Noradrenergic modulation of somatosensory cortical neuronal responses to lontophoretically applied putative neurotransmitters

EXPERIMENTAL

NEUROLOGY

69, 30-49 (1980)

Noradrenergic Modulation of Somatosensory Cortical Neuronal Responses to lontophoretically Applied Putative Neurotransmitters BARRY D. WATERHOUSE, HYLAN C. MOISES, AND DONALD J. WOODWARD’ Department of Cell Biology, The University of Texas Health Sciences Center, Dallas. Texas 75235 Received June 22, 1979; revision received December 17, 1979 We examined the interaction of norepinephrine (NE) applied iontophoretically in small doses with the responsiveness of somatosensory cortical neurons to the putative neurotransmitter substances acetylcholine (ACh) and gamma-aminobutyric acid (GABA). Neuronal responses to microiontophoretic pulses (8- to 10-s duration at 45-s intervals) of ACh and GABA were examined before, during, and after NE iontophoresis. Computer-generated histograms used for quantitation of drug responses revealed a NE-induced enhancement of neuronal responsiveness to both ACh and GABA. NE differentially suppressed the spontaneous tiring rate more than activity during ACh-induced excitation such that the excitatory response was enhanced relative to background discharge in 86% of the cells tested. In 13 of 35 cells tested, ACh responses were potentiated above control values. GABA-induced inhibition of cortical neuron spontaneous discharge was augmented during iontophoretic application of NE in 94% of the cells examined. Dopamine, even at doses sufficient to depress background firing rate, was not effective in facilitating responses to either ACh or GABA. These results are consistent with the hypothesis that a primary function of the central noradrenergic system is to enhance the efficacy of postsynaptically acting neurotransmitters.

INTRODUCTION Most studies using iontophoretic techniques have revealed a suppression of cortical neuron spontaneous discharge (18,28,33), as a primary action of Abbreviations: ACh-acetylcholine, NE-norepinephrine, GABA-gamma-aminobutyric acid, DA-dopamine, FPZ-fluphenazine, SA-spontaneous activity, R-response. ’ This work was supported by grants from the National Institutes of Health (1 F32 NS05699-1) to B.D.W. and the National Science Foundation (BNS77-01174), National Institute of Drug Abuse (DA-02338), and the Biological Humanics Foundation to D.J.W. We thank Squibb for the generous gift of FPZ. 30 0014~4886/80/070030-20$0200/0 Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any fomt reserved.

NOREPINEPHRINE

POTENTIATES

ACh

AND

GABA

31

norepinephrine (NE). Activation of the ceruleus-cortical noradrenergic pathway (1, 10) was also reported to produce a similar simple slowing or cessation of the spontaneous firing of cortical cells (4). However, we recently reported experiments which demonstrated that iontophoretically applied NE can enhance both excitatory and inhibitory responses of somatosensory cortical neurons to afferent synaptic input (38). In that study stimulus-bound excitation or inhibition was enhanced during NE application relative to a suppression of background discharge. Moreover, in many cases NE produced absolute increases in evoked spiking, and in this respect, appeared to function more to alter neuronal responsiveness to conventional excitatory afferent input than to yield a unidirectional inhibition. Similar NE-induced increases of evoked excitation or inhibition, i.e., gains in signal to noise ratio, reported in the cerebellum of rat (8) and auditory cortex of monkey (5), provide additional evidence for a potential “modulatory” role for NE in the mammalian central nervous system. The present report describes one phase of a series of experiments aimed at fully characterizing the nature of the postulated synergistic interaction between NE and other synaptic systems in the somatosensory cortex. We examined a series of drug interactions which demonstrated that NE in small doses enhanced the actions of the putative cerebrocortical transmitters gamma-aminobutyric acid (GABA) (3, 15,21,30) and acetylcholine (ACh) (14,19,24). This effect is selective for NE in the sense that dopamine (DA) does not exhibit a similar property. These results also provide strong initial evidence that the NE-induced enhancement of synaptic efficacy can occur at a point subsequent to synaptic release of transmitter, because both GABA and ACh themselves are known to exert direct postsynaptic actions. A preliminary report of this work has appeared (37). METHODS Twenty-seven female Sprague-Dawley rats, weight 180 to 250 g, were anesthetized with halothane (0.5-0.75% in oxygen), intubated, and allowed to breathe spontaneously. Body temperature was monitored by a rectal probe and maintained between 36 and 37°C with a heating lamp. The skull and dura over the forelimb area of the somatosensory cortex (13,39) were removed and the exposed brain tissue was covered with 2% agar in balanced salt solution. Five-barrel micropipets, tip diameter 4 to 6 pm, were used to record extracellularly the spontaneous discharge of somatosensory cortical neurons and to apply chemical substances at the recording site by microiontophoresis. The center barrel, filled with 4 M NaCl, was used for recording. Each of three side barrels, filled by centrifugation, contained

