Brain Research, 86 (1975) 229-242
229
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECTS OF PUTATIVE NEUROTRANSMITTERS ON NEURONAL ACTIVITY IN MONKEY AUDITORY CORTEX
STEPHEN L. FOOTE, R O B E R T F R E E D M A N AND A. P A U L OLIVER
Laboratory of Neuropharmacology, Division of Special Mental Health Research, National Institute o f Mental Health, St. Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.) (Accepted November 6th, 1974)
SUMMARY
The effects of the putative neurotransmitters norepinephrine (NE), gammaaminobutyric acid (GABA), and acetylcholine (ACh) were tested on auditory cortex neurons which were activated acoustically by species-specific vocalizations in awake squirrel monkeys. Five-barrel glass electrodes were used to record the activity of single neurons in the superior temporal gyrus and to apply NE, GABA, or ACh microiontophoretically. Poststimulus time histograms and raster displays of neuronal responses to the vocalizations were computed before, during, and after iontophoresis. Dose-dependent inhibition of spontaneous and vocalization-evoked discharge rates was seen with NE and GABA. Generally, excitation was observed with ACh. A given dose of NE or GABA reduced spontaneous activity by a greater proportion than it reduced activity evoked by the vocalizations. During excitatory responses, segments with lower discharge rates were reduced proportionately more than segments with higher discharge rates. Usually, response 'pattern' was not altered by iontophoresis of any of the substances. However, in some cases the differential inhibition of slow activity produced by NE or GABA did result in a 'pattern' change. The demonstration that small amounts of locally applied NE and GABA substantially alter the specific neuronal activation produced by vocalizations provides additional evidence that these agents may function as neurotransmitters in this neocortical area and offers clues about their functional significance.
I NTRODUCTION
Substantial anatomical and biochemical evidence suggests that norepinephrine (NE) z,tg,2°,28,a°,35, gamma-aminobutyric acid (GABA)3,17,av, and acetylcholine (ACh) 16,21 function as neurotransmitters in neocortex. The technique of microionto-
230 phoresis has permitted electrophysiological measurement of potential postsynaptic effects of these agents. However, utilization of this technique has been limited in at least two major respects. First, it has typically been used in anesthetized, curarized, or brain stem-transected animals. Only two brief reports13, 45 describe the effects of iontophoretically applied agents on neocortical cells in unanesthetized, unparalyzed animals. Second, iontophoresis has generally been used to measure the effects of these agents on 'spontaneous' discharge activity, or activity evoked by unphysiologic electrical or chemical stimulationS-S,12,1s,2a-~5,28,3~,32,~8. Two previous reports '~'2,40 provide only limited descriptions of the effects of these chemicals on specific, patterned neocortical cellular activity evoked by appropriate sensory stimuli. The present report differs from earlier studies in that it describes the effects of iontophoretically applied putative transmitters on physiologic, stimulus-evoked neuronal activity in the unanesthetized, unparalyzed animal. Five-barrel micropipettes were used to record, extracellularly, the discharge activity of single neurons in the auditory cortex of the awake squirrel monkey. The cells were acoustically activated with various vocalizations of this species 43. NE, GABA, or ACh was then applied iontophoretically to the cell, and changes in 'spontaneous' and vocalization-evoked activity were studied. METHODS
Surgery Five 'Roman' and two 'Gothic 'z6 adult, male squirrel monkeys (Saimiri seiureus) were prepared for recording. Aseptic surgery was performed under Halothane anesthesia. A plastic disk, later used to immobilize the animal's head, was rigidly attached to the top of the skull. At the recording site on the superior temporal gyrus, a bone flap 3 mm × 7 mm was removed. The hole was centered at AS, 15 mm above the inter-aural line and was placed so that the antero-dorsal edge of the opening lay over the Sylvianfissure11,14, zg. The dura was excised and the exposed brain covered with 4 ~o agar. A plastic window with a removable cover was cemented over the skull hole. Animals were allowed to recover for at least 2 days before the first recording session. Usually, the recording window remained usable for 2-3 weeks (4-10 recording sessions) before a maximum of 20 penetrations had been made, infection occurred, or scar tissue formed in the window. Subsequent histological examination confirmed the position of the windows with respect to cortical landmarks.
Micropipettes and chemicals Five-barrel micropipettes with 4-6 #m tips were used to record extracellular action potentials and to iontophorese NE, GABA and ACh. Electrode preparation is described in detail elsewhere a4. The center barrel, filled with 5 M NaCl, was used for recording. Three side barrels were filled by centrifugation with: L-NE (Aldrich), 0.5 M, pH 4.5; GABA (Sigma), I M, pH 4.0; ACh chloride (Calbiochem), 2.5 M, pH 3.8. Chemicals were ejected as cations by currents in the nanoamp (nA) range or retained by currents of opposite polarity. The remaining barrel, containing 3 M NaC1,
231 was used to continuously neutralize tip potential through an automatic balancing circuit.
Stimuli Squirrel monkey vocalizations, recorded and formated on magnetic tape by the method of Newman and Wollberg 29, served as stimuli. Successive presentations of the same vocalization occurred at 5-sec intervals. A 'Microstatic' speaker delivered stimuli into a recording chamber within a sound-insulated r o o m at 80 (4- 10) dB SPL. The overall bandwidth of the stimulus recording and presentation system was 1-15 kHz.
Recording and data collection The animal was strapped into a contoured chair, and his head was restrained by bolting the plastic disk to a surrounding framework. The coxrer over the recording site was removed and the agar replaced. Action potentials were monitored by conventional methods. All action potentials were initially negative, at least twice the noise level in amplitude, and stable in waveform, Fig. 1 shows a typical recording o f neuronal activity. An amplitude-sensitive discriminator converted each action potential to a standardized pulse which was relayed to a PDP-12 computer. Responses to a particular vocalization were determined by presenting that vocalization 16 times and computing, on-line, a dot display and peristimulus time histogram (PSTH) with a 3-msec bin width (see Fig. 2). When it had been determined that a cell responded to a vocalization, iontophoresis was begun. When the chemical had produced a stable effect on the rate of spontaneous discharge activity, usually l--2 min later, the stimulus presentation and data collection procedure was repeated. Iontophoresis currents that altered discharge rates by about 50 % of control levels (5-80 nA) were utilized. Post-iontophoresis control histograms were also computed.
