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Mark E. Jackson, Houman Homayoun and Bita Moghaddam- NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex

Mark E. Jackson, Houman Homayoun and Bita Moghaddam- NMDA receptor hypofunction produces concomitant firing rate potentiation and burst activity reduction in the prefrontal cortex

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NMDA receptor hypofunction produces concomitantfiring rate potentiation and burst activity reduction inthe prefrontal cortex
Mark E. Jackson, Houman Homayoun, and Bita Moghaddam*
Department of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260Edited by L. L. Iversen, University of Oxford, Oxford, United Kingdom, and approved April 19, 2004 (received for review December 18, 2003)
Cognitive deficits associated with frontal lobe dysfunction are adeterminant of long-term disability in schizophrenia and are noteffectively treated with available medications. Clinical studiesshow that many aspects of these deficits are transiently induced inhealthy individuals treated with
-methyl-
D
-aspartate (NMDA)antagonists. These findings and recent genetic linkage studiesstrongly implicate NMDA receptor deficiency in schizophrenia andsuggest that reversing this deficiency is pertinent to treating thecognitive symptoms of schizophrenia. Despite the wealth of be-havioral data on the effects of NMDA antagonist treatment inhumans and laboratory animals, there is a fundamental lack ofunderstanding about the mechanisms by which a general state ofNMDAdeficiencyinfluencesthefunctionofcorticalneurons.Usingensemble recording in freely moving rats, we found that NMDAantagonist treatment, at doses that impaired working memory,potentiated the firing rate of most prefrontal cortex neurons. Thispotentiation, which correlated with expression of behavioral ste-reotypy, resulted from an increased number of irregularly dis-charged single spikes. Concurrent with the increase in spike activ-ity,therewasasignificantreductioninorganizedburstingactivity.These results identify two distinct mechanisms by which NMDAreceptor deficiency may disrupt frontal lobe function: an increasein disorganized spike activity, which may enhance cortical noiseand transmission of disinformation; and a decrease in burst activ-ity, which reduces transmission efficacy of cortical neurons. Thesefindings provide a physiological basis for the NMDA receptordeficiency model of schizophrenia and may clarify the nature ofcortical dysfunction in this disease.
N
-methyl-
D
-aspartate (NMDA) receptors are increasinglyimplicated in schizophrenia. The majority of genes thathave so far been linked to increased susceptibility to developschizophrenia can modulate NMDA receptor-mediated signaltransduction (1, 2), suggesting that NMDA receptors are acritical component of genetic vulnerability to develop schizo-phrenia. Recreational or investigator-administered exposure todrugs that have antagonist activity at NMDA receptors producesa transient state of psychosis and schizophrenia-like cognitivedeficits (3–5). In fact, double-blind clinical studies suggest thatthe cognitive deficits produced by NMDA antagonists in healthy volunteersarenearlyidenticaltothedeficitsobservedinpatients with schizophrenia (6). Based on these pharmacological andgenetic linkage studies, the NMDA antagonist treatment isconsidered a mechanistically relevant model for cognitive def-icits of schizophrenia (7–10). Understanding the impact of thistreatment on cellular mechanisms that support the expression of abnormal cognition is critical for understanding the impact of anunderlying NMDA dysfunction on the disease process and fordefining better treatments. However, despite a large body of literature that has characterized the behavioral aspects of thismodel in laboratory animals, there remains a fundamental lackof understanding about the cellular mechanisms that lead to thedisruption of cognitive functioning.Functional imaging studies suggest that, after systemic admin-istration of NMDA antagonists, the most affected brain regionis the prefrontal cortex (PFC) (11, 12). Animal studies alsosuggest that the PFC is a key region responsible for the expres-sion of behaviors that are relevant to schizophrenia, including working memory and behavioral stereotypy (13, 14). This isconsistent with a large body of functional and postmortemliterature implicating PFC abnormalities in schizophrenia (15–17) and the fact that the most prevalent cognitive dysfunctionsreported in schizophrenia involve constructs that depend on thefunctional integrity of