32

WATERHOUSE,

MOISES,

AND

WOODWARD

one of the following drug solutions: 0.5-1.0 M DLnorepinephrine-HCl, pH 4.5 (Sigma); 0.5-1.0 M dopamine-HCl, pH 4.5 (Sigma); 0.25-0.5 M gamma-aminobutyric acid, pH 4.0 (Sigma); 1.0 M acetylcholine chloride, pH 4.5 (Sigma); 0.5 M fluphenazine, pH 4.2 (Squibb). A crystal clock-regulated logic circuit controlled the iontophoresis unit (12) so that constant-current pulses of uniform duration and magnitude could be passed at equal intervals through the drug barrels (7). Tip potentials, which could directly influence cell discharge, were minimized by a current-balancing circuit which automatically passed a current equal but of opposite polarity to the ejection current through a fourth peripheral barrel containing 3 M NaCl. Action potentials of cortical neurons were monitored on an oscilloscope and converted to uniform voltage pulses by a window discriminator. The pulses were integrated for l-s intervals by a ratemeter and displayed on a chart recorder. The window discriminator output was also led to a digital computer (PDP-12, Digital Equipment Corp.) which summed unit activity during regularly spaced pulses of putative cerebrocortical transmitter agents in the manner of a poststimulus time histogram. Histograms were computed before, during, and after a period of continuous iontophoretic administration of NE. Agonist responses were quantitated by comparing the discharge rate during transmitter application with the fning rate between drug pulses, and expressing the difference as a percentage excitation or inhibition of the baseline firing frequency, accordingly. After cessation of NE application, successive histograms were computed until recovery of the control agonist response was observed. Cells not exhibiting recovery were rejected from analysis. As described elsewhere (27, 38), differential changes in agonist responses and spontaneous tiring resulting from NE iontophoresis were quantitated by comparing discharge rates during identical epochs of agonist-induced and spontaneous activity in control and NE histograms. The period of agonist response was selected to begin at the particular bin where counts deviated significantly from baseline and terminate at bins where counts reapproached the baseline. To facilitate comparisons between histograms, equal numbers of agonist applications were used for each. Two-way analysis of variance tests assessed the statistical significance of differences between the percentage spontaneous activity (SA) change and the percentage response (R) change for the entire sample of cells (16). This quantitative approach allowed the data to be more realistically presented as scatter diagrams rather than as an arbitrary summary of positive or negative effects. The following operational definitions were used as before (27, 38) to assess the influence of NE on the actions of GABA. “Augmentation” of GABA-induced inhibition was declared when the firing rate during the drug

NOREPINEPHRINE

POTENTIATES

ACh

AND

GABA

33

pulse was depressed proportionately more during NE administration than was the baseline rate of background firing. Conversely, interactions were when the background rate of firing was defined as “antagonistic” depressed proportionately more by NE than was activity during GABA application. NE was assumed to have no significant differential effect on GABA responses when spontaneous and drug-induced rates of tiring were affected proportionately to the same extent by NE, as represented by the dotted 45” “equivalence line” in the graph of Fig. 8. “Enhancement” of the response to ACh administration relative to background discharge was declared when spontaneous firing was suppressed to a greater extent by NE application than was activity during an ACh-evoked excitation. “Potentiation” of ACh-induced excitation was defined as an absolute increase in the ACh response above control, accompanied by a decrease or no change in the amount of spontaneous discharge. NE-ACh interactions were termed antagonistic when the excitatory response to ACh was suppressed proportionately more than background discharge. Such definitions create a useful terminology in the absence of any generally accepted description of transmitter interactions. By using them, no assumptions are, or can be, made concerning linear or nonlinear summation effects. RESULTS Norepinephrine Effects on Acetylcholine-induced Excitation. Application of uniform pulses of ACh at regular intervals produced consistent increases in cortical neuron discharge which were selectively preserved relative to suppression of spontaneous firing rate during application of NE. Figure 1 shows the ratemeter (Fig. 1, 1) and corresponding computergenerated histogram (1) records of the control response from a typical cell whose tiring rate was increased 104% above spontaneous values after ACh administration. Histogram analysis revealed that during NE iontophoresis spontaneous activity was suppressed 50%, from 5.0 (histogram 1) to 2.5 spikes/s (histogram 2), whereas activity during the ACh-induced response decreased only 25%, from 10.2 (histogram 1) to 7.7 spikes/s (histogram 2); yielding a twofold increase (from 104% to 208% excitation) in “signal to noise” ratio (mean rate during the evoked response divided by background tiring rate). Continued application of NE (histogram 3) produced a further increase in signal to noise ratio by a factor of 6.1 times greater than the initial control value. Recovery toward the control response was observed during a 4-min period (histograms 4 and 5) following cessation of NE administration. In 17 of 35 cortical neurons tested, NE application suppressed spontaneous discharge more than the ACh-induced excitation, in a manner similar to that shown in Fig. 1.