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Fig. 2. A: responses of a neuron to repeated presentations of an 'isolation peep' before (left), during (center), and after (right) the ejection of N E with 5 nA of current. A horizontal sweep of the dot display was begun 200 msec before each of the 16 presentations of the vocalization which is represented in the bottom trace. Each display sweep lasted 1.5 sec. Each action potential generated a dot and added a count to the appropriate histogram bin. The histogram thus shows the summed activity for all trials. The histogram bin width is 3 msec, and each graduation on the ordiuate represents one count. Note that NE inhibited discharge activity both before and during the vocalization. Pre-vocalization activity (PVA) was reduced by 68 ~ of control while vocalization-evoked activity (VA) was reduced by 41 ~ . B: complex response of another neuron to the 'isolation peep' before, during, and after the ejection of 10 nA of GABA. PVA was reduced by 67 ~ while VA was reduced by 36 ~ ,
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233 RESULTS
In agreement with previous reports z9,44, we found that over 80 ~ of the neurons tested responded to one or more vocalizations. Thirty-four responsive cells were analyzed with at least one vocalization and one chemical. Most cells were studied with repeated tests for 1-3 h. Typically, a given neuron produced similar responses to successive presentations of a particular vocalization, but responses of different strength, latency, and pattern were obtained upon presentation of a different vocalization. Figs. 2 and 3 show various response patterns before, during, and after iontophoresis. Effects of chemicals on discharge rates The effects of the putative transmitters on spontaneous or pre-vocalization activity (PVA) and vocalization-evoked activity (VA) were quantified. We defined PVA as the total number of counts in a 16-trial histogram during the 200-msec interval prior to stimulus onset and VA as total counts during the following 1100 msec (see Fig. 3). Vocalizations lasted 300-950 msec, and the 1100-msec interval was chosen to include all observed neuronal responses. To determine whether an agent reduced or enhanced either PVA or VA in a selective fashion, the percentage change in each was calculated for each cell, for each agent tested. To assess statistical significance for the entire sample of cells, the difference for each cell between percentage PVA change and percentage VA change was computed and used as a score in a two-tailed t-test for correlated means. Since a given neuron was often studied for a period of hours, with several applications of different chemicals,we compared control histograms computed throughout this period to determine whether the two types of activity changed over time. Although discharge rates fluctuated slightly over the recording period, PVA and VA varied by similar proportions. Changes induced in spontaneous activity, as measured by 30-sec samples collected during periods when vocalizations were not being presented, verified the general magnitude of chemical effects as measured by changes in PVA. NE. NE inhibited both PVA and VA in each of the 28 neurons tested. For most cells, 5-10 nA applied for about 1 min was sufficient to reduce discharge activity by 50 70. This inhibitory effect, and subsequent recovery, was obtained consistently despite the wide variety of responses to vocalizations. Greater reduction of PVA was evident in 21 of the neurons and was statistically significant for the 28-cei1 sample (P < 0.002, two-tailed t-test). The NE-induced inhibition illustrated in Fig. 2A was typical. Differential inhibition was most pronounced for neurons which exhibited vigorous excitatory vocalization responses. The results for the 15 most extensively tested cells with such responses are shown in Fig. 4. A separate analysis of these 15 cells indicated that NE application enhanced the ratio of VA rate to PVA rate by a factor of 1.8. GABA. Each of the 19 cells tested with GABA was inhibited, usually to 5070 of control rates, with 5-10 nA ejection currents. GABA acted more rapidly than NE, and recovery occurred within about 1 rain. PVA was inhibited by a greater proportion
234
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Fig. 4. Percent inhibition of PVA and VA by NE in 15 cells with excitatory vocalization responses. Each dot represents one test of one cell. Note that all but one of the dots fall below the 45° line demonstrating that PVA was inhibited by a greater percentage than VA. The insets at the right are plotted in the same fashion as the large graph and represent tests of 4 neurons (A-D) with varying doses of NE. At each dose VA was more resistant than PVA to NE inhibition. The histograms at the upper left show a cell with a typical sustained excitatory response. NE (60 nA) reduced PVA by 86 ~ and VArby 71 ~. The arrow indicates the dot representing this test. than VA (17 of 19 cells, P < 0.002, two-tailed t-test). Fig. 2B shows a typical case: PVA was reduced by 67 ~ while VA was reduced by 36 ~ , and the VA rate/PVA rate ratio was increased by a factor of 2.0. This later figure equalled the mean ratio increase for the entire sample. ACh. Of the 7 cells tested with ACh, 6 were excited to 1.3-2.0 times their original discharge rate by 5-10 nA applied for 1-2 min (see Fig. 3). PVA and VA were not differentially enhanced (P > 0.1, two-tailed t-test). Other cells exhibited enhanced discharge rates during ACh ejection (20-60 hA), but action potential amplitude was concomitantly decreased to the extent that reliable discrimination was not possible.
Effects of chemicals on segments of vocalization responses The next analysis was designed to determine whether those portions of VA characterized by high discharge rates responded differently to a given agent than did portions characterized by low discharge rates. We divided the 1-sec interval following the onset of the vocalization into 7 144-msec segments and determined the change iontophoresis induced in each in terms of the absolute number of counts added o r subtracted. We also computed the proportion of control segment counts this difference represented (see Fig. 5). For each iontophoresis test we then calculated correlation coefficients (r) between control segment counts and (I) the absolute change during iontophoresis for each segment, and (2) the proportionel change for each segment. The statistical significance of the r scores for the entire sample was assessed by transforming them into Z scores 15 which were used in a two-tailed t-test.
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Fig. 5. Example of the histogram segment analysis for one cell showing responses to the 'trill' vocalization before (top) and during the ejection of 5 nA of NE. The histograms and the bar graph below are divided into seven 144-msec segments during VA. Within each of these segments in the graph there are two bars, one (white) whose height indicates the number of counts 'subtracted' from that segment by NE application and one (black) indicating the proportional reduction for the same segment. The bar graph demonstrates that the VA segments of most intense neuronal activity (i.e., segments 1 and 2) had the largest absolute reductions and the smallest proportional reductions with NE application. The NE-induced decrease in the number of counts in a segment (white bars) was positively correlated with the number of counts in that segment in the control histogram (r = 0.96). Proportional Change in each segment (black bars) was inversely correlated with the number of counts in a segment (r = ---0.77). The vertical lines during the PVA interval show the proportional (solid) and absolute (broken) changes for the first 144 msec of this period. The PSTH display shows that this interval contained fewer counts than any VA segment, and the graph indicates that PVA was reduced by the largest proportion and by the second smallest absolute number. F o r this m o r e detailed analysis we selected the n e u r o n s we h a d tested m o s t extensively, which h a d exhibited a stable degree o f i o n t o p h o r e t i c i n h i b i t i o n o r excit a t i o n to a fixed ejection current, a n d which h a d e x h i b i t e d consistent responses to the test vocalization. N E . H i s t o g r a m segments exhibiting the m o s t intense activity were r e d u c e d b y the largest n u m b e r o f counts when N E was applied. F o r each o f the 20 cells tested with N E there was a positive c o r r e l a t i o n between the n u m b e r o f c o u n t s in a c o n t r o l h i s t o g r a m segment and the n u m b e r o f c o u n t s ' s u b t r a c t e d ' f r o m t h a t segment b y the a p p l i c a t i o n o f N E ( m e d i a n r value = 0.88, range = 0.40--0.99, P < 0.002). F o r e x a m ple, the r value for the cell s h o w n in Fig. 2 A was 0.92 a n d for the cell s h o w n in Fig. 5 was 0.96. T h e large r values o b t a i n e d d i d n o t result f r o m the i m p o s s i b i l i t y o f the segm e n t s c o n t a i n i n g few c o u n t s d e c r e a s i n g b e l o w 0 a n d thus h a v i n g a n artificially low difference score. L a r g e r values were o b t a i n e d for h i s t o g r a m p a i r s where even ind i v i d u a l N E h i s t o g r a m segments h a d m a n y counts. F u r t h e r m o r e , in h i s t o g r a m s c o n t a i n i n g few counts in s o m e segments, results were s i m i l a r w h e t h e r o r n o t these segments were i n c l u d e d in the analysis. T h e values r e p o r t e d here include o n l y segments which h a d one o r m o r e c o u n t s d u r i n g the a p p l i c a t i o n o f N E . A l t h o u g h r e s p o n s e pea ks lost the largest n u m b e r o f c o u n t s when N E was a p p l i e d ,
236 proportionately they were least affected. For each of the 15 cells with a predominantly excitatory vocalization response, there was a negative relationship between the number of spikes in a control segment and the percent inhibition of activity in that segment (median r =- --0.42, range -: --0.04 to --0.90, P < 0.002). For example, the cell shown in Fig. 2A yielded an r value o f - - 0 . 8 3 while the cell shown in Fig. 5 attained a value o f - - 0 . 7 7 . The results obtained with the 5 predominantly inhibitory cells indicate a complementary process: there is a tendency for periods of more intense activity to be reduced by a larger proportion (median r = 0.54, range -= 0.500.85, P 0.8). However, each of the neurons exhibited a negative r value when segment rate was correlated with the percentage rate change induced in that segment (median r value ~= --0.65, range = --0.14 to - - 0.88, P < 0.02). Thus, as with NE and GABA, ACh produced its most profound proportional changes in those histogram segments characterized by low rates.
