Differential effects of ascending neurons containing dopamine and noradrenaline in the control of spontaneous activity and of evoked responses in the rat prefrontal cortex

0306-4522/88 $3.00 + 0.00 Pergamon Press plc 0 1988 IBRO

Neuroscience Vol. 27, No. 2, PP. 517-526, 1988 Printed in Great Britain

DIFFERENTIAL EFFECTS OF ASCENDING NEURONS CONTAINING DOPAMINE AND NORADRENALINE IN THE CONTROL OF SPONTANEOUS ACTIVITY AND OF EVOKED RESPONSES IN THE RAT PREFRONTAL CORTEX J. MANTZ, Chaire de Neuropharmacologie,

C. MILLA,

J. GLOWINSKI

and A. M.

THIERRY*

INSERM U.114, Collige de France, 11 place Marcelin Eierthelot, 75231 Paris Cedex 05, France

Abstract-The

medial prefrontal cortex receives converging projections from the mediodorsal thalamic nucleus, dopaminergic cells from the ventral tegmental area and noradrenergic cells from the locus coeruleus. Stimulation of the ventral tegmental area inhibits the spontaneous activity of prefrontal cortical neurons and blocks the excitatory response evoked by stimulation of the mediodorsal thalamic nucleus (10 Hz). The aim of the present study was to compare the influence of dopaminergic and noradrenergic afferents on the spontaneous and evoked activity of medial prefrontal cortical neurons. In ketamineanaesthetized rats, repetitive stimulation (20 Hz, 10 s) of the locus coeruleus produced a long-lasting post-stimulus inhibition (mean duration: 45 s) of the spontaneous activity of 56% of the tested cells. This effect was decreased markedly following selective destruction of the ascending noradrenergic pathways (local 6-hydroxy-dopamine injection) or depletion of cortical catecholamines by a-methyl-para-tyrosine pretreatment, suggesting that these inhibitory responses are mediated by noradrenergic neurons. The excitatory response to mediodorsal thalamus nucleus stimulation (10 Hz) could still be evoked during the post-stimulus inhibitory period induced by locus coeruleus stimulation (20 Hz, 10 s) resulting in the enhancement of signal-to-noise ratio. On the other hand, a population of prefrontal cortex neurons (26%) was found to be reproducibly activated by noxious tail pinch. This evoked response was still present during the post-stimulus inhibitory period induced by locus coeruleus stimulation but was completely suppressed during stimulation of the ventral tegmental area (10 Hz). In conclusion, these results indicate that the dopaminergic and noradrenergic systems exert a completely distinct control of information transfer in the medial prefrontal cortex.

INTRODUCTION In

the rat, as in other mammals, the dopaminergic innervation of the cerebral cortex is restricted to discrete areas. The medial prefrontal cortex is the cortical zone that receives the heaviest network of DA nerve terminalsI which are distributed mainly in the deep layers (V and VI) and more scarcely in layer III.4,8*16 These fibres originate from the A10 group of DA cells located in the ventral tegmental area (VTA).“j In a previous study, we have shown that activation of the mesocortico-prefrontal system by electrical stimulation of the VTA induces an inhibition of the spontaneous activity of target cortical cells and also blocks the excitatory responses evoked by stimulation of the mediodorsal nucleus of the thalamus (MD).” In contrast to the DA innervation, noradrenergic (NA) fibres, which originate from the locus coeruleus (LC), are distributed in all regions and layers of the neocortex.16 It has been reported (DA)

*To whom correspondence should be addressed. DA, dopaminergic/dopamine; HPLC, highperformance liquid chromatography; LC, locus coeruleus; MD, mediodorsal thalamic nucleus; Me5, trigeminal mesencephalic nucleus; a-MpT, a-methylparu-tyrosine; NA, noradrenergic/noradrenaline; 6OHDA, 6-hydroxydopamine; VTA, ventral tegmental area.

Abbreuiarions:

517

that iontophoretic application of NA or stimulation of the LC induce a strong inhibition of spontaneous firing of cingulate cortical cells.’ Moreover, in the somatosensory cortex, iontophoretic application of NA suppresses the spontaneous discharge of the cells to a greater extent than the evoked responses to natural stimulations (foot-tap).33 Microiontophoretic investigations have also indicated that the spontaneous activity of a population of cells in the medial prefrontal cortex is inhibited by both NA and DA, suggesting that ascending NA and DA systems have common target ~~11s.~In fact, it has been shown recently that NA neurons exert a permissive role on the denervation-induced supersensitivity of D, receptors in the prefrontal cortex, suggesting an interaction between NA and DA afferents on certain cortical cell~.~~Finally, the turnover of DA and NA is increased in the cerebral cortex in rats submitted to stressful situations.‘4*30 Thus the aim of the present study was to analyse: (1) the effects of electrical stimulation of the LC on the spontaneous activity of cells in the media1 prefrontal cortex of the rat and determine whether these effects were mediated by NA neurons; (2) the effects of LC stimulation on the excitatory responses induced either by stimulation of the MD or by painful stimuli (indeed, we have observed in a

preliminary study that the firing rate of some cortical cells is increased following the application of a noxious tail pinch); (3) finally, the effects of LC and VTA stimulation on the two types of evoked responses (MD, painful stimulus) and the convergence of these effects on the same cortical cell were investigated. EXPERIMENTAL

