spike train.
rat brain
Primary olfactory nerve
(PON) axons synapse on
mitral (M) and tufted (T)
projection neurons.
Periglomerular (P) and
granule (G) neurons
provide lateral interactions.
Interspike Intervals
1
2
3
4
5
Lateral negative feedback sharpens
chemotopic image of signal from
olfactory receptors.
Evidence:
- PG cells are GABAergic and GABA
is an inhibitory neurotransmitter
- paired-pulse attenuation of evoked
potentials to strong PON stimulation
time (5 msec ticks)
Subjects: Male Sprague-Dawley rats (Rattus norvegicus) from 180 to 450g
TGase1 Positive
A cross-correlogram is a histogram of cross
intervals between two unit spike trains recorded
concurrently. Intervals corresponding to excitatory
synaptic delays appear as “linkage peaks” above
the background correlational “noise level”.
METHODS
Data Criteria
GL
2
11
1
2
RTHM Correlogram
enhanced
linkage peak
2
A RTHM scatterplot plots ranked local
interspike interval averages against RTHM
cross-intervals. Excitatory synaptic linkages
appear as vertical bands, with density reflecting
activity-dependent synaptic gain. Excitatory
gain drops off at high and low local firing rates.
c
9-1
d
a, c: Interspike interval histograms from mitral cells approximate
Poisson distributions truncated by an initial refractory period.
b, d: A stochastic model was used to generate ISIHs for artificial
spike trains (light traces) with identical mean firing rates and
refractory periods to the original real spike trains (bold traces).
The real spike trains show a higher number of very short intervals,
as would be expected for units participating in a mutually
excitatory neuronal ensemble.
RESULTS
RTHM Plots
a
b
c
d
ABSTRACT
In the mammalian olfactory bulb coordinated
neural activity associated with odor
discrimination is dominated by narrow-band
oscillations in the gamma EEG range (30-80 Hz),
gated by respiratory inspiration. Mutual
excitation among the mitral cell projection
neurons is a required feature of successful bulbar
models, but has not been conclusively
demonstrated by either direct histological or
electrophysiological evidence. In the present
study tungsten-steel microelectrodes were
lowered to the mitral cell layer of the olfactory
bulb in urethane- or pentobarbital-anesthetized
rats, and positioned using electrophysiological
response criteria. Ten to twenty minute samples
of resting neuronal activity were recorded to
analog tape. A template matching system was
used to isolate and extract multiple, simultaneous
single-unit spike trains. Temporal linkages
between spike trains were evaluated using
recurrence-time history matching (RTHM), a
algorithm for enhancing conventional conditional
cross-correlograms. In the RTHM analysis plots
of some pairs of mitral cells apparent mutual
excitation was evidenced by the presence of highdensity, short-latency linkage bands and a withinband pattern suggesting activity-dependent gain.
The validity of inferences based on RTHM was
verified using artificially-generated spike trains
with stochastically-defined firing patterns and
inter-neuronal linkages.
ranked mean local ISI
A unit spike train is a temporal series of
sequential occurrence times of action potentials
extracted for a single neuron.
excitatory
linkage
band
MCL
ML Cells With Strong Excitatory Linkages
Evidence Activity-Dependent Excitatory Gain
METHODS
Recording and Analysis
a
b
c
d
Analog Recording
Spike Train Extraction
.
One or more single-unit spike trains
were extracted from analog
recordings using a an adaptive template-matching system
(Cambridge Electronic Design 1401/Spike-2).
Correlational Analysis
cross-interval in msec
REFERENCES
a, c: RTHM correlogram and RTHM scatterplot for two MCL units
(cells 11-3 & 11-4). The density peaks and bands at the arrows
suggest short-latency mutual excitatory interactions. The void
surrounding the zero latency line is an artifact of extracting two
unit trains from a single-electrode recording.
b, d: Shuffled ISI sequence versions of a,c with the central region
masked, to demonstrate the uniform RTHM distributions for
expressly uncorrelated units.
Selected 5-10 minute epochs meeting the criteria for MCL
activity were recorded to analog cassette tapes (RCA SCT-510).
RTHM Scatterplot
time (5 msec ticks)
11-1
b
1 second
cross-interval in msec
2
a
MCL Cells Evidence Weak Mutual Excitation
As the microelectrode was lowered into the OB, the glomerular
layer (GL) was identified by multicellular
burst firing (arrows) ,
.
synchronized to the respiratory wave. The underlying mitral cell
layer (MCL) was characterized by isolated unit APs, with low
background activity, and non-synchronized, non-bursting firing.
1) Interspike Interval Histograms provide weak
evidence for excitatory input to MCL cells.
2) RTHM plots of simultaneously recorded
spiking cells from the. MCL show evidence for
mutual synaptic excitation.
3) RTHM plots do not provide any evidence for
lateral inhibition between mitral cells.
GL
Action potential occurrence times may be
extracted from multi-neuronal extracellular
recordings by a template matching process.
2
Single Unit ISIHs Provide Weak Evidence
for Excitatory Input to MCL Cells
Identification of Mitral Cells for Recording
Recurrence time history matching (RTHM) uses
a similarity algorithm related to the Hausdorff
distance to enhance linkage peak resolution.
