nicotinic receptors in the rat prefrontal cortex: increase in glutamate release and facilitation of...

European Journal of Neuroscience, Vol. 11, pp. 18–30, 1999 © European Neuroscience Association

Nicotinic receptors in the rat prefrontal cortex: increase inglutamate release and facilitation of mediodorsalthalamo-cortical transmission

Y. Gioanni, C. Rougeot,3 P. B. S. Clarke,1 C. Lepouse, A. M. Thierry and C. Vidal2INSERM U114, College de France, 11 Place Marcelin-Berthelot, Paris 75231 Cedex 05, France1Pharmacology and Therapeutics, McGill University, 3655 Drummond St. Room 1325, Montreal, Canada H3G 1Y62Molecular Virology, Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France3Genetics and Biochemistry of Development, Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France

Keywords: in vivo electrophysiology, microdialysis, nicotine binding

Abstract

The modulatory influence of nicotinic acetylcholine receptor (nAChRs) on thalamocortical transmission was characterized in theprelimbic area (PrL) of the rat prefrontal cortex. In the first experiment, rats received a unilateral excitotoxic lesion centred on themediodorsal thalamic nucleus (MD), and were sacrificed 1 week later. The lesion resulted in a 40% reduction of 3H-nicotineautoradiographic labelling in the ipsilateral prefrontal cortex, particularly in areas that are innervated by the MD. Electrophysiologicalexperiments were subsequently performed in non-lesioned anaesthetized animals, in order to study modulation of short- and long-latency responses of PrL neurons evoked by electrical stimulation of the MD. The short-latency responses result from activationof the MD–PrL pathway and are mediated via AMPA-type glutamatergic receptors, whereas the long-latency responses reflectactivation of the recurrent collaterals of cortical pyramidal neurons. Iontophoretic application of nicotinic agonists (nicotine, DMPP)facilitated both types of response. Local application of the nAChR antagonists dihydro-beta-erythroidine, mecamylamine andmethyllycaconitine, prevented both kinds of facilitation. Finally, intracerebral microdialysis experiments were performed in order totest for nicotinic modulation of extracellular glutamate concentrations in the PrL. Direct application of nicotine via the dialysis probeincreased glutamate levels in a dose-dependent manner. This effect was blocked by local perfusion of dihydro-beta-erythroidine.These findings therefore provide anatomical and functional evidence for nAChR-mediated modulation of thalamocortical input tothe prefrontal cortex. Such a mechanism may be relevant to the cognitive effects of nicotine and nicotinic antagonists.

Introduction

Evidence from human and animal studies suggests that cholinergicneurotransmission mediated by nicotinic acetylcholine receptors(nAChRs) is important in higher cognitive functions. In particular,attentional processes associated with working memory are markedlyimproved following nicotine administration (Levin, 1992; Arnericet al., 1995; Muir et al., 1995). Similar attentional functions arealtered after lesions of cortical prefrontal areas in humans and inexperimental animals (Fuster, 1989; Owenet al., 1991; Granonet al.,1995). These observations raise the possibility that the prefrontalcortex (PFC) may be an important target for the memory-enhancingeffects of nicotine (Vidal, 1994).

Two main types of nicotinic radioligand binding sites have beenthus far identified in mammalian brain. One population is labelledby 125I-α-bungarotoxin (Seguelaet al., 1993), the other by nicotinicagonists such as3H-nicotine,3H-acetylcholine, and3H-cytisine (Marks& Collins, 1982; Schwartzet al., 1982; Happeet al., 1994). Bothpopulations are expressed in the cerebral cortex of the rat (Clarkeet al., 1985). Convergent evidence suggests that nAChRs labelled by3H-nicotine are localized on the terminals of many thalamocorticalprojection neurons. First,3H-nicotine binding is enriched in cortical

Correspondence:Dr Yves Gioanni, as above. E-mail: [email protected]

Received 12 March 1998, revised 21 July 1998, accepted 22 July 1998

laminae that receive the densest thalamic input, both in the cat(Prusky et al., 1987; Parkinsonet al., 1988) and in the rat (Clarkeet al., 1984; Zilleset al., 1989; Happeet al., 1994); this is not thecase for125I-α-bungarotoxin binding (Clarkeet al., 1985; Fuchs &Schwark, 1993). Second, cortical3H-nicotine labelling is little alteredby local excitotoxic lesions, but is reduced following excitoxic lesionsof the thalamus (Pruskyet al., 1987; Parkinsonet al., 1988; Sahinet al., 1992; Lavineet al., 1997), reflecting a reduction of receptordensity rather than affinity (Lavineet al., 1997). Analogous experi-ments have not been performed with125I-α-bungarotoxin. Third, theareal and laminal pattern of reduced binding closely matches theextent of deafferentation (Pruskyet al., 1987; Lavineet al., 1997).Fourth, many thalamic nuclei strongly express message for alpha4and beta2 nAChR subunits (Wadaet al., 1989), which are commonlyassociated with3H-nicotinic agonist binding sites (Whiting & Lind-strom, 1988; Floreset al., 1992). In contrast, thalamic expression ofalpha7 subunit mRNA is very low, suggesting that whatever nAChRsubtype(s) may expressed on thalamocortical terminals, they areunlikely to bindα-bungarotoxin (Seguelaet al., 1993).