PROCEDURES

Experiments were performed on 34 SpragueeDawley male rats (25&300 g). Rats were anaesthetized with ketamine (80 mg/kg i.p.); additional injections (80 mg/kg i.m.) were made to maintain a stable level of anaesthesia. Animals were placed in a conventional stereotaxic head frame (Horsley Clarke apparatus). Body temperature was monitored with a rectal thermometer and kept between 37 and 38 ‘C. Bipolar co-axial stimulating electrodes (distance tipbarrel 300 pm; diameter 2OOpm) were positioned in the LC (A, -0.8 mm; L, I .I mm; H, 2.7 mm); VTA (A. 3.7 mm; L, 0.5 mm; H, 2.0mm) and MD (A, 6.7mm; L, 0.8mm: H, 4.6mm) according to the atlas of Paxinos and Watson,*” Electrical stimuli consisted of square wave pulses (0.5-ms duration, 5&150-PA intensity) delivered at a frequency of IllOHz for VTA or MD stimulation. The stimulating electrode was positioned in the LC according to previously reported aid criteria.’ In most cases. the LC was stimulated either with a single pulse (0.4-ms duration, 5&lOO-pA intensity, I Hz) or with a 20-Hz train of pulses applied for 10s. Noxious (non-traumatic) tail pinch was applied for IO s with surgical forceps. The activity of medial prefrontal-cortical neurons located deeper than 800 pm (layers IIIIVI) was recorded with glass micropipettes filled with 4% Pontamine Sky Blue dissolved in 0.4 M NaCl solution (impedance 610 Ma). Extracellular activity was amplified and displayed on a memory oscilloscope. The action potentials were separated from the noise by means of a window discriminator. Output from the window discriminator was fed to a digital computer (CED 1401 interface connected to an IBM-PC) to generate on-line rate or peri-stimulus time histograms. Histograms were stored on floppy discs for further statistical analysis. The electrocortigram was also recorded longitudinally by two silver ball electrodes on the dura mater to control the level of vigilance, particularly during noxious stimulations. For each studied cortical cell, basal firing was first recorded over a period of 60 s and its mean basal discharge determined. Cortical cells exhibited broad patterns of spontaneous activity (mean k S.E.M. 6.6 + 3.8 spikes/s, n = 222) from almost silent to 30 spikes/s. Thus inhibitory responses were analysed only on neurons which had a spontaneous activity higher than 0.5 spike/s. The effect of LC stimulation (20 Hz, 10 s) on the cell firing was analysed on spontaneous rate histograms using successive 5-s bins after LC stimulation since this stimulation tended to produce effects of long duration. The average number of action potentials was calculated for each bin and compared with the prestimulus mean basal firing. The duration of inhibition was defined as the sum of consecutive bins of 5 s each for which the activity of the cell was decreased by at least 50% when compared with the prestimulus basal firing. To assess the influence of tail pinch on the activity of cortical neurons, rate histograms were analysed using l-s bins from the onset of tail pinch application. A neuron was considered as activated (or inhibited) when the mean firing rate during the 10-s tail pinch application was increased (or decreased respectively) bv at least 50%. The inhibitory . responses induced by VTA _ stimulation (1 Hz) were analysed on peri-stimulus time histograms using IO-ms bins, corresponding to the cumulative responses of the cortical neurons to I50 VTA stimu-

lations. The stimulus-evoked tnhibitmn pcrrod U;IS dctrncd as the duration of complete cessation of firing after the stimulus. VTA stimulations were considered IO inhibit evoked responses when they reduced by at Icast 5()“,, the increased firing rate of cortical cells induced by tail pmch application, or the mean number of spikes evoked hv hlD stimulation. At the end of each experiment, the tip of each stunulattng electrode was marked by electrical deposits of iron (IO /tA anodal. 15s) and observed on histological slice, followrng a ferri-ferrocyanide reaction. Results obtained wtth stimulating electrodes placed outside the LC. VTA or MD wcrc excluded from the analysis. The tip of the recording macroelectrode was marked by iontophoretic ejection of Pontamine Sky Blue (8 /IA cathodal, 20 min) to determmc the position of the recorded cortical cells. At the end of the experiments, animals were perfused through the heart with a solution of IO% formalin. The localization of blue points was observed on serial frozen sections (80 pm) stained with Cresyl Violet as shown in Fig. I. Catecholamine (DA and NA) depletion was obtained tn eight animals by two injections of r-methyl-pirrlr-t~r[~~ine (a-MpT, a catecholamine synthesis inhibitor) I4 and 2 h before the recording session at a dose of 2OOmg,‘kg i.p. Selective NA denervation was made in eight rats by bilateral microinjections of 6-hydroxydopamine (6-OHDA) (3 )cg in 1 ~1 of isotonic saline solution containing I % ascorbic acid delivered at a speed of I pIi min) into the ascending NA pathways laterally to the pedunculus cerebellaris superior (A. I.2 mm; L, 1.4 mm: H, 2.X mm. according to the atla\ of Paxinos and Watson”‘). a site where the :txon\ 01’ the ventral and dorsal NA pathways run together. These animals were kept for at least 2 weeks before the recording session. Following electrophysiological recordings. each animal was killed and its brain removed and rapidly frozen for the determination of NA and DA content. Endogenous levels of DA and NA in the prefrontal cortex were estimated by high-performance liquid chromatography (HPLC) with electrochemical detection according to the method of Wagner et al.”