34

WATERHOUSE,

MOSES,

2

1 CONTROL

4

RECOVERY

I

207

NE

AND WOODWARD

I

3

5

RECOVERY

NE

II

II

76

FIG. 1. Enhancement of acetylcholine-induced excitation by norepinephrine (NE) relative to inhibition of spontaneous discharge. Continuous ratemeter record (above) and peridrug histograms (below) show responses of a single somatosensory cortical unit to iontophoretic application of acetylcholine (ACh), before, during, and after NE administration. In the ratemeter record, ACh pulses are indicated by solid bars and the period of NE ejection by the broken bar. Drug response histograms were computed at various times indicated by arrows and numbers beneath the ratemeterrecord. In each histogram, the excitatory response to ACh (indicated by horizontal line beneath histogram) is expressed as a percentage excitation above background discharge. During NE administration the excitatory response to ACh was preserved relative to the inhibition of background discharge, yielding a twofold increase in the “signal to noise” ratio (histogram 2). Note that signal to noise ratio continued to improve with further application of NE (histogram 3). After termination of NE iontophoresis, spontaneous and ACh-evoked activity returned toward control values in both ratemeter and histogram records. Calibrations: ratemeter, horizontal, 30 s; vertical, spikes per second. Histograms, horizontal, 5 s; vertical, counts per address.

Application of NE potentiated (Figs. 2 and 3) the ACh-induced excitation above control values with no change or a decrease in spontaneous discharge in 13 of the 35 neurons tested (Table 1). For the cell illustrated in Fig. 2, a marked potentiation of the excitatory response, from 53 to 981% above background discharge, was observed during NE application. In many cells, the NE-induced facilitation of ACh excitation persisted for several minutes after the cessation of NE administration. As shown in Fig. 3, for example, the excitatory response to ACh, although less than during NE application, was still greater than the control response in histograms computed at 1 (recovery I) and 4 (recovery II) min after termination of NE iontophoresis. Of the 35 cells tested, NE antagonized or had no significant effect on ACh excitation in five cases.

NOREPINEPHRINE

POTENTIATES

ACh AND GABA

35

FIG. 2. Potentiation of acetylcholine-induced excitation during norepinephrine (NE) administration. The continuous ratemeter (left) and histogram (right) records show the response of a cortical neuron to periodic administration of ACh 50 nA (solid bars) before, during, and after NE (broken bar) iontophoresis. Drug response histograms were constructed during the periods indicated by adjacent ratemeter records. Numbers above each histogram indicate the percentage ACh-induced excitation (dashed-dotted line beneath histograms) over background tiring rate. During NE application, responses to ACh were increased by a factor of 17.51, from 53 to 981%, and spontaneous activity was depressed. Recovery toward control activity was observed after termination of NE application. Calibrations: ratemeter, horizontal, 30 s; vertical, spikes per second. Histograms, horizontal, 5 s; vertical, counts per address.

For all cells studied, quantitative analysis of the histograms revealed a differential effect of NE on spontaneous and ACh-evoked discharge. The graph at right in Fig. 4 shows the percentage change in ACh-evoked and spontaneous activity produced by NE in all 35 units @led circles) studied. The majority (94%) of points lie below the dotted 45” equivalence line indicating cells in which spontaneous discharge was suppressed proportionately more than the agonist-evoked response during NE iontophoresis, (an effect statistically significant for the 35cell sample; t (68) = 3.59, P < 0.001). In 12 cells, plotted below the abscissa, the agonist response was potentiated above the control value by NE. In five of these neurons (points adjacent the Y-axis), potentiation of the ACh-induced response was observed at NE doses which caused little or no effect on the baseline firing rate. In the representative example shown in Fig. 4 (left), spontaneous activity (dashed line) was depressed 34% from 9.7 (SA 1) to 6.9 spikes/s (SA 2) during NE application, whereas activity during the ACh response (solid bar) was inhibited by only 19% from 20.1 (R 1) to 16.3 spikes/s (R 2). Thus,

36

WATERHOUSE, Ach

40 nA

MOISES, AND WOODWARD Control

NE

Recovery

Recovery

I -

105

-

73

II IO

-Ilr

.-.-.-.-.

5s

FIG. 3. Time course of norepinephrine (NE) effects on cortical neuron responsiveness to periodic administration of acetylcholine (ACh). The continuous ratemeter (left) and histogram (right) records illustrate the response of one cortical neuron to iontophoretic application of ACh (solid bars); before, during, and several minutes after NE (dashed line) administration. Conventions are the same as in Fig. 2. Two to three minutes after the onset of NE ejection, background activity was depressed by 51% from 8.7 to 4.3 spikes per second, whereas the ACh-induced excitation was increased by 27% from 13.5 to 17.2 spikes per second. These changes in spontaneous and evoked activity yielded a net increase (445%) in the ACh response from a 55 to 300% excitation above background discharge. One minute after cessation of NE iontophoresis, responses to ACh remained potentiated (91%) above control, as shown in both ratemeter and histogram (Recovery I) records. After 4 min, a gradual return toward control values of spontaneous and evoked activity was observed (Recovery II). Calibrations: ratemeter, horizontal, 30 s; vertical, spikes per second. Histograms, horizontal, 5 s; vertical, counts per address.