In the rat, the association of3H-nicotine binding with thalamocort-ical pathways has been seen not only within sensory and motor relayprojections, but also in associational projections arising from theanterior thalamic group (i.e. anterodorsal, anteromedial and anteroven-tral nuclei) (Lavineet al., 1997). Although high-affinity3H-agonist

Nicotinic receptors in the rat prefrontal cortex 19

binding sites have also been described in the rat PFC (Happeet al.,1994), it remains to be established whether these nAChRs or anysubtype of nAChRs are also located on thalamic afferents in thiscortical area.

Electrophysiologicalin vitro experiments suggest that nicotine canincrease excitatory transmission in the PFC by enhancing the releaseof glutamate (Vidal & Changeux, 1993). In this earlier study, short-latency excitatory postsynaptic potentials (EPSPs) were evoked inlayers II–III of the PFC by electrical stimulation of the superficialcortical layers. These EPSPs were mediated by glutamatergic AMPA-type receptors, and were increased by application of nicotine. Evidencesuggested that the facilitatory effect of nicotine was due to theactivation of presynaptic nAChRs located on glutamatergic terminals.

The PFC is reciprocally connected to the mediodorsal thalamicnucleus (MD) (Rose & Woolsey, 1948). MD afferents are mainlylocated in superficial layers (I–III) of the PFC, whereas PFC neuronsprojecting to the MD are located in layer VI and, to a lesser extent,in layer V (Krettek & Price, 1977; Groenewegen, 1988). On the basisof cytoarchitectonic and anatomical connections, the PFC has beensubdivided into several areas (Krettek & Price, 1977; Groenewegen,1988). In a previous work, we have shown that electrical stimulationof the rostral part of the MD induces two main types of excitatoryresponses in the prelimbic area of the PFC (PrL), differing in latency(Pirot et al., 1994). The short-latency responses (, 4 ms) are evokedby low frequency stimulation (0.3–1 Hz) of the MD and are observedmainly in superficial layers of the PrL; these responses result fromactivation of the MD–PrL pathway. In contrast, the long-latencyresponses (. 10 ms), induced by stimulating the MD at a frequencyof 3–10 Hz, are observed more frequently in deep than in superficiallayers. These long-latency responses probably result from activationof recurrent collaterals of the PrL–MD pathway which originatesfrom cortical layers V and VI. Both types of excitatory responseswere blocked by the application of CNQX, an AMPA receptorantagonist (Pirotet al., 1994, 1995).

The aim of the present study was to determine the effect of theactivation of nAChRs on thalamocortical transmission in the PrL.Three approaches were used. First,3H-nicotine autoradiography wascombined with excitotoxic lesions of the MD, in order to test forthe presence of nAChRs on MD–PrL afferent fibres. Second, anelectrophysiological approach was used, to determine the effect oflocal applications of nicotinic agonists on the excitatory responsesinduced in the PrL by MD stimulation. Third, the effect of nicotineon extracellular levels of glutamate in the PrL was investigated, usingthe microdialysis technique in freely moving rats.

Materials and methods

All surgical and animal care procedures adhered to the Society forNeuroscience Handbook (1991) guidelines.

Autoradiographic experiments

Excitotoxic lesions

Male adult Long-Evans rats (Charles River, St. Constant, Quebec),weighing 230–270 g, were anaesthetized for surgery with pentobarbital(65 mg/kg i.p.). A unilateral stereotaxic infusion ofN-methyl-D-aspartate (NMDA) or vehicle (control) was made into the MD via a30-gauge stainless steel cannula attached by polyethylene tubing toa 5µL Hamilton syringe driven by a syringe pump. The stereotaxiccoordinates were: AP: – 2.8 mm from bregma, L: 0.7 mm and DV:5.4 mm from skull (Paxinos & Watson, 1986). NMDA (0.12M) wasdissolved in vehicle consisting of 0.1M Na phosphate buffer (pH 7.4).

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 18–30

A volume of 0.4µL was infused over 4 min, followed by a further4 min delay before cannula removal. Animals were killed by decapita-tion 7–8 days after surgery.

3H-nicotine autoradiography and histology

To assess the extent of the excitotoxic lesion, coronal tissue sections(20 µm thick) were taken at intervals of 0.5 mm through the anteropos-terior extent of the thalamus. Two sections at each level were Nissl-stained with thionine. Thalamic nuclei were distinguished by Nisslstaining patterns, examined at3 50–3 400 magnification. Thelesioned area was identified by loss of neurons and by gliosis. Onlyrats that had received accurately located lesions were processed forautoradiography.