the PFC, such as working memory (18).The aim of the present study was to define the mechanisms by which behaviorally relevant reductions in NMDA receptor func-tion may disrupt the pattern of activity of PFC neurons. We usedensemble recording in freely moving animals and post hocanalysis of spike activity to determine the effect of the use-dependent NMDA antagonist MK801, at doses that we found toimpair working memory, on the firing characteristics of PFCneurons. Considering that behavioral stereotypy, which is in-duced by NMDA antagonist treatment (9, 19), is a criticalcomponent of cognitive and motor abnormalities in schizophre-nia, we also quantified behavioral stereotypy during the record-ing sessions.
Experimental Procedures and Methods
General Animal Use and Electrophysiology Procedure.
Male Spra-gue–Dawley rats (340–420 g) were used. All procedures wereapproved by the Institutional Animal Care and Use Committee. Arrays of eight stainless steel Teflon-insulated microwires(50-
m diameter, NB Labs, Denison, TX) were implanted underhalothane anesthesia in the prelimbic PFC: 3.0–3.6 mm anteriorand 0.5–0.8 lateral to Bregma, and 3.5 mm ventral to the dorsalsurface of the brain (20). Animals were allowed to recover for1 week. They were then acclimated to handling and recordingprocedures for 1 week before the first recording session. Re-cordings were performed in the animal’s home cage (clearpolycarbonate 44
22
42 cm), placed inside a Faraday cage. Animals were connected to an eight-channel connecter contain-ing FET headstages (NB Labs) via a commutator (NB Labs) thatpermitted unrestricted movement during recording.Spontaneous unit activity was recorded by using multiplechannel amplifiers with 500
gain and 220- to 5.9-kHz bandpassfilters (Plexon, Dallas). The amplified signal from each electrode was digitized (30 kHz), and continuous data files were saved ona PC for spike sorting using
OFF
-
LINE SORTER
software (Plexon).Neurons were selected as single units only if autocorrelogramsand interspike interval histograms indicated there were nosignificant errors in sorting due to noise or similar waveforms.Typically, two to three neurons were isolated from eachelectrode.
This paper was submitted directly (Track II) to the PNAS office.Abbreviations: NMDA,
N
-methyl-
D
-aspartate; PFC, prefrontal cortex; FS, fast spiking; RS,regular spiking.*To whom correspondence should be addressed. E-mail: moghaddam@bns.pitt.edu.© 2004 by The National Academy of Sciences of the USA
www.pnas.org
cgi
doi
10.1073
pnas.0308455101 PNAS
June 1, 2004
vol. 101
no. 22
8467–8472
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 All rats received, in random order, a maximum of four of fivepossible i.p. injections, which included saline and four doses of MK801 (0.01, 0.05, 0.1, and 0.3 mg
kg). Injections were made atleast 1 week apart.
Analysis of Electrophysiology Data.
General information.
Electrophys-iological data were imported into
NEUROEXPLORER
(Plexon) foranalysis. Only putative single units with autocorrelograms andinterspike interval histograms consistent with an absolute re-fractory period of at least 1.1 msec were considered
‘‘
singleneurons.
’’
Based on established criteria of firing rates andautocorrelogram statistics (21
23), we divided units into twogroups that correspond to descriptions (24) of fast-spikingneurons (FS, putative interneurons) and regular spiking neurons(RS, putative pyramidal neurons). In general, FS cells had spike waveforms with large after-hyperpolarizations (unlike RS cells),high firing rates (
10 Hz), and autocorrelograms correspondingto a tonic firing pattern. RS neurons had lower firing rates (
5Hz) and autocorrelograms consistent with a sporadic firingpattern.
Firing rate analysis.
Firing rate statistics were calculated by usingfiring rate histograms (5-min bins) produced in
NEUROEX
-
PLORER
, and the results were imported into
MATLAB
(Math-Works, Natick, MA) for further analysis. For each unit, meanbaseline spontaneous firing rates and 99% confidence intervals were calculated for the 30-min baseline period. Neurons werethengroupedintooneofthreeresponsecategoriesdependingon whether they had a significant increase (type 1), decrease (type2),ornochange(type3)inspontaneousfiringrateaftersystemicinjection. Criterion for determining significant changes frombaseline firing rates was two consecutive 5-min bins exceedingthe 99% confidence intervals (greater than for type 1, less thanfor type 2). Criterion for terminating a significant change frombaseline was two consecutive bins within the 99% confidenceinterval.
 