the overall effect of NE was to increase the signal to noise ratio of the excitatory response by a factor of 1.27. Effects of Dopamine. In 14 cells tested, DA produced no consistent enhancement of the ACh-induced excitation despite causing a routine depression of background discharge (Table 1). Analysis of variance of the 14-cell sample revealed only a moderate trend toward a differential action of DA on ACh-induced excitation versus background discharge (t (26) = 1.83, P between 0.1 and 0.05). The graph in Fig. 4 illustrates the percentage changes in spontaneous and ACh-induced activity observed during DA iontophoresis (open squares) for 12 of the 14 neurons studied. Note that the data points are scattered with no tendency to be above or below the 45” equivalence line, and, more importantly, that in no case did

NOREPINEPHRINE

POTENTIATES TABLE

37

ACh AND GABA

1

Effects of Catecholamines on Somatosensory Cortical Neuronal Responses to Acetylcholine Drug-induced

change in ACh response (No. of cells)

Agent tested

Potentiation

Relative increase signal to noise

Antagonism

No effect

NE DA

13 (37%) 0 (0%)

17 (49%) 6 (43%)

2 (6%) 5 (36%)

3 (8%) 3 (21%)

DA potentiate ACh excitation above control values. Two of 14 cells (not plotted) appeared to be anomalous in that DA produced a substantial increase in spontaneous discharge (38 and 25%) accompanied by increases in the ACh-evoked responses (29 and 19%). A direct comparison of the effects of DA and NE on neuronal responsiveness to ACh excitatory actions was achieved by interacting both catecholamines with ACh in six neurons. DA was found to be more potent than NE in facilitating the excitatory response to ACh in two units, less effective in two other units, and antagonistic to the ACh response in two cases. Similar iontophoretic currents of NE potentiated ACh excitation in all six cases. An example of these differences in the actions of NE and DA on the same neuron is illustrated in Fig. 5. During NE iontophoresis (25 nA), the response to ACh was facilitated 15% (from 278 to 3 19% excitation above background) relative to an inhibition of spontaneous discharge. A similar ejection current of DA (20 nA) gradually suppressed spontaneous activity and, after 2 to 3 min, completely abolished the excitation induced by pulsatile applications of ACh. No enhancement of signal to noise ratio was observed in this cell during the period of DA iontophoresis. Blockade of Norepinephrine Fluphenazine. It was previously

Effects on Acetylcholine

Excitation

Dy

shown that fluphenazine (FPZ), a potent and specific blocker of NE-induced depression of Purkinje cell discharge (6), can reversibly antagonize the facilitating effects of NE on GABAinduced inhibition in the cerebellum (27). In the present study, iontophoretic application of FPZ (Fig. 6) also reversed the NE-induced potentiation of ACh excitation in cortical neurons. For the cell shown in Fig. 6, ACh excitation (dashed-dotted lines beneath histograms) was initially potentiated from 59 to 243% excitation above background discharge (histogram 2) during NE application. In the presence of FPZ (histogram 3), the ACh response returned to control values, but was again potentiated from 52 to 176% excitation after cessation of phenothiazine administration (histogram 4). Recovery of the ACh response toward the

38

WATERHOUSE,

MOISES, AND WOODWARD

CONTROL

SAl

Rl

I ZPL 0-

50

NE 2OnA

RECOVERY

ap

. .

.

.

% INlilDlllON OF SPONTANEOUS ACTIVITY

l q

During During

NE DA

ISA)

FIG. 4. Differential action of NE on spontaneous activity and ACh-induced excitation. Histogram records (left) show the response of a single cortical neuron to ACh before, during, and after NE iontophoresis. NE-induced changes in spontaneous activity (SA 1, SA 2) and the excitatory response (Rl, R2) were expressed as percentage increase or decrease from control values. For this cell, spontaneous discharge (dashed line) was inhibited 34% from 9.7 @A 1) to 6.4 spikes per second (SA 2), whereas ACh-induced excitation was suppressed only 19% from 20.1 (R 1) to 16.3 spikes per second (R 2). The graph at right summarizes the results of similar experiments with NE @led circles) on 35 somatosensory cortical neurons. In 33 cells, plotted below the dotted 45” equivalence line, NE suppressed background discharge more than activity during the ACh-induced excitation, yielding net increases in signal to noise ratio. Note that in 12 cells (points below the abscissa) an actual potentiation of the ACh response was observed during NE administration. The arrow corresponds to data from the cell illustrated at left. Overall, dopamine (DA, open squares) produced effects different from NE when interacted with ACh. DA facilitated ACh-induced excitation by suppressing background more than evoked activity in only 6 of 12 cases. Note also that potentiation of the agonist response above control values was never observed with DA. Calibrations: horizontal, 30 s; vertical, counts

per address.