Stalislical analysi,v Data were expressed as mean _+ S.E.M. x1 test was used to compare: (I) the number of cortical cells inhibited by LC stimulation in control and a-MpTor 6-OHDA-treated animals, (2) the number of cortical neurons for which the excitatory response to MD stimulation was present before or after VTA or LC stimulation, and (3) the number of cortical cells inhibited following LC stimulation and following stimulations made in the vicinity of the LC. Comparisons of cortical DA and NA levels between control and lesioned animals were made using the Mann-Whitney test. ANOVA was used to test the influence of tail pinch, VT.4 and LC stimulation on the spontaneous firing of cortical neurons. The magnitude of these effects on cortical ceil discharge and the enhancement of signal-to-noise ratio under LC stimulation were assessed by the Wilcoxon’s test for paired comparisons. P < 0.05 was considered as significant.

RESULTS

Effects of locus coeruleus stimulation on the spooltaneous activity of prefrontal-cortical cells Single-pulse stimulation (1 Hz) of the LC did not result in reliable modification of the activity of cells in the medioprefrontal cortex. However, when a higher frequency of stimulation (> 10 Hz) was used, a marked decrease in the firing rate of cortical neurons was observed. Trains of 0.4-ms pulses

Rat prefrontal cortex dopaminergic and noradrenergic systems

519

Fig. I. Photomicrographs of recording and stimulating sites visualized on Nissl-stained brain sections. In (a), the arrow indicates a recording site in the medial prefrontal cortex (CPF). In (b)-(d), the thick arrows indicate the localization of the tip of the stimulating electrodes in the mediodorsal thalamic nucleus (MD), the ventral tegmental area (VTA) and the locus coeruleus (LC). SN, Substantia nigra; DTg, dorsal tegmental nucleus; Me5, trigeminal mesencephalic nucleus.

at a frequency of 20 Hz applied for 10 s were found to be particularly effective. Therefore, this stimulation paradigm was used routinely. It produced a reproducible long-lasting post-stimulus inhibition of the spontaneous discharge in 57% of the cells tested (n = 222) in layers III-VI of the medial prefrontal cortex (Table 1). These inhibitions lasted for 20-75 s, their average duration being 45 f 6 s. In addition, the

mean decrease in the firing rate of cortical cells during this inhibitory period was 70% as compared with the prestimulus basal firing (2.2 k 0.37 vs 7.4 + 0.97 spikes/s, P < 0.01). In most cases, the cell discharge slowed down for l-5 s immediately after the end of the stimulation. A complete cessation of the firing then occurred for 10-20 s and the cell activity then gradually returned to the prestimulus firing rate

Table 1. Effect of a -methyl-para-tyrosine and 6-hydroxydopamine pretreatment on the responses induced by locus coeruleus stimulation (20 Hz, 10 s) in medial prefrontal cortex Group of rats Control a-MpT 6-OHDA

No. of cell tested

No effect

Excitation

222 41 72

84 (38%) 35(85%) 57 (79%)

11 (5%) 0 2 (3%)

Inhibition 127 (57%) 6* (15%) 13* (18%)

a-MpT: Rats were treated with a-MpT (200 mg/kg) 19 and 2 h before the recording session. 6-OHDA: Animals in which NA ascending systems were lesioned by local 6-OHDA microinjection. *P < 0.001 (x2 test).

J. MANTZ et al.

520

LC

LC

U oJ

LU Q.

13

1 nl~

200

300

41:10

500

TIME (sec) Fig. 2. Time-frequency histogram showing a typical example of the inhibitory response induced by LC stimulation (20 Hz, I0 s) on the spontaneous activity of a prefrontal cortical neuron. The periods of stimulation are indicated by horizontal bars and the concomitant peaks correspond to stimulus artefacts. Note the long-lasting post-stimulus inhibition and the progressive recovery of the spontaneous firing.