Pattern changes Since many vocalization responses were composed of successive, brief excitatory or inhibitory components, an analysis of the distribution of counts within the histograms was performed to determine whether the chemicals induced significant changes in response patterns. Normalized, cumulative histograms ofiontophoretic and control responses were compared using the Kolmogorov-Smirnov (K-S) non-parametric test ~6 (see Fig. 6). We evaluated the data used for the histogram segment analysis. For the K-S test, the maximum difference between the two cumulative distributions was determined and divided by a variance term. The resulting value was compared against a probability table which indicated the probability of obtaining a difference that large by chance. NE. In most cases, N E did not produce a significant change in the distribution of counts in the response portion of the histogram. For most cells, areas of low discharge activity before and after response peaks were disproportionately depressed but not sufficiently to produce a statistically significant change. With larger doses than were typically used, however, the complete inhibition of all but response peaks sometimes led to a significant change (see Fig. 6). For the 20 cells tested (5 of which were tested twice), 18 of the NE histograms were not significantly different from control while 7 were (P 0.10) whereas NE 2 did (P < 0.025, two-tailed).
GABA. S i m i l a r results were o b t a i n e d with G A B A . O f the 16 n e u r o n s e v a l u a t e d , 11 d i d n o t exhibit statistically significant ( P > 0.05) p a t t e r n changes with doses o f G A B A t h a t r e d u c e d discharge rates b y at least 30 %. L i k e N E , G A B A p r o d u c e d s o m e significant changes because o f its d i s p r o p o r t i o n a t e i n h i b i t i o n o f slower r e s p o n s e segments (see Fig. 7). ACh. O n l y one o f the 6 cells e v a l u a t e d achieved statistical significance ( P < 0.05) on the K - S test. W e next c o m p a r e d v o c a l i z a t i o n responses o b t a i n e d while i o n t o p h o r e s i n g one c h e m i c a l with those o b t a i n e d while i o n t o p h o r e s i n g a n o t h e r chemical. W h e n N E responses were c o m p a r e d with G A B A responses for the s a m e cell a n d v o c a l i z a t i o n , no significant difference was o b t a i n e d in 8 o f 11 cases (see Fig. 8). C o m p a r i s o n o f A C h a n d G A B A tests s h o w e d no statistically significant difference for 4 o f 5 cells. F i v e o f the 6 n e u r o n s tested with A C h a n d N E r e s p o n d e d differently ( P < 0.05) to the two
238 CUMULATIVE %
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Fig. 7. The same neuron and vocalization as in Fig. 6. The PSTH displays at the left were computed before (top), during (center), and after (bottom) the application of 5 nA GABA. During GABA ejection, pre- and post-vocalization activity were reduced by 59 ~ and 69 % respectively. However, activity during the vocalization was reduced by only 7 %. The cumulative histograms to the right illustrate the similarity of the pre- and post-iontophoresis controls, which did not differ significantly (P > 0.10). The steeper slope of the GABA distribution resulted from the increased percentage of counts in the response peak. The GABA histogram differed from both controls (P < 0.001, pre and 0.01, post), and the point of maximum difference, indicated by the arrow, was similar for each comparison. Note the similarity of the control and NE 1 displays in Fig. 6 to the control and GABA displays, respectively, in this figure. These similarities illustrate the stability of control responses and the similarity of NE and GABA effects.
chemicals. This resulted from the opposite effects of the chemicals on slower segments of responses. DISCUSSION
Numerous studies4,1°, 4z of various cortical regions indicate that neocortical neuronal discharge activity is precisely determined by changes in the animal's sensory environment and by motor activity. Therefore, specific, stimulus-evoked activity was used to assay the effects of putative cortical transmitters in this study. The auditory cortex on the surface of the superior temporal gyrus is a secondary sensory area zl,27 in which a large proportion of neurons respond to one or more calls of the species TM 29,42,44. These previously described vocalization responses are usually complex and frequently cannot be predicted from the response characteristics evident upon testing
239 CUMULATIVE ~0
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Fig. 8. Responses of the same cell as shown in Figs. 6 and 7 when tested with the 'err-chuck' vocalization. The PSTH displays at the left were computed during a control period (top) and during the ejection of GABA (5 nA, center) and NE (5 nA, bottom). Pre- and post-iontophoresis PSTHs were collected for each test. Control histograms did not differ from one another (P > 0.10) when evaluated with the K-S test. Each iontophoresis histogram differed from control (GABA, P < 0,025; NE, P < 0.05), and the point of maximum difference between the cumulative distributions (shown by the arrows) was similar in each case. The NE and GABA histograms did not differ (P > 0.10). During NE ejection, the response peaks were inhibited by 46% of control activity levels while the surrounding areas of low activity were inhibited by 95 %. For GABA these figures were 15 ~ and 76 ~ . This relative resistance of the response peaks to inhibition is manifested in the cumulative histograms as sharpened inflection points and steeper slopes during the response peaks.
the neuron with simple stimuli~9, 42. These findings suggest that vocalizations initiate unique, complex activity patterns in auditory neocortical neurons, providing a possible neuronal substrate for the specific alterations of behavior caused by certain vocalizations in the wildg, sg. The unanesthetized animal was utilized in this study to allow this vocalization-evoked activity to attain physiological levels. In previous studies in unanesthetized squirrel monkeys~9,44, over 80 ~o of neurons in this area were found to be responsive to vocalizations, whereas in animals receiving minimal doses of barbiturate this figure was reduced to 41 ~14. There is also evidence that anesthesia depresses spontaneous activity and alters auditory responses in the medial geniculate body of cats 1. Thus, the vocalizations served not only to identify the cells as auditory neurons but also to activate them in a behaviorally relevant, entirely physiological manner so that iontophoretic effects on the presumed functional activities of the cells could be studied.