control value was observed upon termination of NE iontophoresis (histograms 5 and 6). Reversible antagonism of the noradrenergic potentiation of ACh excitation by FPZ was found in all three neurons studied. Norepinephrine Effects on GABA-Znduced, Inhibition. Iontophoretic administration of small doses of NE also increased the magnitude and duration of cortical cell inhibition produced by brief (8 to 10 s)

NOREPINEPHRINE

CONTROL

POTENTIATES

DURING

DA

39

ACh AND GABA

RECOVERY

30

FIG. 5. Differential effects of dopamine (DA) and norepinephrine (NE) iontophoresis on somatosensory cortical neuron responsiveness to acetylcholine (ACh) application. Ratemeter and histogram records in A and B show the responses of the same cortical cell to iontophoretic application of ACh 50 nA (solid bars) before, during, and after catecholamine administration. Excitation produced by ACh (dashed-dotted lines beneath histograms) was expressed as a percentage excitation above background discharge. During NE iontophoresis (A) the evoked excitation increased from 278 to 319%, a 15% potentiation of the ACh response. Ratemeter and corresponding histogram records in B show that the ACh-induced excitation decreased from 141 to 81% during DA administration, a 43% antagonism of the ACh-induced response. Note that, at the doses used, both NE and DA suppressed spontaneous discharge. Calibrations: ratemeter, horizontal, 30 s; vertical, spikes per second. Histograms, horizontal, 10 s; vertical, counts per address.

microiontophoretic pulses of GABA. In the example shown in Fig. 7, the response to GABA was augmented during NE administration from 61% (control, histogram 1) to 79% (during NE, histogram 2) inhibition of spontaneous firing rate. The GABA inhibitory response increased further to 91% with continued application of NE (histogram 3), followed by a return to control values after cessation of NE iontophoresis (histogram 4). The inhibitory action of GABA was augmented in a similar manner during NE administration in 16 of the 17 cortical neurons tested (Table 2). In 12 of these 16 cells, GABA-induced inhibition was augmented for several minutes after the termination of NE iontophoresis. The recovery histogram in Fig. 7, for example, was computed 10 min after cessation of NE administration, during which time GABA responses were less than during NE application, but still elevated above control values. As summarized in Fig. 8, the rate of firing during the GABA response

40

WATERHOUSE,

MOISES,

I Control

2 NE

4 NE

5 Rscowry

AND WOODWARD

3 NE- FPZ

I

6 Ftscovsry

II

a&

m.-.-.-.-.

-.-.-.-._ 5s

FIG. 6. Blockade of norepinephrine (NE) potentiation of acetylcholine responses by iontophoretically applied fluphenazine (FPZ). Continuous ratemeter and corresponding histogram records illustrate the response of a single cortical cell to ACh 40 nA (solid bars) applied before, during, and after application of NE; and during application of both NE and FPZ. Histograms were computed during the periods bounded by arrows. The ratemeter record and histograms show that excitation produced by periodic ACh application was potentiated during NE iontophoresis 5 nA (dotted line). This potentiation was reversibly antagonized (histogram 3) by microiontophoretic administration of the phenothiazine derivative, FPZ 30 nA (dashed line) which, at this dose, had no effect on spontaneous or ACh-evoked activity. Note the return of NE-induced potentiation of the ACh response following the cessation of FPZ application (histogram 4) and gradual recovery to the control agonist response after termination of NE administration (histograms 5 and 6). Calibrations: ratemeter, horizontal, 30 s; vertical spikes per second. Histograms, horizontal, 5 s; vertical, counts per address.

was depressed to a greater extent by NE than was the rate of spontaneous discharge in the majority of cortical neurons tested (filled circles). Sixteen points (94% of the cells) lie above the 45” line, indicating that for these units the percentage augmentation of the inhibition during the GABA response period was greater than the NE-induced depression of spontaneous activity. This differential effect of NE on spontaneous discharge and activity during a GABA-induced inhibition was statistically significant for the 17-cell sample (t (32) = 3.39, P C 0.001). Five of the 16 cells lie to the left of the Y-axis indicating, that, in these cases, augmentation of the GABA

NOREPINEPHRINE

1 CONTROL

3

NE ,I

POTENTIATES

2

4

ACh AND GABA

41

NE I

RECOVERY

FIG. 7. Effects of norepinephrine (NE) iontophoresis on GABA-induced inhibition of somatosensory cortical neuron spontaneous discharge. Continuous ratemeter record shows the response of a single unit to periodic pulses of GABA 18 nA (solid bars) applied before, during, and after NE iontophoresis (dotted line). Drug response histograms (l-4) were constructed during the periods bounded by arrows beneath the ratemeter record. Inhibition produced by GABA ejection was quantitated by comparing firing rate during the GABA response (dashed-dotted line under histogram) to spontaneous discharge preceding drug application. Numbers above each histogram indicate the percentage inhibition of spontaneous activity induced by GABA. Solid bar above histograms indicates the period and duration of GABA ejection. During NE administration (2 and 3), both the magnitude and duration of the GABA-induced inhibition were augmented. Recovery to the control GABA response was observed 10 min after termination of NE iontophoresis (4). Calibrations: ratemeter, horizontal, 30 s; vertical, spikes per second. Histogram, horizontal, 5 s: vertical, counts per address.