For 3H-nicotine autoradiography, coronal sections were taken attwo anteroposterior levels of the frontal cortex, corresponding to 12and 12.8 anterior to interaural zero according to the atlas of Paxinos& Watson (1986). Slide-mounted sections were processed for3H-nicotine autoradiography, as previously described (Lavineet al.,1997). Briefly, thawed sections were first fixed in 10% formalin, andrinsed in distilled water. Previous tests have shown that this procedurepreserves tissue quality but has no discernible effect on3H-nicotinebinding (Lavine et al., 1997). Sections were then incubated witha non-saturating concentration of3H-nicotine (4.1 nM) in 50 mM

trishydroxymethyl-aminomethane (Tris)/8 mM Ca21 buffer (pH 7.4)containing 0.1µM mercaptoacetic acid (antioxidant), for 20 min atRT, in a nitrogen atmosphere protected from light. At each level, twopairs of sections were taken for3H-nicotine labelling (one pair todefine non-displaceable binding, defined by the addition of 10–5 M L-nicotine bitartrate). Sections were removed and gently agitated infour consecutive washes of 30 s each, in Tris/Ca21 buffer (pH 7.4 at4 °C), and immediately dried under a stream of cold air. Sectionswere subsequently apposed tightly against tritium-sensitive film(Hyperfilm, Amersham, Oakville, Ontario, Canada), together withtritium-impregnated methylacrylate standards (range: 0.69–58 fmol/mg wet tissue;3H-Microscales, Amersham). Film exposure was of6 months duration at RT. Films were processed in Kodak D19developer at RT for 5 min, then fixed for 4 min. Finally, the radiolabel-led tissue sections were stained with thionine.

Image analysis was performed using an MCID M4 microcomputer-based system (Imaging Research, St. Catherine’s, Ontario, Canada).Binding of 3H-nicotine was measured from two tissue sections perrat per condition. Brain structures were identified from Nissl-stainedsections with reference to the stereotaxic atlas of Paxinos & Watson(1986). In each area, a rectangular sampling frame (0.2 mm wide)was placed tangentially over the band of highest3H-nicotine labelling(Fig. 2). Optical density measurements were converted into wet tissueequivalent binding (fmol/mg), with reference to the tritium standards.

Data analysis

Statistical analyses were performed using commercial software (Sys-tat, Evanston, IL, USA). As NMDA-associated changes in bindingwere found to be minimal or absent on the unlesioned side, theprincipal dependent variable was the percentage of binding on thelesioned side compared with the unlesioned side, using each rat asits own control. Comparisons between lesioned and sham groupswere made by Student’st-tests with the Bonferroni correction(Glantz, 1992).

In vivo electrophysiological experimentsPreparation

Experiments were performed in Sprague–Dawley rats (250–300 g,Charles River, Saint-Aubin les Elbeuf, France) under ketamine

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(Imalgene 500, Rhoˆne-Merieux, France) anaesthesia (80 mg/kg, i.p.).Additional i.m. injections (80 mg/kg) were made to maintain a stablelevel of anaesthesia. The animals were placed in a stereotaxic frame(Unimecanique, Epinay sur Seine, France). Two craniotomies wereperformed on the same side, one exposing the PFC and the otherallowing the introduction of the stimulating electrode into the MD.

Electrical stimulation and extracellular recording

A bipolar coaxial stimulating electrode was positioned in the MD(A: 1 6.4 mm, L: 0.4 mm, H:1 4.6 mm, with respect to interauralzero; Paxinos & Watson, 1986). Electrical stimuli consisted of squarewave pulses (0.3 ms duration, 0.2–0.8 mA intensity) delivered at afrequency of 0.3–10 Hz. Single unit activity of PrL neurons locatedin layers III–VI (A: 1 12–13 mm, L: 0.4–1 mm, H: 2–4 mm fromthe cortical surface) was recorded with glass micropipettes filled with4% pontamine sky blue dissolved in 0.4M NaCl solution (impedance6–10 MΩ). Neuronal activity was logged on a computer (IBM PC)connected to a CED 1401 interface (Cambridge Electronic DesignLtd, Cambridge, UK), to generate on line peristimulus time histo-grams (PSTHs).

Iontophoresis and pressure applications

A five-barrel electrode was glued to the recording electrode (tip-tipdistance of 10–15µm), this particular disposition prevents currentartefacts (Crossmanet al., 1974). Individual barrels were filled withone of the following solutions: nicotine tartrate (100 mM, pH 4),1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP, 100 mM, pH 4),mecamylamine hydrochloride (10 mM, pH 4), dihydro-beta-eryth-roidine hydrobromide (DHBE, 50 mM, pH 7), methyllycaconitinecitrate (MLA, 10 mM, pH 7) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1 mM) (RBI, Natick, MA, USA). All compounds werediluted in NaCl 0.9%. Nicotine, DMPP, mecamylamine and CNQXwere applied by iontophoresis. A retaining current of 5–15 nA wasapplied to each barrel and drugs were expelled either with positivecurrents (nicotine, DMPP, mecamylamine,1 50 to 1 90 nA), ornegative currents (CNQX, – 60 to – 70 nA), (Bionic Instruments, Briisur Forge, France). DHBE and MLA were applied by micropressure(pneumatic picopump, PV 800, WPI), using pulses of 50–100 msduration, at a frequency of 0.2–0.5 Hz, and the pressure of ejectionwas 10–20 psi. In the experiments where nicotinic antagonists weretested, a nicotinic antagonist (mecamylamine, DHBE or MLA) wasejected (during 2–4 min) and a nicotinic agonist (DMPP or nicotine)was then applied. The ejection of the nicotinic antagonist wasmaintained during the application of the nicotinic agonist.