240 Application of NE or GABA inhibited the spontaneous discharge of all cells tested, a finding in substantial agreement with other iontophoretic studies of neocortical neurons in anesthetized rat3S; anesthetized6,8,2~-23,25, 40, cerveau isoh; 2'",23, and awake 13 cat, and anesthetized squirrel monkey eS. As in these other preparations, NE inhibition had a longer latency and duration than GABA inhibition. PVA, which was almost always slower than evoked activity, was inhibited proportionately more than VA during NE or GABA application. One possible role of such an effect might be to enhance VA/PVA ratios in the output of these cells. Examination of excitatory responses showed that although response peaks lost a larger number of counts than other poststimulus segments, proportionately they were less inhibited. Although there was a statistically significant tendency toward a negative linear relationship between rate and percent inhibition, the data were too variable to exclude the possibility that this relationship was systematic but non-linear. This variability and limited sample size also precluded a statistically appropriate, direct comparison of the percent inhibition of PVA with VA segments of the same control discharge rate to determine whether rate p e r se was adequate to predict NE or GABA effects, or whether PVA was more strongly inhibited, independent of rate. Thus, for excitatory responses both averaged poststimulus activity and, in particular, specific peaks of excitation are inhibited proportionally less by NE or GABA than is spontaneous activity. The results of the K-S analysis suggest that response latency and the pattern of excitatory peaks and inhibitory troughs in the poststimulus histogram did not substantially change with NE, GABA, or ACh administration. The significant changes that were observed can be most parsimoniously explained as resulting from the transmitters' marked effects on slower portions of the response. Thus, although putative transmitter effects were assessed with a complex neuronal response, only relatively simple, rate-dependent effects were observed. These results may reflect limitations of the iontophoretic technique as used here. Although iontophoretic application of NE is an adequate model for evaluating some of the postsynaptic effects of this putative transmitter, these experiments could not fully mimic synaptic NE inhibition. First, since the presumed NE synapses in auditory cortex may originate in cells which project widely over neocortex 28 as well as to otber areas of the brain, activation of NE cells may affect auditory neurons not only directly, but also indirectly, lontophoresis thus mimics the monosynaptic effects of NE while artificially excluding polysynaptic NE influences which may normally accompany them. Second, the ejection of NE over 3-5-min periods, while it may mimic tonic NE inhibition, is not an adequate model of rapid, phasic NE release. Third, the NE doses used here were chosen to produce readily observable decreases in neuronal activity and may not reflect physiological levels. Despite these limitations, it is cle3r that application of NE with small ejection currents is capable of substantially altering, in a consistent fashion, the presumed functional activities of these neurons. In the analyses detailed above, no significant differences between the effects of GABA and NE were found. We did observe a shorter latency of onset and shorter duration of effect for GABA. Although this was possibly due to differences in the iontophoretic release of the two agents, it may reflect differences in the functions of
241 these p u t a t i v e t r a n s m i t t e r s . S y n a p t i c G A B A release m a y cause the b r i e f i n h i b i t o r y pauses o b s e r v e d in a u d i t o r y responses. Such pauses a p p e a r to result f r o m active inh i b i t i o n 88. N E m a y p r o v i d e a tonic, low-level i n h i b i t i o n a c c o u n t i n g for p r e v i o u s l y o b s e r v e d s p o n t a n e o u s I P S P s aa a n d the resulting low s p o n t a n e o u s activity levels in these ceils. Such t o n i c i n h i b i t i o n , as shown in this study, w o u l d e n h a n c e the difference between b a c k g r o u n d a n d s t i m u l u s - b o u n d activity. ACKNOWLEDGEMENTS T h e a u t h o r s wish to a c k n o w l e d g e the generous assistance o f J o h n D. N e w m a n who p r o v i d e d c o m p u t e r p r o g r a m s , v o c a l i z a t i o n tapes, a n d i m p o r t a n t technical suggestions. D a v i d S y m m e s wrote a n d s u p p l i e d c o m p u t e r p r o g r a m s . B. Hoffer m a d e helpful technical suggestions. E. S t r a u g h n p r o v i d e d technical assistance. M. Segal, F. E. B l o o m , J. D. N e w m a n , a n d D, S y m m e s m a d e helpful suggestions c o n c e r n i n g the m a n u s c r i p t . J. Peay c o n t r i b u t e d e d i t o r i a l a n d clerical assistance.
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242 18 JOHNSON, E. S., ROBERTS, M. H. T., SOBIESZEK,A., AND STRAUGHAN, D. W., Noradrenaline sensitive cells in cat cerebral cortex, Int. J. Neuropharmacol., 8 (1969) 549-566. 19 KORF, J., AGHAJANIAN, G. K., AND ROTH, R. H., Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of the locus coeruleus, Neuropharmacology, 12 (1973) 933-938, 20 KORE, J., ROTH, R. H., AND AGHAJANIAN, G. K., Alterations in turnover and endogenous levels of norepinephrine in the cerebral cortex following electrical stimulation and acute axotomy of cerebral noradrenergic pathways, Europ. J. Pharmacol., 23 (1973) 276-282. 21 KRNJEVI(:'~.,K., Microiontophoretic studies on cortical neurons, hzt. Rev. Neurobiol., 7 (1964) 41 98. 22 KRNJEVIC, K., AND PH1LLIS, J. W., Actions of certain amines on cerebral cortical neurones, Brit. J. Pharmacol., 20 (1963) 471~90. 23 KRNJEVI~, K., AND PHILLIS, J. W., lontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol. (Lond.), 165 (1963) 274-304, 24 KRNJEVI6, K., PUMAIN, R., AND RENAOD, L., The mechanism of excitation by acetylcholine in the cerebral cortex, J. PhysioL (Lond.), 215 (1971) 247-268. 25 KRNJEVlt~, K., AND SCHWARTZ, S., The action of gamma-aminobutyric acid on cortical neurones, Exp. Brain Res., 3 (1967) 320-336. 26 MACLEAN, P. D., Mirror display in the squirrel monkey, Saimiri sciureus, Science, 146 (1964) 950-952. 27 MESULAM, M.-M., AND PANDVA, D. N., The projections of the medial geniculate complex within the sylvian fissure of the rhesus monkey, Brain Research, 60 (1973) 315-333. 28 NELSON, C, N., HOFFER, B. J., CHU, N.-S., AND BLOOM, F. E., Cytochemical and pharmacological studies on polysensory neurons in the primate frontal cortex, Brain Research, 62 (1973) 115-133. 29 NEWMAN, J. D., AND WOLLBERG, Z., Multiple coding of species-specific vocalizations in the auditory cortex of squirrel monkeys, Brain Research, 54 (1973) 287-304. 30 NVSTRt3M, B., OLSON, L., AND UNGERSTEDT, U,, Noradrenaline nerve terminals in human cerebral cortices: first histochemical evidence, Science, ! 76 (1972) 924-926. 