inhibitory action occurred despite elevation in the rate of spontaneous discharge during NE microiontophoresis. Potentiation of GABA inhibition by NE occurred independent of direct noradrenergic depressant effects on spontaneous neuronal firing. Synergistic effects between NE and GABA were demonstrated after deliberately varying the NE dose to yield both minimal and substantial suppression of background activity. In three cells, marked augmentation of the GABA

42

WATERHOUSE,

MOISES, AND WOODWARD TABLE

2

Effects of Catecholamines on Somatosensory Cortical Neuronal Responses to GABA Drug-induced Agent tested NE DA

change in GABA response (No. of cells)

Augmentation 16 (94%) 2 (22%)

Antagonism

No effect

1 (6%) 6 (67%)

0 (0%) 1 (11%)

response occurred even at concentrations of NE which caused little or no depression of background discharge (Fig. 8). For the cell shown in Fig. 8 (left), spontaneous activity (SA 1, SA 2) did not change, yet GABAinduced inhibition (R 1, R 2) was augmented 55% from the control response. CONTROL

NE

IOnA

RECOVERY

I’

q -40

%

INl,ISITlON

SPONTANEOUS

OF

ACTIVITY

ISA1

FIG. 8. Differential effects of norepinephrine (NE) and dopamine (DA) on spontaneous activity and GABA-induced inhibition of somatosensory cortical neuron discharge. Histograms (left) illustrate the inhibitory response to periodic GABA pulses 15 nA applied before, during, and after NE iontophoresis. Comparison of firing rates during the period of spontaneous activity (dotted bars) and the GABA-induced inhibition (solid line) revealed that the inhibitory response to GABA was augmented 55% during NE application, despite no change in background discharge. As summarized by the graph at right, in 16 of 17 cortical neurons tested, NE enhanced the GABA response relative to inhibition of spontaneous discharge. The arrow denotes the data plotted from histograms at left. In contrast to the effects found with NE, DA antagonized the inhibitory response to GABA in six of nine units tested (open squares). Calibration: horizontal, 30 s; vertical, counts per address.

NOREPINEPHRINE

POTENTIATES

ACh

AND

GABA

43

Interactions of Dopamine with GABA. In contrast to the action of NE, DA in subthreshold doses, or doses adequate to suppress background discharge, produced no consistent potentiation of GABA inhibition. In fact, the inhibitory response to GABA application was antagonized in six of nine cells despite some depression of spontaneous discharge rate during DA administration (Table 2, Fig. 8). Analysis of variance of the nine-cell sample revealed no trend at all for DA to exert a differential effect on activity during a GABA-induced inhibition (t (8) = 0.51, P between 0.7 and 0.5) relative to its direct depressant effect on spontaneous discharge. In the graph at right in Fig. 8, DA antagonism of the GABA inhibitory response is indicated by the six points (open squares) which lie below the 45” equivalence line. The effects of DA and NE were directly compared on six cells where each catecholamine was interacted with GABA. GABA responses were augmented in all six cells during NE iontophoresis, yet DA at similar ejection currents antagonized GABA inhibition in four of the six cases. Depth ofCells versus Effects of NE. Previous reports (9,22) suggested an uneven distribution of noradrenergic terminals among the six layers of the cerebral cortex. Therefore, in the present study particular attention was paid to the interaction of iontophoretically applied NE and putative transmitter substances with respect to depth from the cortical surface. The majority (N = 41,79%) of neurons tested were found in the middle cortical layers between 0.6 and 1.2 mm deep from the pial surface. NE-induced enhancement of cortical unit responses to ACh was observed (left in Fig. 9) in all layers of the somatosensory cortex; however, the absolute potentiation of ACh excitation (solid line) induced by NE was restricted to 13 of the 28 (46%) cells tested between 0.5 and 1.2 mm from the cortical surface. Potentiation of the ACh-induced response by NE was not observed in any cells situated deeper than 1.2 mm. Chi-square analysis supported the hypothesis that NE-induced potentiation of ACh excitation occurs preferentially in cells situated less than 1.2 mm (x2 (1) = 5.17, P < 0.05). Although the preponderance of cells tested for NE interactions with GABA were in layer IV, augmentation of GABA-induced inhibition was observed in cells situated throughout the vertical extent of the cortical tissue (Fig. 9, right graph).