Data analysis

Excitatory responses induced by MD stimulation were quantified onPSTHs (1 ms bins, 50 cumulative sweeps). These excitations wereconsidered to be facilitated by the application of a nicotinic agonistwhen the response was increased by at least 50%. The increase ofresponses was calculated as follows: [(number of spikes undernicotinic agonist – number of spikes without nicotinic agonist)/numberof spikes without nicotinic agonist]3 100. It was considered thatapplication of nicotinic or glutamatergic antagonists blocked theagonist-induced facilitation when this facilitatory effect was reducedby at least 60%.

Histological verifications

At the end of each experiment, the position of the stimulatingelectrode was marked by electrical deposit of iron (8µA positive


current, 20 s) and observed on histological sections following a ferri-ferrocyanide reaction. In order to determine the position of recordedcortical cells, the tip of the recording microelectrode was marked byiontophoretic ejection of pontamine sky blue. Blue points wereobserved on coronal brain sections (80µm) stained with safranine.

In vivo microdialysis experiments

Adult male Sprague–Dawley rats (150–200 g) were anaesthetizedwith pentobarbital and implanted with a guide cannula in the PrL(AP: 1 3.7 mm from bregma, L: 0.5 mm from midline and DV:– 4 mm from skull; Paxinos & Watson, 1986). The guide cannulawas secured with dental cement to anchoring screws fixed in theskull. The rats were allowed to recover for 3 days before theexperiments.

The microdialysis probes were 2 mm effective length, 0.24 mmouter diameter, 20 kDa cut-off (CMA/11, Carnegie Medicine). Onthe day of the experiment, the rat was placed in a Plexiglas cagewith free access to food and water. The probes were inserted into thebrain cannulas, and perfused at a rate of 1µL/min with artificialcerebrospinal fluid (ACSF) containing (in mM): NaCl, 124; KCl, 3.3;CaCl2, 1.2; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 0.4. Fractions werecollected every 10 or 20 min. Prior to sample collection, the animalswere dialysed for 90 min to allow the perfusate to reach equilibriumconcentration.

At the end of the experiments, the rats were killed and the cannulainjected with methylene blue dye. The brain was removed and fixedin 10% formalin for gross histological confirmation of cannulaplacement. Only animals with cannulas located in the PrL wereincluded in the present study.

TheL-glutamate content of the dialysates was determined by high-performance liquid chromatography using precolumn derivatizationwith ortho-phtaldialdehyde and fluorescence detection as describedby Skilling et al. (1988). In vitro relative recovery by the dialysisprobe, determined at RT, was 106 0.4% for glutamate (n 5 3) at1 µL/min flow rate. All data are uncorrected for recovery and aredisplayed as the percentage of the mean6 SEM of the first threebaseline samples. At least three rats were tested for each treatment.Analysis of variance was used for statistical analysis of drug effectsas compared with baseline values. A confidence level of 95% wasaccepted as significant.

Drugs and suppliers were as follows: mecamylamine,L-nicotinehydrogen tartrate and tetrodotoxin (TTX) (Sigma, St Quentin, France);1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), dihydro-beta-erythroidine (DHBE), methyllycaconitine citrate (MLA), and NMDA(Research Biochemicals International, Natick, MA, USA).

Results


Histology

Control (i.e. vehicle-infused) rats sustained only a small degree ofgliosis along the cannula tract. Infusion of NMDA resulted in a majorloss of neuronal cell bodies local to the injection site, with associatedgliosis. On the basis of histology, seven lesioned rats were selectedfor autoradiography. In all seven rats, the zone of neuronal lossincluded virtually the entire MD and a ventromedial portion of theanterior hippocampus (Fig. 1). Partial damage occurred in severaladjacent thalamic nuclei: anterodorsal (five of seven rats), mediodorsalpart of laterodorsal (five of seven), mediorostral part of lateroposterior(four of seven), centrolateral (four of seven), parafascicular (threeof seven), anteroventral (two of seven), anteromedial, paratenial,


FIG. 1. Extent of lesions determined by histological examination. Excitotoxic (NMDA) lesions were centred on the mediodorsal thalamic nucleus (MD). Coronalsections were taken at different levels and were Nissl-stained with thionine to assess the extent of neuronal loss. The panels and abbreviations are based on theatlas of Paxinos & Watson (1997), with distances shown in millimetres anterior to interaural zero. The cross-hatching depicts, at each level, the smallest lesion(if present) and the largest lesion.