(Fig. 2). In a few cases, this inhibition was seen during the application of the stimulus train. A small population of cells (5%) were excited while the remaining cells (38%) were not affected by the LC stimulation. When the stimulating electrode was placed in the vicinity but outside the LC, the number of inhibited cortical cells was dramatically decreased (62 units tested, P < 0.01). More precisely, when the electrode was located close to the ventral part or posterior to the LC, 8% of cortical cells only were still inhibited (38 cells tested). In one case where the electrode was positioned in the lateral part of the dorsal tegmental nucleus, 20% of cortical cells were still inhibited, but 16% were excited (24 cells tested). The inhibitory responses evoked by LC stimulation were reduced markedly after pharmacological (~-MpT) depletion of catecholamines or selective destruction (6-OHDA) of the ascending N A pathways (Table 1); ct-MpT pretreatment resulted in a marked decrease of cortical N A (80%) and D A (64%) levels (Table 2). In these animals, a small percentage (15%) of the cortical units tested responded to LC stimulation with a mean duration of inhibition of 2 6 + 5s. Two to 3 weeks after near-complete 6 - O H D A destruction of the ascending N A pathways (92% decrease in cortical N A levels and no significant change in cortical D A levels; see Table 2), few (18%) cortical cells displayed inhibitory responses, the mean duration of these responses being 31 + 5 s.

Effect o f noxious tail pinch on the activity of neurons in the medial prefrontal cortex The application of a noxious pinch to the tail for 10 s led to an activation of 26% of the population of

cells (n = 289) tested in the medial prefrontal cortex. Activated cells were found in all cortical layers examined (layers III-VI). In addition, an inverse correlation was found between the amplitude of the response and the level of the basal firing rate of the cells; indeed, cells with low basal firing rate exhibited higher magnitude of responses (r = - 0 . 6 0 , P < 0.01). The usual pattern of the response was a marked increase in the discharge frequency occurring 1-5 s after the onset of the pinch; this higher firing rate lasted throughout the pinch application and in some cases for a longer period (2-20 s). The response to tail pinch was reproducible when the noxious stimuli were applied consecutively at intervals of at least 2 min (Fig. 3). In a few cases (7%) inhibitory responses were observed, but a large population Table 2. Mean levels of noradrenaline and dopamine in the prefrontal cortex NA Control 2115+370 c~-MpT 429 __+47* (20%) 6-OHDA 185 +68* (8%)

DA 800+ 129 203 + 41" (36%) 711__+101NS

Results are expressed as mean __+S.E.M. (pg/mg protein). Control animals (n = 8) and both 6-OHDA- and ct-MpT-treated rats were anaesthetized with ketamine and used in recording sessions before biochemical assays. 6-OHDA (NA): Animals in which ascending NA pathways were destroyed by bilateral microinjections of 6-OHDA into the ascending NA pathways laterally to the pedunculus cerebellaris superior (n = 8). c~-MpT: Animals depleted in NA and DA by i.p. injections of ct-MpT (200 mg/kg) 19 and 2 h before the recording sessions (n = 8). *P < 0.01 (Mann-Whitney test). NS: not significant.

Rat prefrontal cortex dopaminergic and noradrenergic systems

Fig. 3. Example of a prefrontal cortical neuron excited by both tail pinch and heat. Noxious tail pinch (TP) was applied for 10 s; hot water was applied on the tail at increasing temperatures (51-53°C) for 20 s.

(67%) of the cells tested was not affected by the noxious stimuli. Units activated by noxious tail pinch also responded to noxious thermic stimulus. In six cells examined, immersion of the tail in a hot-water bath (51-53°C) for 20 s also induced a marked activation of the firing rate (Fig. 3). On the other hand, for no cell tested did hair movement, light touch, slight persistent pressure or passive joint movements affect the firing rate. Finally, no modification in the electrocortigram was observed during or after the application of the noxious tail pinch. Eflects of stimulation of the ventral tegmental area on evoked responses in the prefrontal cortex

Confirming previous results, the stimulation of the VTA (1 Hz) inhibited most (88%) of the cells recorded (n = 45) in the medial prefrontal cortex.rO This inhibitory response lasted for 110 f 24 ms with a mean latency of 18 f 3.5 ms. A complete cessation of cortical cell firing was observed when VTA stimulation was made at a higher frequency (10 Hz) but, contrasting with results obtained following LC stimulation (20 Hz), this inhibitory effect occurred only during and not after the period of VTA stimulation. As previously described,1° most of the cortical cells inhibited by VTA stimulation (l-10 Hz) could be excited by stimulation of the MD (5-10 Hz). VTA stimulation applied before that of MD markedly reduced the excitatory response in 60% of the units tested (n = 27) (Fig. 4). In these cells, as indicated by the number of evoked responses resulting from 15 successive MD stimulations, the probability of the appearance of evoked responses was reduced by 80% (P < 0.01) under VTA stimulation. VTA stimulation (10 Hz) inhibited completely the spontaneous activity of most cortical cells sensitive to tail pinch. For all neurons tested (n = 17), the excitatory response evoked by painful stimulus was completely blocked

when the stimulus was applied during VTA stimulation (10 Hz) but reappeared afterwards (Fig. 5). Effects of locus coeruleus stimulation on evoked responses in the medial prefrontal cortex