DISCUSSION In our view, previous descriptions (18,28,33) of the inhibitory effects of NE on spontaneous firing rates do not readily lead to an accurate prediction of how NE should interact with the operation of local cerebrocortical neuronal circuits. Our contention is that an interrelated sequence of studies using both iontophoretic and synaptic release of NE in a variety of

44

WATERHOUSE,

0 2 -g

MOUES,

AND WOODWARD 0

NE -ACH

.=

NE-GABA

.2

NUMBER

OF

CELLS

FIG. 9. Distribution by depth of cortical units whose responses to GABA or acetylcholine were interacted with norepinephrine (NE) iontophoresis. In both graphs, depth from the cortical surface is indicated in millimeters along the ordinate and number of cells tested or modulated by NE are plotted along the abscissa. The approximate vertical extent of layer IV is indicated by the stipling. NE enhancement of neuronal responses to putative transmitter substances was observed at all levels of the somatosensory cortex where units were isolated.

paradigms is required to reveal the predominant physiological influence of the noradrenergic system on activity evoked in target regions of the cerebral cortex. The present investigation demonstrates that NE, but not DA, at small amounts can enhance both the relative and absolute excitatory responses to ACh and the inhibitory response to GABA in cerebrocortical neurons. Sire of Noradrenergic Action. Previously (37), it was shown that NE could enhance the synaptically evoked responses of somatosensory cortical neurons to naturally stimulated afferent inputs in a manner that suggested a modulatory action of NE in addition to its previously demonstrated inhibitory effect on cerebrocortical units (7, 18,28,33). The present study shows that such effects can be mimicked by interactions of NE with directly applied putative neurotransmitters. It is important to note that an effect of NE on presynaptic transmitter release can largely be ruled out as the basis for increased synaptic efficacy if the same NE modulatory actions can be demonstrated on cortical neuronal responses to iontophoretically applied putative cerebrocortical transmitter agents such as ACh (14, 19, 34) and GABA (3, 15, 21, 30).

NOREPINEPHRINE

POTENTIATES

ACh

AND

GABA

45

Considerable evidence suggests that the response to ACh, GABA, or NE when released directly onto neurons from a micropipet results from a predominant postsynaptic action of these agents. Intracellular studies on cortical cells have shown that microiontophoretic administration of ACh causes a slow membrane depolarization in association with a decrease in potassium conductance (20), whereas GABA produces hyperpolarization and a decrease in membrane resistance (21). Furthermore, inhibition of cerebellar Purkinje cell discharge by iontophoretic application of NE or GABA was consistently demonstrated in preparations where presynaptic elements were absent or markedly reduced (11, 32, 40, 41). There is no conclusive evidence that ACh and GABA are in fact the substances which mediate afferently evoked responses in the cortex. However, the present results provide an important demonstration that NE can alter somatosensory cortical neuronal responsiveness to likely transmitter candidates which have direct effects on postsynaptic membranes. Still to be resolved, however, is the issue of whether such facilitating actions of NE result from a direct alteration in membrane responsivity, or reflect changes in either agonist receptor binding or transmitter inactivation processes. Selectivity of Norepinephrine Effects. Specificity of the NE action on neuronal responsiveness to ACh was demonstrated by the failure of DA to mimic NE effects. DA did not routinely enhance the ACh-induced excitation relative to its direct suppression of background activity, and was never observed to potentiate the ACh response above control values. These results confirm and extend similar findings observed previously with cerebellar Purkinje cells (27). In the cerebral cortex and cerebellum, both DA and NE depress spontaneous activity, but at equivalent values of spike suppression only NE exhibits the modulatory effect; hence, the facilitation by NE is not likely to derive solely from its direct depressant actions. Evidence for specific receptor mediation of the observed modulatory actions of NE was provided by the finding that fluphenazine reversibly blocked the NE-induced enhancement of ACh excitation. Previous studies showed that fluphenazine specifically antagonized NE but not GABAinduced inhibition of Purkinje cell discharge (6) and blocked catecholamine receptor actions in peripheral (23) and central nervous system (24, 3 1, 36) tissues. Thus, blockade by fluphenazine suggests that NE produces its facilitating effect on ACh by way of specific activation of an adrenergic receptor rather than through interaction with a nonspecific spike suppression mechanism. The finding that NE augments the ACh-induced excitatory responses of neurons located predominantly in the upper 1.2 mm of the cortex (see Fig. 9), suggests that this particular facilitating action of NE might be associated with a specific, but as yet unidentified, morphological characteristic of