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FIG. 2. Upper panel: cortical areas sampled for quantification of3H-nicotinelabelling, abbreviated as follows: prelimbic (PrL), cingulate area 1 (Cg1),frontal areas 2 and 3 (Fr2, Fr3), agranular insular area (AI) and lateral orbital(LO). The rostrocaudal level is 12.0 mm anterior to lambda (Paxinos &Watson, 1997). Lower panel:3H-nicotine autoradiograph from a representativerat, 1 week after a unilateral (left side) excitotoxic NMDA lesion of themediodorsal thalamic nucleus (MD). Coronal sections were incubated with anon-saturating concentration of3H-nicotine. Note the reduction of labellingipsilateral to the lesion in PrL, Cg1 and LO.

paracentral, and posterior (one of seven each). The anterior lateralhabenula sustained damage in three of seven rats. There was nodetectable cell loss in cerebral cortex.

3H-nicotine autoradiography

Autoradiographs obtained from both anteroposterior levels (i.e.µ 12.0and 12.8 anterior to interaural zero) revealed similar patterns oflesion-induced change. Quantitative analysis was restricted to theposterior level (Figs 2 and 3). Six cortical areas were sampled (Fig. 2):PrL, cingulate area 1 (Cg1), frontal areas 2 and 3 (Fr2, Fr3), agranularinsular area (AI), and lateral orbital (LO). Nomenclature was basedon the stereotaxic atlas of Paxinos & Watson (1986) except for PrL(Paxinos & Watson, 1997). Of these, all but Fr3 received a projectionfrom the MD (Groenewegen, 1988).

3H-nicotine labelling was heaviest in layers I and III. Administrationof the excitotoxin NMDA did not appreciably affect contralateralbinding in any of the six areas sampled; thus, sham and lesion groupsdid not differ significantly (Bonferronit-test,P . 0.5 for all areas),and group mean values were similar. Mean6 SEM (fmol/mg wet


FIG. 3. Quantification of 3H-nicotine autoradiography. Rats received aunilateral excitotoxic (NMDA) lesion (n 5 7) or control (vehicle) infusion(n 5 4) into the mediodorsal thalamic nucleus (MD). After 1 week survival,coronal sections were incubated with a non-saturating concentration of3H-nicotine. Labelling was quantified by reference to3H-impregnated plasticstandards. Labelling ipsilateral to the lesion is expressed as a percentage ofcontralateral labelling, which was not affected by the lesion. *P , 0.01,** P , 0.001 vs. vehicle group (Bonferronit-test). Cortical areas areabbreviated as follows: prelimbic (PrL), cingulate area 1 (Cg1), frontal areas2 and 3 (Fr2, Fr3), agranular insular area (AI), and lateral orbital (LO).

TABLE 1. Effects of iontophoretic application of nicotine or DMPP on theshort- and long-latency excitatory responses evoked in the prelimbic corticalarea by electrical stimulation of MD

Short-latency responses Long-latency responses

Agonists n* no change facilitation n no change facilitation

Nicotine 55 14 41 91 40 51DMPP 25 5 20 37 19 18Total 80 19 61 128 59 69

*n 5 number of prelimbic cortical cells tested, unaltered, or facilitated byagonist, respectively.

FIG. 4. Effect of iontophoretic application of nicotine on a short-latencyexcitatory response induced by 0.5 Hz electrical stimulation of the MD in aprelimbic cortical cell. Post-stimulus time histograms are shown, eachcomprising 50 cumulative sweeps. The first bar of each histogram correspondsto the stimulus artefact. The application of nicotine (50 nA) increased theexcitatory response by 76%. Numbers in parentheses indicate the number ofspikes. The experimental design is represented in the top right diagram.


FIG. 5. Effect of iontophoretic application of nicotine on a long-latencyexcitatory response induced by 3 Hz stimulation of the MD in a prelimbiccortical cell. Post-stimulus time histograms are shown, each comprising 50cumulative sweeps. The first bar of each histogram corresponds to the stimulusartefact. The application of nicotine (80 nA) increased the excitatory responseby 172%. Numbers in parentheses indicate the number of spikes. Theexperimental design is represented in the top right diagram.

weight equivalent) values were as follows (sham vs. lesion groups):30.16 2.3 vs. 34.66 6.2 (PrL), 29.86 1.7 vs. 31.26 3.3 (Cg1),28.36 1.7 vs. 29.26 3.0 (Fr2), 27.56 1.5 vs. 29.06 4.3 (Fr3),29.36 1.7 vs. 30.36 4.0 (LO), and 22.36 1.6 vs. 21.56 2.2 (AI).

As shown in Fig. 3, NMDA infusion significantly reduced bindingon the ipsilateral side in certain cortical areas that receive a projectionfrom the MD: PrL (by 41%), Cg1 (by 35%), LO (by 40%). Bindingalso appeared reduced in Fr2 (by 15%), but this did not reachsignificance after the Bonferroni adjustment had been made(P , 0.087). No change was seen in AI, which receives an inputfrom MD, or in Fr3, which does not.

In vivo electrophysiological experiments

The effects of iontophoretic application of nicotinic agonists (nicotine,DMPP) in the PFC were investigated on the short-latencies and onthe long-latency responses evoked by the MD stimulation. Then, theaptitude of several nicotinic antagonists (DHBE, mecamylamine orMLA) to prevent the facilitatory effect of the nicotinic agonists wastested. Finally, the effect of the AMPA receptor antagonist CNQXon the MD evoked responses and on their facilitation by nicotinewas analysed.