Of the 54 cortical cells tested, 31 were activated by MD stimulation (I-10 Hz) and 30 inhibited by LC stimulation (20 Hz, 10 s) while 18 of these cells responded to both MD and LC stimulations. In order to determine whether or not the inhibitory effect of the LC stimulation could block the excitatory response induced by MD stimulation, the MD was stimulated at different times after LC stimulation. Under these conditions, for all cells tested (n = 12), the MD-evoked excitatory response were always preserved (no significant change for 15 MD stimulations) even though the background activity of the cell was greatly reduced or completely blocked (Fig. 4). Similarly, in cells responding both to noxious tail pinch and to the LC stimulation, the excitatory response induced by the noxious stimulus was always preserved when tail pinch was applied during the post-stimulus inhibition induced by LC stimulation (Fig. 6). Such an effect resulted in an enhanced signal-to-noise ratio. Indeed, the ratio of the mean firing rate during tail pinch stimulation over the mean basal firing of the cells was 2.6 + 0.7 before LC stimulation and 10.3 + 5.6 (n = 11, P < 0.01) when tail pinch was applied during the post-stimulus inhibitory period induced by LC stimulation. Convergence of the effects of ventral tegmental area and locus coeruleus stimulations on prefrontal cortical neurons

Of 45 cortical cells tested, 18 showed typical inhibitory responses to both VTA and LC stimulations. Less than 50% of these cells (n = 8) could also be excited by the MD stimulation. These MD-evoked excitatory responses could be blocked by stimulation

J. MAXTZ (‘i d

MD

Mofaftef LC stlmularlon)

Ot

0

20

40

m sec.

eo

eo

Fig. 4. Effect of VTA and LC stimulation on the excitatory response induced by MD stimulation on a prefrontal cortical cell. Results are displayed as peri-stimulus-time histograms computed from I5 stimulus repetitions. From top to bottom: MD stimulation (10 Hz) induced an excitatory response (mean latency: 21 ms); VTA stimulation applied before that of MD blocked the excitatory response; by contrast, when MD stimulation was applied during the long-lasting post-stimulus inhibitory period induced by LC stimuIation (20 Hz, IOs), the excitatory response to MD stimulation was still present. Broken vertical bars correspond to VTA and MD stimulation artefacts.

of the VTA (for the eight cells tested) but not of the LC (for the eight cells tested} (Fig. 4). Of the eight cells which were sensitive to VTA, LC and MD stimulations, six of them also responded to noxious tail pinch. In these cases, the excitatory-evoked response was blocked when the noxious stimulus was applied during VTA stimulation. On the other hand, the excitatory-evoked response was preserved when the noxious tail pinch was applied during the post-

stimulus inhibition of the background by LC stimula~on.

firing induced

DISCUSSION

Several original observations were made in the present study. They can be surn~~~z~ as follows: (1) Repetitive stimulation (20 Hz, 10 s) of LC produced a long-lasting peri-stimulus inhibition of

523

Rat prefrontal cortex dopaminergic and noradrenergic systems

TP

TP -

t

LU)

TIME

TP

300

(set)

Fig. 5. Effect of VTA stimulation on the excitatory response evoked by noxious tail pinch on a prefrontal cortical cell. During VTA stimulation at IO Hz (horizontal bar) the spontaneous activity of the cell was inhibited completely and the excitatory response induced by tail pinch (TP, 10 s) could no longer be observed.

activity in 57% of the neurons tested in the medial prefrontal cortex of the rat. These inhibitory responses seem to be mediated by NA neurons since they were reduced markedly following pharmacological depletion of cortical catecholamines or selective 6-OHDA-indu~ degeneration of the ascending NA pathways. (2) A popuIation of ceils (26% of the cells tested) in the medial prefrontal cortex could be activated reproducibly by a noxious tail pinch. (3) The excitatory responses induced by noxious spontaneous

TP -

stimulus or stimulation of the MD (10 Hz) were suppressed completely during activation of the afferent DA neurons resulting from the VTA stimulation. In contrast, these two types of evoked responses were still present during the inhibitory period following LC stimulation. For comparison with our previous studies on the properties of mesocortico-p~fronta1 DA neurons, all experiments were made in ketamine-anaesthetized rats. This choice of anaesthetic can be challenged since ketamine interacts with several types of neuro-

TP -

TP -

lC(2OHzI .....