46

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AND

WOODWARD

more superficial somatosensory cortical cells. In our previous study (38), NE-induced increases in synaptically evoked spiking occurred in cells no deeper than 1.3 mm from the pial surface. Thus, in both the present and previous studies, potentiation of excitatory responses by NE occurred in the region corresponding to layers I through V of the cortex. In the prefrontal cortex of rat, deeper cortical neurons were shown to be less sensitive to the inhibitory actions of iontophoretically applied NE (2), thus demonstrating pharmacological differences in the noradrenergic responsiveness of superficial and deep neurons. Furthermore, ACh excitation in the rat somatosensory cortex is thought to be mediated by both nicotinic receptors on superficial cortical cells and by muscarinic receptors on deeper pyramidal tract neurons (34). Although still incomplete, such evidence suggests that the interactions of NE and ACh could arise from two populations of neurons with pharmacologically distinct cholinergic receptors. The results reported here also suggest a selective interaction between NE and GABA, confirming previous findings in cerebellar Purkinje cells (27). In the present study, both DA and NE appeared similar in their ability to depress background discharge; yet, the failure of DA to potentiate GABA, even at catecholamine doses which directly suppress spontaneous activity, argues strongly against the contention that NE acts as a nonspecific depressant to algebraically sum with and augment the direct inhibitory action of GABA. The specificity of this facilitating action of NE on GABA is further emphasized by the previous finding in the cerebellum (7) that neither manganese, cobalt, nor verapamil, each a putative calcium antagonist, alters the inhibitory effect of GABA on Purkinje neuron firing despite a marked depression of background discharge. Moreover, Stone and Taylor (35) showed that GABA-induced inhibition of cortical unit discharge was unaffected by iontophoretic doses of adenosine which by themselves caused depression of background spontaneous activity. On the other hand, the benzodiazepine drugs (10) and pentobarbital (29) were shown to selectively amplify GABA inhibition in the cerebellum and superior cervical ganglion, respectively, One interpretation of these findings is that NE may represent an endogenous substance which is capable of regulating the GABA receptor system as part of its normal physiological function. Functional Signijcance. Earlier studies of the action of iontophoretitally applied NE primarily focused upon its ability to depress spontaneous neuronal discharge. The concept of NE as an “inhibitory neurotransmitof Purkinje and ter’ ’ was further supported by the demonstration cerebrocortical neuron inhibition following stimulation of the nucleus locus

NOREPINEPHRINE

POTENTIATES

ACh

AND

GABA

47

ceruleus (4, 17). The demonstration here of direct NE facilitating interactions with putative cerebrocortical transmitters and the previous description of enhanced synaptic efficacy (38) suggests that a more prominent action of NE may be to bias neuronal responsiveness to afferent synaptic input rather than to mediate direct postsynaptic inhibition (42). Such an effect could occur with normal small fluctuations in noradrenergic neuron activity. Although iontophoretic release is useful for an initial survey of an agent’s actions, the reality of any observed effects must be examined under the more natural conditions of synaptic release. The present and previous (38) results lead us to expect that NE released by activation of the ceruleus-cortical noradrenergic pathway should enhance cortical neuron responses to afferent synaptic input. Recent studies in our laboratory, in fact, have shown that a preconditioning locus ceruleus stimulation augments Purkinje cell responses to both iontophoretically applied GABA (26) and conventional cerebellar synaptic inputs (25). Work in progress (Waterhouse, unpublished) has demonstrated similar effects of locus ceruleus activation in cerebral cortex. Further testing of how and when such NE actions appear in unanesthetized states will be critical for establishing the normal physiological role of NE. It should also be noted that clarification of the interactions of NE with cerebrocortical neuronal circuitry is essential for assessing the influence of the many drugs known to interact with central catecholamine systems. REFERENCES 1. ANDEN, N. E., A. DAHLSTROM, K. FUXE, K. LARSON, L. OLSON, AND U. UNGERSTEDT. 1971. Ascending monoamine neurons to the telencephalon and diencephalon. Acta Physiol. &and. 67: 313-326. 2. BUNNEY, B. S., AND G. K. AGHAJANIAN. 1976. Dopamine and norepinephrine innervated cells in the rat prefrontal cortex: pharmacological differentiation using microiontophoretic techniques. Life Sci. 19: 1783-1792. 3. CURTIS, D. R., AND D. FELIX. 1971. The effect of bicuculline upon synaptic inhibition in the cerebral and cerebellar cortices of the cat. Brain Res. 34: 301-321. 4. DILLIER, N., J. LASZLO, B. MULLER, W. P. KOELLA, AND H. -R. OLPE. 1978. Activation of an inhibitory pathway projecting from the locus coeruleus to the cingulate cortex of the rat. Brain Res. 154: 61-68. 5. FOOTE, S. L., R. FREEDMAN, AND A. P. OLIVER. 1975. Effects of putative neurotransmitters on neuronal activity in monkey auditory cortex. Brain Res. 86: 229-242. 6. FREEDMAN, R., AND B. J. HOFFER. 1975. Phenothiazineantagonismofthenoradrenergic inhibition of cerebellar Purkinje neurons. J. Neurobiol. 6: 277-288. 7. FREEDMAN, R., B. J. HOFFER, AND D. J. WOODWARD. 1975. A quantitative microiontophoretic analysis of the responses of central neurones to noradrenaline: interactions with cobalt, manganese, verapamil and dichloroisoprenaline. Br. J. Pharmacol. 54: 529-539.

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