31 PHILLIS, J. W., AND YORK, D. H., Cholinergic inhibition in the cerebral cortex, Brain Research, 5 (1967) 517-520. 32 RANDIC, M., SIMINOFF, R., AND STRAUGHAN, D. W., Acetylcholine depression of cortical neurons, Exp. Neurol., 9 (1964) 236-242. 33 RIBAUPIERBE, F. DE, GOLDSTEIN, M. H., JR., AND YENI-KOMSHIAN, G., lntracetlular study of the cat's primary auditory cortex, Brain Research, 48 (1972) 185-204. 34 SALMOIRAGHI, G. C., AND WEIGHT, F., Micromethods in neuropharmacology, Anesthesiology, 28 (1967) 54-64. 35 SEGAL, M., PICKEL, V., AND BLOOM, F., The projections of the nucleus locus coeruleus: an autoradiographic study, Life Sci., 13 (1973) 817-821, 36 SIEGEL, S., Nonparametric Statistics, McGraw-Hill, New York, 1956, 127 pp. 37 SNODGRASS,S. R., HEDLEY-WHYTE,E. T., AND LORENZO, A. V., GABA transport by nerve ending fractions of cat brain, J. Neurochem., 20 (1973) 771-782. 38 STONE, T. W., Pharmacology of pyramidal tract cells in the cerebral cortex: noradrenaline and related substances, Naunyn-Sehmiedeberg's Arch. exp. Path. Pharmak., 278 (1973) 333-346. 39 THORINGTON, R. W., Observations of squirrel monkeys in a Colombian forest. In L. ROSENBLUM AND R. COOPER (Eds.), The Squirrel Monkey, Academic Press, New York, 1968, pp. 69-85. 40 WALLINGEORD,E., OSTDAHL,R., ZARZECKI,P., KAUFMAN,P., AND SOMJEN,G., Optical and pharmacological stimulation of visual cortical neurones, Nature New Biol., 242 (1973) 210-212. 41 WERNER, G., AND WH1TSEL, B. L., Functional organization of the somatosensory cortex. In A. IGGO (Ed.), Handbook of Sensory Physiology, Vol. 11, Springer, New York, 1973, pp. 621-700. 42 WINTER,P., AND FUNKENSTEIN,H. H., The effect of species-specific vocalization on the discharge of auditory cortical cells in the awake squirrel monkey (Saimiri sciureus), Exp. Brain Res., 18 (1973) 489-504. 43 WINTER, P., PLOOG, D., AND LATTA, J., Vocal repertoire of the squirrel monkey (Sairnirisciureus), its analysis and significance, Exp. Brain Res., 1 (1966) 359-384. 44 WOLLBERG, Z., AND NEWMAN, J., Auditory cortex of squirrel monkey: response patterns of single cells to species-specific vocalizations, Science, 175 (1972) 212-214. 45 WOODY,C. D., CARPENTER,D., KNISPEL, J. D., CROW, T., AND BLACK-CLEWORTH,P., Prolonged increases in resistance of neurons in cat motor cortex following extracellular iontophoretic application of acetylcholine (ACh) and intracellular current injection, Fed. Proc., 33 (1974) 399.
229
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECTS OF PUTATIVE NEUROTRANSMITTERS ON NEURONAL ACTIVITY IN MONKEY AUDITORY CORTEX
STEPHEN L. FOOTE, R O B E R T F R E E D M A N AND A. P A U L OLIVER
Laboratory of Neuropharmacology, Division of Special Mental Health Research, National Institute o f Mental Health, St. Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.) (Accepted November 6th, 1974)
SUMMARY
The effects of the putative neurotransmitters norepinephrine (NE), gammaaminobutyric acid (GABA), and acetylcholine (ACh) were tested on auditory cortex neurons which were activated acoustically by species-specific vocalizations in awake squirrel monkeys. Five-barrel glass electrodes were used to record the activity of single neurons in the superior temporal gyrus and to apply NE, GABA, or ACh microiontophoretically. Poststimulus time histograms and raster displays of neuronal responses to the vocalizations were computed before, during, and after iontophoresis. Dose-dependent inhibition of spontaneous and vocalization-evoked discharge rates was seen with NE and GABA. Generally, excitation was observed with ACh. A given dose of NE or GABA reduced spontaneous activity by a greater proportion than it reduced activity evoked by the vocalizations. During excitatory responses, segments with lower discharge rates were reduced proportionately more than segments with higher discharge rates. Usually, response 'pattern' was not altered by iontophoresis of any of the substances. However, in some cases the differential inhibition of slow activity produced by NE or GABA did result in a 'pattern' change. The demonstration that small amounts of locally applied NE and GABA substantially alter the specific neuronal activation produced by vocalizations provides additional evidence that these agents may function as neurotransmitters in this neocortical area and offers clues about their functional significance.
I NTRODUCTION
Substantial anatomical and biochemical evidence suggests that norepinephrine (NE) z,tg,2°,28,a°,35, gamma-aminobutyric acid (GABA)3,17,av, and acetylcholine (ACh) 16,21 function as neurotransmitters in neocortex. The technique of microionto-
230 phoresis has permitted electrophysiological measurement of potential postsynaptic effects of these agents. However, utilization of this technique has been limited in at least two major respects. First, it has typically been used in anesthetized, curarized, or brain stem-transected animals. Only two brief reports13, 45 describe the effects of iontophoretically applied agents on neocortical cells in unanesthetized, unparalyzed animals. Second, iontophoresis has generally been used to measure the effects of these agents on 'spontaneous' discharge activity, or activity evoked by unphysiologic electrical or chemical stimulationS-S,12,1s,2a-~5,28,3~,32,~8. Two previous reports '~'2,40 provide only limited descriptions of the effects of these chemicals on specific, patterned neocortical cellular activity evoked by appropriate sensory stimuli. The present report differs from earlier studies in that it describes the effects of iontophoretically applied putative transmitters on physiologic, stimulus-evoked neuronal activity in the unanesthetized, unparalyzed animal. Five-barrel micropipettes were used to record, extracellularly, the discharge activity of single neurons in the auditory cortex of the awake squirrel monkey. The cells were acoustically activated with various vocalizations of this species 43. NE, GABA, or ACh was then applied iontophoretically to the cell, and changes in 'spontaneous' and vocalization-evoked activity were studied. METHODS
Surgery Five 'Roman' and two 'Gothic 'z6 adult, male squirrel monkeys (Saimiri seiureus) were prepared for recording. Aseptic surgery was performed under Halothane anesthesia. A plastic disk, later used to immobilize the animal's head, was rigidly attached to the top of the skull. At the recording site on the superior temporal gyrus, a bone flap 3 mm × 7 mm was removed. The hole was centered at AS, 15 mm above the inter-aural line and was placed so that the antero-dorsal edge of the opening lay over the Sylvianfissure11,14, zg. The dura was excised and the exposed brain covered with 4 ~o agar. A plastic window with a removable cover was cemented over the skull hole. Animals were allowed to recover for at least 2 days before the first recording session. Usually, the recording window remained usable for 2-3 weeks (4-10 recording sessions) before a maximum of 20 penetrations had been made, infection occurred, or scar tissue formed in the window. Subsequent histological examination confirmed the position of the windows with respect to cortical landmarks.