TIME

(see)

Fig. 6. Effect of LC stimulation on the excitatory response evoked by noxious tail pinch on a prefrontal cortical cell. When tail pinch (TP) was appiied during the long-lasting post-stimulus inhibitory period induced by LC stimulation (20 Hz, 10 s), the excitatory response to tail pinch was still present. The period of LC st~m~ation is indicated by dashed lines and the concomitant peak corresponds to the stimulus artefact.

transmissions and acts particularly as a noncompetitive antagonist of glutamate for receptors of the N-methyl-D-aspartate type. However, preliminary results similar to those described here have been recently obtained in halothane-anaesthetized rats.

somatosensory stimuli. This could be related to the type or the depth of anaesthesia used in our experiments7 Moreover. it cannot be excluded that nonnoxious peripheral stimulation might be effective tn the absence of anaesthesia. The responses induced by noxious stimulation cannot be attributed to a nonInfluence of the ascending noradrenergic neurons on the specific arousal of the animals since no modification sponta~eo~ activity of cells in the medial prefrontal of the el~trocorticogram nor signs of arousal were cortex observed during nociceptive stimulations. The most obvious effect of LC stimulation in As observed by other authors in the somatosensory ketamine-anaesthetized rats was a reduction in the cortex of halothane-anaesthetized rats, in our experispontaneous discharge rate of a population of cells ments, sensitive cells in the medial prefrontal cortex located in layers III-Vi of the media1 prefrontal of ketamine-anaesthetized rats were in most cases cortex. This inhibition was particularly pronounced excited by intense tail pinch.i5 Most neurons which and of long duration (up to 75 s) when repetitive were activated by noxious tail pinch were also restimulations of the LC were made (20 Hz, 10 s). This sponsive to noxious heat. Little is known concerning pattern of inhibitory response induced by stimulation the identity of the afferent fibres by which noxious of the LC was first observed on cerebettar Purkinje inputs are delivered to the rat cerebral cortex. Howcells and on hippocampal pyramidal neurons.‘3*27 ever, the midline thalamic nuclei which innervate the It has also been seen in other cortical areas of medial prefrontal cortex receive info~ation from chloral-hydrate-anaesthetized rats such as the cinboth the spinothalamic tract and the reticular forgulate, parietal, visual or sensorimotor cortex.9~i9,23*2Ymation and have been shown to respond to noxious stimuli.2,5*?1 This effect has been generally attributed to activation of ascending NA neurons. In fact, iontophoretic Respective roles of the ascending dopaminergic and application of NA on cortical, cerebehar or hippo~ora~energic neurom in the control of evoked recampal neurons mainly results in an inhibition of the spontaneous activity of the cell~.i~~~~~~~ In addition, the sponses in the mediaI prefrontal cortex The mesocortic~prefrontal DA neurons and the inhibitory responses induced by locai application of NA or the stimulation of the LC were shown to be LC-cortical NA neurons can modify markedfy the antagonized by iontophoretic application of activity of cells in the medial prefrontal cortex. /I-adrenergic blocking agents.iq Our results are in However, under appropriate stimulating conditions, most cortical neurons can be controlled by DA agreement with these findings, since the inhibitory neurons (88%) while NA neurons seem to regulate effect of LC stimulation on cells in the medial prethe activity of a more restricted population of cortical frontal cortex was reduced markedly in rats precells (56% of the celB tested}. Confirming the results treated with a-MpT to deplete the endogenous stores obtained in ~croiontophoretic studies which have of cat~holamines, or in animals with selective indicated that some cortical cells are sensitive both to 6-OHDA-induced lesions of the ascending NA DA and NA,6 the present study demonstrates that the neurons. The few remaining inhibitory responses activity of some cortical cells can be affected by observed in our expe~ments may be due either to the remaining stores of NA (ar-MpT-treated rats) or to activation of DA or NA neurons. It seems clear, the remaining NA Gbres (6-OHDA-treated rats) or to however, that the ascending DA and NA systems exert a completely distinct control in the transfer of a spread of the stimulating current to adjacent areas information in the prefrontal cortex. such as the latero-dorsal tegmental nucleus, a strucAs previously described,‘“*22 activation of the ture which also innervates the prefrontai cortex.” mesocortico-prefrontal DA neurons resulting from However, no inhibitory response could be observed when the stimulating electrode was placed 0.5 mm VTA stimulation inhibits the spontaneous activity of cortical neurons both in ketamine- and chloralventrally to the LC. Interestingly, and further SUPporting the involvement of NA neurons, the most hydrate-anaesthetized rats. In addition, VTA stimuefficient stimulation sites were found to be located in lation blocks excitatory inputs delivered from the the central part of the LC, in agreement with the mediodorsal thatamic nucleus.iO These effects can be observed under VTA stimulations made at a frelocalization of the NA cells projecting to the cerebral quency of 1 or 10 Hz. Interestingly, as shown in the cortex.32 present study, the activation of cortical cells evoked Activation by peripheral noxious stimuli of a popu- by noxious tail pinch was no longer observed when lation of cells in the medial prefrontal cortex the peripheral stimulus was applied during the VTA The present study indicates that a population of stimulation. In contrast to the results obtained following eleccells in the medial prefrontai cortex of the rat responds to noxious stimulations such as intense tail trical stimulation at 1 Hz of the VTA, the stimulation pinch or immersion of the tail in hot water. These of the LC with a frequency of 1 Hz did not induce a consistent inhibitory response on cortical ceils. AS cells, however, were not activated by mild peripheral