Effects of nicotinic agonists

Short-latency responses

The effects of iontophoretic applications of nicotine or DMPP weretested on short-latency excitatory responses (, 4 ms) evoked by MDstimulation (0.3–1 Hz) in 80 cells located in superficial layers(II–III) of the PrL. Iontophoretic application of nicotine and DMPPproduced an increase in the excitatory responses (mean value: 86%)in 76% and 80% of cells, respectively (Table 1). This facilitatoryeffect was observed within 1–3 min following the beginning of theiontophoretic application of the agonists, and recovered to controlvalues 4–6 min after cessation of the application. No difference wasseen between the actions of nicotine and DMPP. Typical examplesof the effects of nicotine and DMPP are illustrated in Figs 4 and 6,respectively.


Long-latency responses

Iontophoretic applications of nicotine (Fig. 5) or DMPP (Fig. 7)induced a facilitation of the long-latency excitatory responses(. 10 ms) evoked by MD stimulation (2–10 Hz) as seen in 54% ofthe PrL cells recorded in layers V–VI (n 5 128, Table 1). Theseexcitatory responses were increased by 160% on average. The timecourse of the facilitatory effect was similar to the one observed forthe short-latency responses, i.e. onset within 1–3 min and 4–6 minfor recovery.

Spontaneous activity

Most of the recorded cells had a low spontaneous activity or weresilent. In the case of 11 cells which had a spontaneous activity higherthan 1 Hz, nicotine application did not modify their rate of discharge.Moreover, the application of nicotinic agonists never produced spikingof the silent neurons.

Effects of the nicotinic antagonists

Short-latency responses

The action of local application of the nicotinic antagonists, DHBE,mecamylamine and MLA was tested on the facilitatory effects ofnicotine and DMPP on short-latency responses evoked by MDstimulation. Blockade of facilitation by nicotine or DMPP wasobserved in four of the six cells tested with DHBE, and in all thecells tested with mecamylamine (n 5 9) or MLA (n 5 6) (Table 2).Altogether, 88% of the 17 cells tested were sensitive to the antagonists.A typical experiment is depicted in Fig. 6, which shows the blockadeby DHBE of the facilitation induced by DMPP.

Long-latency responses

Application of DHBE, mecamylamine or MLA prevented the facilita-tory effect induced by nicotine or DMPP on the long-latency excitatoryresponses in 69% of the cases (n 5 32, Table 2). A representativeexperiment showing the blockade by MLA of the facilitatory actionof DMPP is presented on Fig. 7. The facilitatory effect of nicotineor DMPP was blocked in most cells tested with mecamylamine (sixof eight cells), DHBE (eight of 11 cells) and MLA (eight of 13cells) (Table 2).

In a few cases, the application of a nicotinic antagonist alonemodified the short- or the long-latency excitatory responses evokedby MD stimulation. DHBE increased the excitatory responses in fourof 17 cells (70–90% of increase), while mecamylamine had variableeffects, as it was inhibitory in three cells (60–90% of decrease) andfacilitatory in three (80–90% of increase) of 13 tested cells. Incontrast, MLA rarely modified the excitatory responses (inhibitory intwo cells, 60–80% of decrease, and facilitatory in one cell, 80% ofincrease), of 19 tested cells.

Effect of 6-cyano-7-nitroquinoxaline-2,3-dione

The effect of the AMPA receptor antagonist CNQX was tested onthe short-latency (n 5 8) and on the long-latency (n 5 9) responsesevoked by MD stimulation, and on the facilitation of these responsesby nicotine. In all the cases the short-latency (Fig. 8) and the long-latency (Fig. 9) responses evoked by MD stimulation were blocked1–3 min following the iontophoretic application of CNQX. UnderCNQX, the application of nicotine no more induced any increase ofthese short-latency (Fig. 8) or long-latency (Fig. 9) responses.

In vivo microdialysis experiments

In 22 of 25 rats tested, basal levels of extracellular glutamate in thePrL were stable throughout the experiment (2–6 h). The direct

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FIG. 6. Blockade by DHBE of the facilitatory effect induced by DMPP on a short-latency excitatory response evoked by MD stimulation in a PrL cell. Post-stimulus time histograms are shown, each comprising 50 cumulative sweeps. The first bar of each histogram corresponds to the stimulus artefact. The upperpanels show responses in the absence of antagonist. Control: short-latency excitatory response evoked by MD stimulation (1 Hz) in a PrL cell. DMPP:iontophoretic application of DMPP (70 nA, 2 min) increased the excitatory response by 100%. Recovery: return of the predrug excitatory response 4 min aftercessation of DMPP application. The lower panels show responses in the presence of antagonist. DHBE (pressure): micropressure application of DHBE (pulsesof 100 ms, 0.5 Hz, 20 psi, during 2 min) did not modify the excitatory response. DHBE1 DMPP: iontophoretic application of DMPP (70 nA, 2 min), starting3.5 min after the onset of DHBE application, no longer induced any facilitation of the excitatory response. Control: excitatory response 8 min after the cessationof DHBE and DMPP application. Numbers in parentheses indicate the number of spikes.

application of nicotine (20, 100, 1000µM) via the microdialysisprobe increased the glutamate release in a dose-dependent manner(Fig. 10A). Peak effects occurred within the first 10 or 20 minof nicotine application. Return to baseline levels occurred after10–20 min

The effect of nicotine was prevented by the addition of 10–5 M

TTX in the dialysate (Fig. 10B); TTX blocks voltage-dependentsodium channels, and thus action potentials. TTX had no effect onbasal levels of glutamate. Replacement of Ca21 with Mg21 in theperfusate also blocked the effect of nicotine, without affecting basallevels of glutamate (Fig. 10C).