Micropipettes and chemicals Five-barrel micropipettes with 4-6 #m tips were used to record extracellular action potentials and to iontophorese NE, GABA and ACh. Electrode preparation is described in detail elsewhere a4. The center barrel, filled with 5 M NaCl, was used for recording. Three side barrels were filled by centrifugation with: L-NE (Aldrich), 0.5 M, pH 4.5; GABA (Sigma), I M, pH 4.0; ACh chloride (Calbiochem), 2.5 M, pH 3.8. Chemicals were ejected as cations by currents in the nanoamp (nA) range or retained by currents of opposite polarity. The remaining barrel, containing 3 M NaC1,
231 was used to continuously neutralize tip potential through an automatic balancing circuit.
Stimuli Squirrel monkey vocalizations, recorded and formated on magnetic tape by the method of Newman and Wollberg 29, served as stimuli. Successive presentations of the same vocalization occurred at 5-sec intervals. A 'Microstatic' speaker delivered stimuli into a recording chamber within a sound-insulated r o o m at 80 (4- 10) dB SPL. The overall bandwidth of the stimulus recording and presentation system was 1-15 kHz.
Recording and data collection The animal was strapped into a contoured chair, and his head was restrained by bolting the plastic disk to a surrounding framework. The coxrer over the recording site was removed and the agar replaced. Action potentials were monitored by conventional methods. All action potentials were initially negative, at least twice the noise level in amplitude, and stable in waveform, Fig. 1 shows a typical recording o f neuronal activity. An amplitude-sensitive discriminator converted each action potential to a standardized pulse which was relayed to a PDP-12 computer. Responses to a particular vocalization were determined by presenting that vocalization 16 times and computing, on-line, a dot display and peristimulus time histogram (PSTH) with a 3-msec bin width (see Fig. 2). When it had been determined that a cell responded to a vocalization, iontophoresis was begun. When the chemical had produced a stable effect on the rate of spontaneous discharge activity, usually l--2 min later, the stimulus presentation and data collection procedure was repeated. Iontophoresis currents that altered discharge rates by about 50 % of control levels (5-80 nA) were utilized. Post-iontophoresis control histograms were also computed.
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233 RESULTS
In agreement with previous reports z9,44, we found that over 80 ~ of the neurons tested responded to one or more vocalizations. Thirty-four responsive cells were analyzed with at least one vocalization and one chemical. Most cells were studied with repeated tests for 1-3 h. Typically, a given neuron produced similar responses to successive presentations of a particular vocalization, but responses of different strength, latency, and pattern were obtained upon presentation of a different vocalization. Figs. 2 and 3 show various response patterns before, during, and after iontophoresis. Effects of chemicals on discharge rates The effects of the putative transmitters on spontaneous or pre-vocalization activity (PVA) and vocalization-evoked activity (VA) were quantified. We defined PVA as the total number of counts in a 16-trial histogram during the 200-msec interval prior to stimulus onset and VA as total counts during the following 1100 msec (see Fig. 3). Vocalizations lasted 300-950 msec, and the 1100-msec interval was chosen to include all observed neuronal responses. To determine whether an agent reduced or enhanced either PVA or VA in a selective fashion, the percentage change in each was calculated for each cell, for each agent tested. To assess statistical significance for the entire sample of cells, the difference for each cell between percentage PVA change and percentage VA change was computed and used as a score in a two-tailed t-test for correlated means. Since a given neuron was often studied for a period of hours, with several applications of different chemicals,we compared control histograms computed throughout this period to determine whether the two types of activity changed over time. Although discharge rates fluctuated slightly over the recording period, PVA and VA varied by similar proportions. Changes induced in spontaneous activity, as measured by 30-sec samples collected during periods when vocalizations were not being presented, verified the general magnitude of chemical effects as measured by changes in PVA. NE. NE inhibited both PVA and VA in each of the 28 neurons tested. For most cells, 5-10 nA applied for about 1 min was sufficient to reduce discharge activity by 50 70. This inhibitory effect, and subsequent recovery, was obtained consistently despite the wide variety of responses to vocalizations. Greater reduction of PVA was evident in 21 of the neurons and was statistically significant for the 28-cei1 sample (P < 0.002, two-tailed t-test). The NE-induced inhibition illustrated in Fig. 2A was typical. Differential inhibition was most pronounced for neurons which exhibited vigorous excitatory vocalization responses. The results for the 15 most extensively tested cells with such responses are shown in Fig. 4. A separate analysis of these 15 cells indicated that NE application enhanced the ratio of VA rate to PVA rate by a factor of 1.8. GABA. Each of the 19 cells tested with GABA was inhibited, usually to 5070 of control rates, with 5-10 nA ejection currents. GABA acted more rapidly than NE, and recovery occurred within about 1 rain. PVA was inhibited by a greater proportion
234
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Fig. 4. Percent inhibition of PVA and VA by NE in 15 cells with excitatory vocalization responses. Each dot represents one test of one cell. Note that all but one of the dots fall below the 45° line demonstrating that PVA was inhibited by a greater percentage than VA. The insets at the right are plotted in the same fashion as the large graph and represent tests of 4 neurons (A-D) with varying doses of NE. At each dose VA was more resistant than PVA to NE inhibition. The histograms at the upper left show a cell with a typical sustained excitatory response. NE (60 nA) reduced PVA by 86 ~ and VArby 71 ~. The arrow indicates the dot representing this test. than VA (17 of 19 cells, P < 0.002, two-tailed t-test). Fig. 2B shows a typical case: PVA was reduced by 67 ~ while VA was reduced by 36 ~ , and the VA rate/PVA rate ratio was increased by a factor of 2.0. This later figure equalled the mean ratio increase for the entire sample. ACh. Of the 7 cells tested with ACh, 6 were excited to 1.3-2.0 times their original discharge rate by 5-10 nA applied for 1-2 min (see Fig. 3). PVA and VA were not differentially enhanced (P > 0.1, two-tailed t-test). Other cells exhibited enhanced discharge rates during ACh ejection (20-60 hA), but action potential amplitude was concomitantly decreased to the extent that reliable discrimination was not possible.
Effects of chemicals on segments of vocalization responses The next analysis was designed to determine whether those portions of VA characterized by high discharge rates responded differently to a given agent than did portions characterized by low discharge rates. We divided the 1-sec interval following the onset of the vocalization into 7 144-msec segments and determined the change iontophoresis induced in each in terms of the absolute number of counts added o r subtracted. We also computed the proportion of control segment counts this difference represented (see Fig. 5). For each iontophoresis test we then calculated correlation coefficients (r) between control segment counts and (I) the absolute change during iontophoresis for each segment, and (2) the proportionel change for each segment. The statistical significance of the r scores for the entire sample was assessed by transforming them into Z scores 15 which were used in a two-tailed t-test.