2
tests were used to determine significant changes inthe proportion of response types for different groups.To compare neurons with different baseline firing rates, ratehistograms were normalized by dividing each 5-min bin by theaverage of the bins during the 30-min baseline period for eachindividual neuron. Normalized rate histograms were comparedacross different groups by using one-way repeated-measures ANOVA with time as the repeated measure.For neurons with type 1 or type 2 responses, we also measuredthe mean area of the normalized rate histogram for the durationof the response for each group. Changes in the mean area werecompared across groups by a one-way ANOVA with the Bon-ferroni post hoc test.
Spontaneous burst analysis.
The Poisson surprise method of Leg-endy
et al.
(25), as implemented in
NEUROEXPLORER
was used todetect burst activity. The Poisson surprise method detects burstsbyfindingconsecutiveinterspikeintervals(ISI)thatarelessthanhalf the mean ISI, and testing whether these ISI would beexpected if the spike train were a Poisson process with randomtemporal patterning. Thus, the Poison surprise algorithm allowsfor differentiation between irregular, single spike patterns andbursting patterns contained within the same spike train.Several parameters of bursts were measured for each spiketrain, including percentage of spikes that occur within bursts,mean number of bursts per minute, mean number of spikes perburst, and mean intraburst frequency. Changes in burst param-eters were measured in two ways. First, burst parameters weremeasured in each individual spike train during the 2-h post-injection period. Comparisons of burst parameters betweengroupsandresponsetypesweremadebyusingANOVAwiththeBonferroni post hoc test. Second, a more detailed temporalanalysis was done by measuring burst parameters for eachneuron during the 30-min preinjection baseline period, andduring four sequential 30-min postinjection periods. For eachgroup, data pairs were produced for each neuron by using the value of one burst parameter during the baseline period (inde-pendent variable) and the value for the same parameter duringone of the four postinjection periods (dependent variable).First-order linear regression was used to determine whetherthere were significant changes between baseline and postinjec-tion burst parameters. The constant term was set to zero to forcethe regression line to pass through the origin. The quality of fitby linear regression was measured by Student
s
t
test (
 P
0.05).Changes in burst parameters relative to the baseline period weremeasured by changes in the regression coefficient (
SEM). Aregression coefficient of 1.0 indicates that there is no change inthat parameter compared to the baseline measure, whereas values
1.0 indicate a decrease, and values
1.0 indicate asignificant increase. Regression coefficients were calculatedduring each of the four sequential 30-min postinjection periodsrelative to baseline so that the time course of response could beevaluated.
Recording and Analysis of Behavioral Stereotypy.
Throughout therecording period, behavioral stereotypy was rated every 5-min. Animals received a score of 
‘‘
0
’’
for absence or
‘‘
1
’’
for presenceofeachofthefollowingbehaviors:ambulation,freezing,turning,grooming, sniffing up, sniffing down, mouth movements, jawtremor, bed digging, head wagging, and rearing. Behaviors werescored if they were expressed for
30 s. The rater was not blindto the experimental manipulations. Stereotypy scores werecalculated by summing scores for individual behaviors duringeach 5-min block of time for each rat. Total stereotypy scores were calculated by summing scores across all time blocks.Differences between groups were analyzed by ANOVA withrepeated measures. Stereotypy scores recorded during each5-min bin were compared with firing rate changes during thesame 5-min bins for each rat by using Pearson correlation withthe Bartlett
 
2
test for significance.
Spontaneous Alternation Test.
Animals were habituated by dailyhandling for 1 week. They were injected with vehicle or MK80140minbeforetesting,whichwasconductedbyplacingtheanimalon the center platform of a plus maze and allowing 12 min of unimpeded exploration. The plus maze was constructed of Plexiglas (0.63 cm thick) and painted gray. It consisted of acentral platform (14 cm
14 cm) to which four arms (14.0 cm wide, 40.6 cm long, and 12.7 cm high) were joined with no spacebetweenadjacentarms.Thenumberandsequenceofarmentries was recorded for calculation of a percent alternation score. Analternation consisted of four different arm choices out of fiveconsecutive arm entries. A 4
5 alternation score was computedbydividingthenumberofalternationsinoverlappingquintupletsby the total number of possible alternations, and multiplying thequotient by 100. These data were analyzed by one-way ANOVA with Bonferroni-adjusted post hoc tests.
Histology.
Animals were anesthetized with chloral hydrate andperfused with saline followed by 10% buffered formalin. Brainsections (150
m) were stained with cresyl violet for electrodeplacement verification. All recordings reported here were fromlayers V and VI of ventral prelimbic cortex.
Results
Baseline and Postinjection Characteristics of PFC Neurons.
A total of 518 neurons were isolated from eight rats in 27 recordingsessions. Neurons were separated into 510 RS neurons (putativepyramidal neurons) and eight FS neurons (putative interneu-rons), based on established criteria (21
23). The mean baselinefiring rates were 1.32
0.07 Hz for RS neurons and 13.6
1.04Hz for FS neurons. Because of the overall paucity of FS cells, wecould not perform an accurate statistical analysis of their
8468
www.pnas.org
cgi
doi
10.1073
pnas.0308455101 Jackson
et al.
 