Rat prefrontal cortex dopaminergic and noradrenergic systems previously noted, activation of the NA system induced by stimulation of the LC at 20 Hz for 10 s did produce a long-lasting inhibition of the spontaneous firing of cortical cells. The efficiency of this particular pattern of stimulation of LC NA neurons could be related to the characteristics of NA fibres: they are thin and slowly conducting fibres, show abundant collateralization, variability in the excitability of NA cell bodies and fibres, and finally the presence of inhibitory recurrent collaterals.‘~3”~‘6~‘*However, despite its inhibitory action on the basal firing rate of target cortical cells, this activation of NA neurons did not block excitatory responses evoked from the MD or induced by noxious tail pinch. These results are in agreement with previous observations made in the monkey auditory cerebral cortex which first revealed a differential effect of microiontophoretic application of NA on spontaneous and evoked activity.‘* Such an enhancement of the signal-to-noise ratio has been reported following microiontophoretic application of NA or LC stimulation not only in the cerebral cortex but also in the hippocampus and the cerebellum, which also receive NA fibres originating from the LC.ll.34

Biochemical studies were the first to demonstrate

525

that the mesocortico-prefrontal DA neurons and the LC NA neurons can be activated under stressful situations.‘4*30 In addition, electrophysiological investigations have indicated that the NA cells of the LC are activated by noxious stimuli in anaesthetized rats and by somatic stimuli in awake animals.” Such an activation of NA neurons could amplify the effect of LC stimulation on the cortical tail pinch response. On the other hand, it has been reported that some VTA DA cells in anaesthetized animals respond to noxious stimuli.“,*’ It thus remains to be established whether the population of DA cells which innervate the medial prefrontal cortex may be affected by noxious peripheral stimuli such as tail pinch and to determine the temporal relationships between the changes in the activity of DA and NA neurons in order to delineate their respective involvement in the modulation of transfer of nociceptive inputs in the prefrontal cortex.

Acknowledgemenu-The

authors thank Monique Saffroy and Anne-Marie Godeheu for histological assistance. This study was supported by grants from INSERM and RhBnePoulenc Sante. J.M. is recipient of a fellowship from the Fondation pour la Recherche Medicale.

REFERENCES

1. Aghajanian G. K., Cedarbaum J. M. and Wang R. Y. (1977) Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons. Brain Res. 136, 57&577. 2. Albe-Fessard D., Berkley K. J., Kruger L., Ralston H. J. and Willis D. (1985) Diencephalic mechanisms of pain sensation. Brain Res. Rev. 9, 217-296. 3. Aston-Jones G., Segal M. and Bloom F. E. (1980) Brain aminergic axons exhibit marked variability in conduction velocity. Brain Res. 195, 215-222. 4. Berger B., Thierry A. M., Tassin J. P. and Moyne M. A. (1976) Dopaminergic innervation of the rat prefrontal cortex: a fluorescence his&hen-&l study. Brain Res. 106, 133-145. 5. Besson J. M., Guilbaud G., Abdelmoumene M. and Chaouch A. (1982) Physiologie de la nociception. J. Physiol. (Paris) 78, 7-107. 6. Bunney B. S. and Aghajanian G. K. (1976) Dopamine and norepinephrine innervated cells in the rat prefrontal cortex: pharmacological differentiation using microiontophoretic techniques. Life Sci. 19, 17831792. 7. Chapin J. K., Waterhouse B. D. and Woodward D. J. (1981) Differences in cutaneous sensory response properties of single somatosensory cortical neurons in awake and halothane anaesthetized rats. Brain Res. Bull. 6, 63-70. 8. Descarries L., Lemay B., Doucet G. and Berger B. (1987) Regional and laminar density of the dopamine innervation in the adult rat cerebral cortex. Neuroscience 21, 807-824. 9. Dillier N., Laszlo J., Milller B., Koella W. P. and Olpe H. R. (1978) Activation of an inhibitory noradrenergic pathway projecting from the locus coeruleus to the cingulate cortex of the rat. Brain Res. 154, 61-68. 10. Ferron A., Thierry A. M., Le Douarin C. and Glowinski J. (1984) Inhibitory influence of the mesocortical dopaminergic system on spontaneous activity or excitatory response induced from the thalamic mediodorsal nucleus in the rat medial prefrontal cortex. Brain Res. 302, 257-265. 11. Foote S. L., Bloom F. E. and Aston-Jones G. (1983) Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol. Rev. 63, 844914. 12. Foote S. L., Freedman R. and Oliver A. P. (1975) Effects of putative neurotransmitters on neuronal activity in monkey auditory cortex. Brain Res. 86, 229-242. 13. Hoffer B. J., S&gins G. R., Oliver A. P. and Bloom F. E. (1973) Activation of the pathway from locus coeruleus to rat cerebellar Purkinje neurons: pharmacological evidence for noradrenergic central inhibition. J. Pharmac. exp. Ther. 184, 553-569.