The selectivity of the effect of nicotine was demonstrated byapplying the competitive nicotinic antagonist DHBE in the perfusionsolution. DHBE alone had no effect on basal glutamate release butdid inhibit the nicotine-induced increase of glutamate (Fig. 11A). Thenicotinic effect was restored after washout of DHBE.

Finally, we investigated the effects of systemic intraperitonealinjection of nicotine (Fig. 11B). The dose of 0.2 mg/kg (i.p.), whichis behaviourally effective, induced a significant increase in theextracellular levels of glutamate, while control injections of salinedid not alter the basal levels of glutamate. Local infusion of DHBEabolished the effect of systemic nicotine.

Discussion

In the present study, the influence of nAChRs on thalamocorticaltransmission in the PrL was characterized using convergent anatomical


and functional approaches. First, autoradiographic experimentsrevealed a loss of3H-nicotine labelling in PrL following destructionof the input from MD. Second, in anaesthetized animals, iontophoreticapplication of nicotinic agonists in the PrL increased the short- andthe long-latency excitatory responses induced by MD stimulation.The facilitation induced by nicotinic agonists on both short- and long-latency responses were blocked by several nicotinic antagonists(DHBE, mecamylamine, MLA). Third, microdialysis experimentsshowed that local or systemic administration of nicotine increasedthe extracellular levels of glutamate in the PrL. As argued below, theseresults suggest that nicotinic agonists facilitate the thalamocorticalglutamatergic transmission by acting on presynaptic nAChRs.


It is important to note that throughout, we have adopted the nomenclat-ure of Paxinos & Watson (1986, 1997). Thus, the sampled areas thatare labelled as agranular insular (AI) and lateral orbital cortex (LO)lie, respectively, within the dorsal and ventral agranular insularcortices as designated by certain previous authors (Krettek & Price,1977; Groenewegen, 1988).

Layers I and III of the PrL receive the densest innervation fromthe MD (Krettek & Price, 1977; Groenewegen, 1988). Excitotoxiclesions of the MD resulted in a decrease in3H-nicotine binding thatappeared confined to ipsilateral layer III. A comparable loss of mid-cortical labelling in other areas has been seen after lesions centredon four other thalamic nuclear groups (Lavineet al., 1997). As the


FIG. 7. Blockade by MLA of the facilitatory effect induced by DMPP on long-latency excitatory response evoked by MD stimulation in a PrL cell. Post-stimulus time histograms are shown, each comprising 50 cumulative sweeps. The first bar of each histogram corresponds to the stimulus artefact. Upper panelsshow responses before application of antagonist. Control: long-latency excitatory response evoked by MD stimulation (3 Hz) in a PrL cell. DMPP: iontophoreticapplication of DMPP (60 nA, 1 min) increased the excitatory response by 100%. Recovery: return of the predrug excitatory response 4 min after cessation ofDMPP application. MLA1 DMPP: DMPP application (60 nA, 1 min), starting 3 min after the onset of MLA application (pulses of 400 ms, 1 Hz, 40 psi, during2 min), no longer induced any facilitation of the excitatory response. Control: excitatory response 2 min after the cessation of DHBE and DMPP application.DMPP: re-application of DMPP (60 nA, 1 min) 1 min after the control period again induced an increase of the excitatory response comparable with the increaseobtained after the first application of DMPP. Numbers in parentheses indicate the number of spikes.

TABLE 2. Antagonism of the facilitatory effect induced by nicotine or DMPPon the excitatory response evoked in prelimbic cortical neurons

Short-latency responses Long-latency responses

Agonists Antagonists* Blockade †n of cells Blockaden of cellsblocked/n tested blocked/n tested

Nicotine DHBE 2/3 6/8MEC 5/5 6/8MLA 2/2 3/6

DMPP DHBE 2/3 2/3MLA 4/4 5/7

Total 15/17 22/32

*DHBE, dihydro-beta-erythroidine; MEC, mecamylamine; MLA,methyllycaconitine.†Number of prelimbic cortical cells blocked by antagonist out of totalnumber tested.

autoradiography was performed at a single, non-saturating concentra-tion of 3H-nicotine, such a change could reflect a change in eitherreceptor density or affinity. However, an analogous lesion effectpreviously observed in the frontoparietal cortex has been shown toresult from decreased receptor density and not affinity (Lavineet al., 1997).