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7
144rnset segments
Fig. 5. Example of the histogram segment analysis for one cell showing responses to the 'trill' vocalization before (top) and during the ejection of 5 nA of NE. The histograms and the bar graph below are divided into seven 144-msec segments during VA. Within each of these segments in the graph there are two bars, one (white) whose height indicates the number of counts 'subtracted' from that segment by NE application and one (black) indicating the proportional reduction for the same segment. The bar graph demonstrates that the VA segments of most intense neuronal activity (i.e., segments 1 and 2) had the largest absolute reductions and the smallest proportional reductions with NE application. The NE-induced decrease in the number of counts in a segment (white bars) was positively correlated with the number of counts in that segment in the control histogram (r = 0.96). Proportional Change in each segment (black bars) was inversely correlated with the number of counts in a segment (r = ---0.77). The vertical lines during the PVA interval show the proportional (solid) and absolute (broken) changes for the first 144 msec of this period. The PSTH display shows that this interval contained fewer counts than any VA segment, and the graph indicates that PVA was reduced by the largest proportion and by the second smallest absolute number. F o r this m o r e detailed analysis we selected the n e u r o n s we h a d tested m o s t extensively, which h a d exhibited a stable degree o f i o n t o p h o r e t i c i n h i b i t i o n o r excit a t i o n to a fixed ejection current, a n d which h a d e x h i b i t e d consistent responses to the test vocalization. N E . H i s t o g r a m segments exhibiting the m o s t intense activity were r e d u c e d b y the largest n u m b e r o f counts when N E was applied. F o r each o f the 20 cells tested with N E there was a positive c o r r e l a t i o n between the n u m b e r o f c o u n t s in a c o n t r o l h i s t o g r a m segment and the n u m b e r o f c o u n t s ' s u b t r a c t e d ' f r o m t h a t segment b y the a p p l i c a t i o n o f N E ( m e d i a n r value = 0.88, range = 0.40--0.99, P < 0.002). F o r e x a m ple, the r value for the cell s h o w n in Fig. 2 A was 0.92 a n d for the cell s h o w n in Fig. 5 was 0.96. T h e large r values o b t a i n e d d i d n o t result f r o m the i m p o s s i b i l i t y o f the segm e n t s c o n t a i n i n g few c o u n t s d e c r e a s i n g b e l o w 0 a n d thus h a v i n g a n artificially low difference score. L a r g e r values were o b t a i n e d for h i s t o g r a m p a i r s where even ind i v i d u a l N E h i s t o g r a m segments h a d m a n y counts. F u r t h e r m o r e , in h i s t o g r a m s c o n t a i n i n g few counts in s o m e segments, results were s i m i l a r w h e t h e r o r n o t these segments were i n c l u d e d in the analysis. T h e values r e p o r t e d here include o n l y segments which h a d one o r m o r e c o u n t s d u r i n g the a p p l i c a t i o n o f N E . A l t h o u g h r e s p o n s e pea ks lost the largest n u m b e r o f c o u n t s when N E was a p p l i e d ,
236 proportionately they were least affected. For each of the 15 cells with a predominantly excitatory vocalization response, there was a negative relationship between the number of spikes in a control segment and the percent inhibition of activity in that segment (median r =- --0.42, range -: --0.04 to --0.90, P < 0.002). For example, the cell shown in Fig. 2A yielded an r value o f - - 0 . 8 3 while the cell shown in Fig. 5 attained a value o f - - 0 . 7 7 . The results obtained with the 5 predominantly inhibitory cells indicate a complementary process: there is a tendency for periods of more intense activity to be reduced by a larger proportion (median r = 0.54, range -= 0.500.85, P 0.8). However, each of the neurons exhibited a negative r value when segment rate was correlated with the percentage rate change induced in that segment (median r value ~= --0.65, range = --0.14 to - - 0.88, P < 0.02). Thus, as with NE and GABA, ACh produced its most profound proportional changes in those histogram segments characterized by low rates.
Pattern changes Since many vocalization responses were composed of successive, brief excitatory or inhibitory components, an analysis of the distribution of counts within the histograms was performed to determine whether the chemicals induced significant changes in response patterns. Normalized, cumulative histograms ofiontophoretic and control responses were compared using the Kolmogorov-Smirnov (K-S) non-parametric test ~6 (see Fig. 6). We evaluated the data used for the histogram segment analysis. For the K-S test, the maximum difference between the two cumulative distributions was determined and divided by a variance term. The resulting value was compared against a probability table which indicated the probability of obtaining a difference that large by chance. NE. In most cases, N E did not produce a significant change in the distribution of counts in the response portion of the histogram. For most cells, areas of low discharge activity before and after response peaks were disproportionately depressed but not sufficiently to produce a statistically significant change. With larger doses than were typically used, however, the complete inhibition of all but response peaks sometimes led to a significant change (see Fig. 6). For the 20 cells tested (5 of which were tested twice), 18 of the NE histograms were not significantly different from control while 7 were (P 0.10) whereas NE 2 did (P < 0.025, two-tailed).
GABA. S i m i l a r results were o b t a i n e d with G A B A . O f the 16 n e u r o n s e v a l u a t e d , 11 d i d n o t exhibit statistically significant ( P > 0.05) p a t t e r n changes with doses o f G A B A t h a t r e d u c e d discharge rates b y at least 30 %. L i k e N E , G A B A p r o d u c e d s o m e significant changes because o f its d i s p r o p o r t i o n a t e i n h i b i t i o n o f slower r e s p o n s e segments (see Fig. 7). ACh. O n l y one o f the 6 cells e v a l u a t e d achieved statistical significance ( P < 0.05) on the K - S test. W e next c o m p a r e d v o c a l i z a t i o n responses o b t a i n e d while i o n t o p h o r e s i n g one c h e m i c a l with those o b t a i n e d while i o n t o p h o r e s i n g a n o t h e r chemical. W h e n N E responses were c o m p a r e d with G A B A responses for the s a m e cell a n d v o c a l i z a t i o n , no significant difference was o b t a i n e d in 8 o f 11 cases (see Fig. 8). C o m p a r i s o n o f A C h a n d G A B A tests s h o w e d no statistically significant difference for 4 o f 5 cells. F i v e o f the 6 n e u r o n s tested with A C h a n d N E r e s p o n d e d differently ( P < 0.05) to the two
238 CUMULATIVE %
f-
100
CONTROL
/// ///
50
f
. /
CONTROL
MSEC
1000
500 '
Ii 00
MSEC
Fig. 7. The same neuron and vocalization as in Fig. 6. The PSTH displays at the left were computed before (top), during (center), and after (bottom) the application of 5 nA GABA. During GABA ejection, pre- and post-vocalization activity were reduced by 59 ~ and 69 % respectively. However, activity during the vocalization was reduced by only 7 %. The cumulative histograms to the right illustrate the similarity of the pre- and post-iontophoresis controls, which did not differ significantly (P > 0.10). The steeper slope of the GABA distribution resulted from the increased percentage of counts in the response peak. The GABA histogram differed from both controls (P < 0.001, pre and 0.01, post), and the point of maximum difference, indicated by the arrow, was similar for each comparison. Note the similarity of the control and NE 1 displays in Fig. 6 to the control and GABA displays, respectively, in this figure. These similarities illustrate the stability of control responses and the similarity of NE and GABA effects.