14. Korf J., Aghajanian G. K. and Roth R. H. (1973) Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of the locus coeruleus. Neuropharmacology 12, 933-938. 15. Lamour Y., Willer J. C. and Guilbaud G. (1983) Rat somatosensory (SmI) cortex: I. Characteristics of neuronal responses to noxious stimulation and comoarison with resoonses to non-noxious stimulation. EXDI Brain Res. 49. 3525. 16. Lindvall 0. and Bjiirklund A. (1984) General organization of cortical monoamine systems. In Monoamine Innervation of Cerebral Cortex (eds Descarries L., Reader T. R. and Jasoer H. H.). DD. 9-40. Alan R. Liss. New York. 17. Maeda H. and Mogenson G. J. (1982) Effects of peripheral stimulation on the activity of neurons in the ventral tegmental area, substantia nigra and midbrain reticular formation of rats. Brain Res. Bull. 8, 7-14. 18. Nakamura S. (1977) Some electrophysiological properties of neurones of rat locus coeruleus. J. Physiof. 267,64L658.

19. Olpe H. R., Glatt A., Laszlo J. and Shallenberg A. (1980) Some electrophysiological and pharmacological prc)pcrtich of the cortical, noradrenergic projection of the locus coeruleus in the rat. Brain Res. 186, 9.~19. 20. Paxinos G. and Watson C. (1982) The Rut Brain in Srerentlr.\-ic.Coordinutes. Academic Press, New York. 21. Pechanski M.. Guilbaud G. and Gautron M. (1981) Posterior int~laminar region in rat: neuronal responses to noxious and non-noxious cutaneous stimuli. E.~pf Neural. 72, 226238. 22. Peterson S. L., St Mary J. S. and Harding N. R. (1987) Cic-flupentixol antagonism of the rat prefrontal cortex ncuronai response to apomorphine and ventral tegmental area input. Brain Res. BUN. 18, 723-729. 23. Phillis J. W. and Kostopoulos G. K. (1977) Activation of a noradrenergic pathway from the brainstem to rat cerebral cortex. Gen. Pharmac. 8, 207-21 I. 24. Sakanaka M., Shiosoka S., Takatsuki K. and Tohyama M. (1983) Evidence for the existence of a substance P containtng pathway from the nucleus latero-dorsalis tegmenti (casteldi) to the medial prefrontal cortex of the rat. Bruin Re,v. 259, 123-126. 25. Schultz W. and Romo R. (1987) Responses of nigrostriatal dopamine neurons to high intensity somatosensory stimulation in the anaestbetized monkey. J. Neurophysiol. 57, 201-217. 26. Segal M. and Bloom F. E. (1974) The action of norepinephrine in the rat hippocampus. 1. Iontophoretic studies. Brtrin Rex 72, 79-97. 27. Segal M. and Bloom F. (19’74) The action of norepinsphrine in the rat hippocampus. 11. Activation of the input pathway. Brain Rex 72, 99-114. 28. Tassin J. P., Studler J. M., Herve D., Blanc G. and Glowinski J. (1986) Contribution of noradrenergic neurons to the regulation of dopaminergic (Dl) receptor denervation supersensitivity in rat prefrontal cortex. J. Neurochem. 46, 243-248. 29. Taylor D. A. and Stone T. W. (1980) The action of adenosine on norad~nergic neuronal inhibition induced by stimulation of locus coeruleus. Brain Res. 183, 367-376. 30. Thierry A. M., Tassin J. P., Blanc G. and Glowinski J. (1976) Selective activation of the mesocortical DA system by stress. Nature 263, 242-244. ot 31. Wagner J., Vitali P., Palfreyman M. G., Zraika M. and Huot S. (1982) Simultaneous determination 3,4-dihydroxyphenylalanine, %hydroxytryptophan, dopamine, 4-hydroxy-3-methoxyphenylalanine, norepinephrine, 3,~dihydrox~henylacetic acid, homovanillic acid, serotonin and 5-hydroxyindoIa~tic acid in rat cerebrospinal fluid and brain by high-~rfo~ance liquid chromatography with electrochemical detection, J. Neuroc~em. 38, 1241~-1260. 32. Waterhouse B. D., Lin C. S., Burne R. A. and Woodward D. J. (1983) The distribution of neocortical projection neurons in the locus coeruleus. J. camp. Neurol. 217, 418-431. 33. Waterhouse B. D. and Woodward D. (1980) Interaction of norepinephrine with cerebrocortical activity evoked by stimulation of somatosensory afferent pathways in the rat. Expr Neural. 67, 1 I -34. actions of 34. Woodward D. J., Moises H. C., Waterhouse B. D., Hoffer B. J. and Freedman R. (1979) M~ulato~ norep~neph~ne in the central nervous system. Fedn Proc. Fe& Am. Sacs exp. Biol. 38, 2109-2116. (Accepted 21 April 1988)