BACKGROUND:
Spike Trains
Five-second epochs of single unit activity show non-bursting firing.
At an appropriate anesthetic level, the OB surface EEG was
characterized by slow (1-3HZ) waves synchronized to
respiratory inspiration, with sinusoidal gamma frequency
oscillations (arrows) riding the recovery phase of most waves.
BACKGROUND:
RTHM Analysis
CONCLUSIONS
11-4
Amplification and Monitoring: The signal from the depth microelectrode was
passed through a microelectrode amplifier (A-M Systems 1800; x1000; 500Hz-5kHz
band-pass filtered) and simultaneously monitored in three ways:
linkage peak
cross-interval in msec
Will enhanced spike train correlational analysis of paired
M cell single unit spike trains (via RTHM) support M<> M
mutual excitation, as evidenced by linkage bands and
activity-dependent gain?
11-3
1) a 250um formvar-insulated stainless steel wire loop was placed against the OB surface to serve
as a surface EEG electrode and presser foot to suppress vascular pulsations
2) a electrolytically-sharpened, parlene-insulated tungsten-steel microelectrode (A-M Systems
#575500, 5Mohm) was lowered to depth through the center of the presser-foot ring
Anesthetic Level
Experimental Question:
11-2
Electrode Placement: Two electrodes were stereotaxically positioned:
time (10 msec ticks)
- averaged evoked potentials (AEP)
indicate P<>P excitation
- current source density analysis indicates
GL excitation
- P<>P excitation mediated by GABA
- conventional correlograms show
M<>M excitation (de la Luz, 2005)
11-1
1) computer video display via an A/D conversion system (ADInstruments PowerLab 8sp;
Scope 5.11 software)
2) audio monitoring (RCA model SA-155 stereo amplifier)
3) analog oscilloscope (GW model GOS-622G)
Cross Intervals
Cross Correlogram
9-2
1) Urethane (125mg/ml in normal saline) IP @ 1200-1500 mg/kg body weight
2) Nembutal (sodium pentobarbital 50mg/ml) IP @ 50-75mg/kg body weight
3) Ketamine cocktail (Ketamine 60mg/ml, Xylazine 6mg/ml, Acepromazine 1mg/ml) IM
@ 0.2-0.4ml plus Nembutal @ 0.2-0.3ml as required.
Electrophysiological Methods
M<>G negative feedback creates
oscillatory activity (~ 60Hz for rat).
P<>P and M<>M mutual lateral
excitation promotes spatial amplitude
patterns with information content.
Evidence:
9-1
Anesthesia: Anesthesia for each rat was maintained at a level characterized by
dilated pupils, regular breathing, insensitivity to deep leg pinch pain, and no
movement, following one of three anesthesia protocols:
1) the rat was placed on a warming pad (Gaymar T/Pump), on a mechanical isolation table within
a grounded mesh Faraday cage
2) the head was secured in the nose clip and ear bars of stereotaxic frame (David Kopf Instr.)
3) the scalp, superficial muscle, and fascia were cut down the midline and reflected
laterally to expose the dorsal skull
4) two stainless steel screws for electrical ground and reference were implanted in the parietal
and/or nasal bones
5) the left orbit was surgically exenterated of all soft tissue
6) a surgical drill was used to open a window encompassing the lateral OB, lateral olfactory tract,
and anterior pole of the frontal cortex
7) the dura mater was stripped off and the exposed brain surface was coated with mineral oil
ISIH
Freeman (1975)
Representative Spike Trains From Two Rats
Surgical Exposure: The following steps were performed in sequence:
Two Theories of OB Function:
Shepherd (1979)
Surgical Methods
RESULTS
Single Units
Correlational analysis, including ISIHs and RTHM plots, was
performed on spike train pairs (after Rhoades et al, 1994).
De la Luz M, Diaz E and Maldonado PE. Simultaneous single
unit recording in the mitral cell layer of the rat olfactory
bulb under nasal and tracheal breathing. Biological
Research. 38: 13-26, 2005.
Freeman WJ. Mass Action in the Nervous System. Academic
Press 1975.
Rhoades BK. Excitatory Actions of GABA in the Olfactory
Bulb. Doctoral Dissertation,University of California at
Berkeley, 1990.
Rhoades BK, Kowalski JM, Mackey HJ and Gross GW.
Graphical measures of spike train similarity and coupling
based on recurrence time history matching (RTHM).
Society for Neuroscience 24th Annual Meeting. Miami
Beach, FL, 1994.
Shepherd GM. The Synaptic Organization of the Brain.
Oxford University Press, 1979.
ACKNOWLEDGEMENTS
a, b: RTHM correlogram and RTHM scatterplot for two ML units
(cells 9-1 & 9-2) with strong excitatory linkages (arrows).
c: Extraction of a linkage band (vertical lines)
d: ISIH for band in c, showing reduction in linkage strength for
short ISIs/high firing rates (activity-dependent gain).
This project was supported, in part, with funds from
NSF/CCLI grant #DUE9950546 and an endowment to
Wesleyan College from the Munroe family of Atlanta, GA.