chemicals. This resulted from the opposite effects of the chemicals on slower segments of responses. DISCUSSION
Numerous studies4,1°, 4z of various cortical regions indicate that neocortical neuronal discharge activity is precisely determined by changes in the animal's sensory environment and by motor activity. Therefore, specific, stimulus-evoked activity was used to assay the effects of putative cortical transmitters in this study. The auditory cortex on the surface of the superior temporal gyrus is a secondary sensory area zl,27 in which a large proportion of neurons respond to one or more calls of the species TM 29,42,44. These previously described vocalization responses are usually complex and frequently cannot be predicted from the response characteristics evident upon testing
239 CUMULATIVE ~0
50
" r I ~ p v '~
o
'
MSEC
,ooo
o
s~o
I~oo
MSEC
Fig. 8. Responses of the same cell as shown in Figs. 6 and 7 when tested with the 'err-chuck' vocalization. The PSTH displays at the left were computed during a control period (top) and during the ejection of GABA (5 nA, center) and NE (5 nA, bottom). Pre- and post-iontophoresis PSTHs were collected for each test. Control histograms did not differ from one another (P > 0.10) when evaluated with the K-S test. Each iontophoresis histogram differed from control (GABA, P < 0,025; NE, P < 0.05), and the point of maximum difference between the cumulative distributions (shown by the arrows) was similar in each case. The NE and GABA histograms did not differ (P > 0.10). During NE ejection, the response peaks were inhibited by 46% of control activity levels while the surrounding areas of low activity were inhibited by 95 %. For GABA these figures were 15 ~ and 76 ~ . This relative resistance of the response peaks to inhibition is manifested in the cumulative histograms as sharpened inflection points and steeper slopes during the response peaks.
the neuron with simple stimuli~9, 42. These findings suggest that vocalizations initiate unique, complex activity patterns in auditory neocortical neurons, providing a possible neuronal substrate for the specific alterations of behavior caused by certain vocalizations in the wildg, sg. The unanesthetized animal was utilized in this study to allow this vocalization-evoked activity to attain physiological levels. In previous studies in unanesthetized squirrel monkeys~9,44, over 80 ~o of neurons in this area were found to be responsive to vocalizations, whereas in animals receiving minimal doses of barbiturate this figure was reduced to 41 ~14. There is also evidence that anesthesia depresses spontaneous activity and alters auditory responses in the medial geniculate body of cats 1. Thus, the vocalizations served not only to identify the cells as auditory neurons but also to activate them in a behaviorally relevant, entirely physiological manner so that iontophoretic effects on the presumed functional activities of the cells could be studied.
240 Application of NE or GABA inhibited the spontaneous discharge of all cells tested, a finding in substantial agreement with other iontophoretic studies of neocortical neurons in anesthetized rat3S; anesthetized6,8,2~-23,25, 40, cerveau isoh; 2'",23, and awake 13 cat, and anesthetized squirrel monkey eS. As in these other preparations, NE inhibition had a longer latency and duration than GABA inhibition. PVA, which was almost always slower than evoked activity, was inhibited proportionately more than VA during NE or GABA application. One possible role of such an effect might be to enhance VA/PVA ratios in the output of these cells. Examination of excitatory responses showed that although response peaks lost a larger number of counts than other poststimulus segments, proportionately they were less inhibited. Although there was a statistically significant tendency toward a negative linear relationship between rate and percent inhibition, the data were too variable to exclude the possibility that this relationship was systematic but non-linear. This variability and limited sample size also precluded a statistically appropriate, direct comparison of the percent inhibition of PVA with VA segments of the same control discharge rate to determine whether rate p e r se was adequate to predict NE or GABA effects, or whether PVA was more strongly inhibited, independent of rate. Thus, for excitatory responses both averaged poststimulus activity and, in particular, specific peaks of excitation are inhibited proportionally less by NE or GABA than is spontaneous activity. The results of the K-S analysis suggest that response latency and the pattern of excitatory peaks and inhibitory troughs in the poststimulus histogram did not substantially change with NE, GABA, or ACh administration. The significant changes that were observed can be most parsimoniously explained as resulting from the transmitters' marked effects on slower portions of the response. Thus, although putative transmitter effects were assessed with a complex neuronal response, only relatively simple, rate-dependent effects were observed. These results may reflect limitations of the iontophoretic technique as used here. Although iontophoretic application of NE is an adequate model for evaluating some of the postsynaptic effects of this putative transmitter, these experiments could not fully mimic synaptic NE inhibition. First, since the presumed NE synapses in auditory cortex may originate in cells which project widely over neocortex 28 as well as to otber areas of the brain, activation of NE cells may affect auditory neurons not only directly, but also indirectly, lontophoresis thus mimics the monosynaptic effects of NE while artificially excluding polysynaptic NE influences which may normally accompany them. Second, the ejection of NE over 3-5-min periods, while it may mimic tonic NE inhibition, is not an adequate model of rapid, phasic NE release. Third, the NE doses used here were chosen to produce readily observable decreases in neuronal activity and may not reflect physiological levels. Despite these limitations, it is cle3r that application of NE with small ejection currents is capable of substantially altering, in a consistent fashion, the presumed functional activities of these neurons. In the analyses detailed above, no significant differences between the effects of GABA and NE were found. We did observe a shorter latency of onset and shorter duration of effect for GABA. Although this was possibly due to differences in the iontophoretic release of the two agents, it may reflect differences in the functions of
241 these p u t a t i v e t r a n s m i t t e r s . S y n a p t i c G A B A release m a y cause the b r i e f i n h i b i t o r y pauses o b s e r v e d in a u d i t o r y responses. Such pauses a p p e a r to result f r o m active inh i b i t i o n 88. N E m a y p r o v i d e a tonic, low-level i n h i b i t i o n a c c o u n t i n g for p r e v i o u s l y o b s e r v e d s p o n t a n e o u s I P S P s aa a n d the resulting low s p o n t a n e o u s activity levels in these ceils. Such t o n i c i n h i b i t i o n , as shown in this study, w o u l d e n h a n c e the difference between b a c k g r o u n d a n d s t i m u l u s - b o u n d activity. ACKNOWLEDGEMENTS T h e a u t h o r s wish to a c k n o w l e d g e the generous assistance o f J o h n D. N e w m a n who p r o v i d e d c o m p u t e r p r o g r a m s , v o c a l i z a t i o n tapes, a n d i m p o r t a n t technical suggestions. D a v i d S y m m e s wrote a n d s u p p l i e d c o m p u t e r p r o g r a m s . B. Hoffer m a d e helpful technical suggestions. E. S t r a u g h n p r o v i d e d technical assistance. M. Segal, F. E. B l o o m , J. D. N e w m a n , a n d D, S y m m e s m a d e helpful suggestions c o n c e r n i n g the m a n u s c r i p t . J. Peay c o n t r i b u t e d e d i t o r i a l a n d clerical assistance.
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