responsetoMK801.Therefore,onlytheresultsfromtheRScellsare depicted in figures.Three types of postinjection responses were identified in RSneurons: increased firing rate (type 1), decreased firing rate(type 2), or no change in firing rate (type 3). After salineinjection, an approximately equal proportion of neurons re-sponded with either an increase or decrease in firing rate, and21% of the neurons had no significant change in firing rate (Fig.1). However, after MK801 injection, there was a greater per-centage of type 1 neurons (
 
2
test
61.08, df 
8,
p
0.00001). All FS cells had type 1 responses.
Temporal Profile of Firing Rate and Behavioral Changes After MK801.
In neurons with type 1 responses (Fig. 2
 A
), there was an overalleffect of dose on firing rate (
 F 
4,312
26.9,
P
0.00001). Whencompared to saline, there was a significant sustained increase infiring rate after MK801 injection at 0.1 mg
kg (
 F 
1,123
4.46,
P
0.037) and 0.3 mg
kg (
 F 
1,144
18.89,
P
0.00003). Low dosesof MK801 were not different from saline. Thus, although agreater number of neurons exhibited increased firing rate in lowdoseMK801groupsascomparedtothesalinegroup(seeFig.1),the magnitude of this increase was similar in both groups.Neurons with type 2 responses were not significantly differentfrom saline (Fig. 2
 B
).MK801 at moderate doses is known to produce behavioralstereotypy in rats involving ambulation, rearing, head wag, andoral movements (Fig. 2
C
). A significant increase in stereotypy was observed in groups that received MK801 at 0.05 mg
kg (
 F 
1,7
5.46,
P
0.05), 0.1 mg
kg (
 F 
1,7
22.15,
P
0.002), and 0.3mg
kg (
 F 
1,7
85.23,
P
0.00005).The mean area under the normalized postinjection rate his-togram (Fig. 3
 A
) significantly increased after the two highestdoses of MK801 (
 F 
4,312
40.35,
P
0.00001). In addition, thetotal stereotypy score also increased at those doses (
 F 
4,16
26.76,
P
0.001). The correlation coefficients depicted in Fig.3
 B
indicate a poor correlation between behavior and firing ratefor saline and MK801 at 0.01 mg
kg and 0.05 mg
kg, but a highcorrelation between firing rate and behavior at the two higherdoses of MK801, suggesting an association between increases inPFC neural activity and increases in stereotypy.
MK801-Induced Changes in Burst Patterns.
As demonstrated on Fig.4, in neurons with increased firing rate after MK801 injection,there was a dose-dependent decrease in the percentage of spikes
Fig. 2.
Temporal pro
le of
ring rate changes and behavioral activity aftersystemic MK801 injection. Peristimulus rate histograms (5-min bins) werenormalized by dividing the postinjection
ring rates by the baseline
ringrate.(
 A
)Neuronswithtype1responseshadasustainedincreasein
ringratefor high doses of MK801. (
B
) Neurons with type 2 responses were not signif-icantly different from saline. (
) MK801 at moderate and high doses signi
-cantly increased stereotypy scores.
Fig.1.
Percentageofneuronswithincrease,decrease,ornochangein
ringrate. There was a signi
cant increase in the percentage of neurons withpostinjectionincreasesin
ringrateafterMK801(0.01mg
kg,0.05mg
kg,0.1mg
kg, and 0.3 mg
kg) compared to saline. Asterisks indicate signi
cantdifference by
 
2
(
0.01) compared to saline. Note that this pattern ofresponsedidnotgeneralizetootherpsychoactivedrugssuchasamphetamine(Fig. 7, which is published as supporting information on the PNAS web site).
Jackson
et al.
PNAS
June 1, 2004
vol. 101
no. 22
8469
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