In the present study, excitotoxic damage was not confined to theMD. However, several considerations suggest that the observeddecrease in3H-nicotine labelling in PrL was indeed the result ofdamage to the MD rather than to adjacent thalamic nuclei. Thalamic


afferents to PrL arise from the MD, ventromedial and certainintralaminar nuclei (Zilles & Wree, 1985; Groenewegen, 1988;Berendse & Groenewegen, 1991). In our experiments, the ventromed-ial nucleus was not damaged. Two intralaminar nuclei (centrolateraland parafascicular) sustained subtotal damage in half the animals;however, neither nucleus projects appreciably to PrL.

Excitotoxic lesions centred on the MD also resulted in reducedcortical labelling in Cg1, LO and possibly in Fr2. Here, too, it islikely that the critical lesion was to MD. All three cortical areas arerecipients of MD input (Krettek & Price, 1977; Zilles & Wree, 1985;Groenewegen, 1988). However, none of these areas receives asignificant input from other thalamic and extra-thalamic nuclei thatsustained partial damage, i.e. anterodorsal nucleus (Sripanidkulchai& Wyss, 1986; Shibata, 1993; van Groen & Wyss, 1995), centrolateraland parafascicular nuclei (Berendse & Groenewegen, 1991), laterodor-sal and lateroposterior nuclei (Faull & Mehler, 1985), ventral anteriorhippocampus and lateral habenula (Faull & Mehler, 1985; Zilles &Wree, 1985).

Based on previous findings, the cortical3H-nicotine labelling thatwas lost following the MD lesion probably represents nAChRs locatedon afferent fibres originating from the MD. Immunoprecipitationstudies suggest that most and possibly all nAChRs that are labelledwith 3H-nicotine contain alpha4 and beta2 subunits (Whiting &Lindstrom, 1988; Floreset al., 1992), and the MD has moderate tostrong levels of markers related to alpha4/beta2-type nAChRs. Thesemarkers comprise alpha4 and beta2 mRNA (Wadaet al., 1989),binding sites for3H-nicotine (Clarkeet al., 1984) and3H-cytisine

26 Y. Gioanniet al.

FIG. 8. Blockade by CNQX of the MD evoked short-latency excitatory response and of its facilitation by nicotine in a PrL cell. Post-stimulus time histogramsare shown, each comprising 50 cumulative sweeps. The first bar of each histogram corresponds to the stimulus artefact. Control: short-latency excitatoryresponse evoked by MD stimulation (1 Hz). Nicotine: iontophoretic application of nicotine (70 nA, 2 min) increased the excitatory response by 94%. Recovery:return to the predrug response 2 min after cessation of nicotine application. CNQX: iontophoretic application of CNQX (70 nA, 2 min) blocked the excitatoryresponse. CNQX1 nicotine: nicotine (70 nA, 2 min) applied 3 min after the onset of CNQX application (70 nA), no longer induced any facilitation of theexcitatory response. Control: excitatory response 10 min after the cessation of CNQX and nicotine application. Numbers in parentheses indicate the number of spikes.

(Happeet al., 1994), and beta2-like immunolabelling (Swansonet al.,1987; Hill et al., 1993). In contrast, a trans-synaptic loss of receptorsseems unlikely, as excitotoxic lesions made locally in other corticalareas had little effect on3H-nicotine labelling (Pruskyet al., 1987;Sahinet al., 1992; Lavineet al., 1997). Taken together, these consid-erations support the existence of presynaptic nAChRs on MD–PrLfibres, but do not provide conclusive evidence.

Electrophysiological experiments

The present data show that local application of nicotinic agonistsinduced a facilitatory effect on the cortical excitatory responsesevoked by MD stimulation. Both short- and long-latency responseswere enhanced. The former responses result from the activation ofthe MD–PrL pathway, whereas the long-latency responses probablyreflect the activation of the recurrent collaterals of pyramidal cellsprojecting to the MD (Pirotet al., 1994).

On the contrary, the spontaneous activity was not modified by theapplication of nicotinic agonists. This result is consistent with earlierin vivo studies in which the effect of iontoporetically appliedacetylcholine or nicotinic agonists was tested in the cerebral cortex(Krnjevic & Phillis, 1963; Lamouret al., 1982).

Nicotinic facilitation of short- and long-latency responses wasblocked by several nicotinic antagonists (DHBE, mecamylamine,MLA) confirming the participation of nAChRs. These antagonistsdiffer in their selectivity for nAChR subtypes. Thus, DHBE appearsto act more potently on putative alpha4/beta2 nAChRs than onpresumed alpha7 nAChRs (Alkondon & Albuquerque, 1993). MLA


has a high affinity for alpha7 subunits (Wonnacottet al., 1993) butcan also block alpha3-containing nAChR, and at high concentrationsalpha4/beta2 subunits (Yumet al., 1996). In our experiments, it ispossible that the local concentration of MLA was not low enough toact in an nAChR subtype-selective manner, and thus that MLAblocked in addition to alpha7 other subunits.

The present autoradiographic experiments suggest that the facilita-tory effects induced by nicotinic agonists on the short-latencyresponses evoked in the PrL are due to the activation of presynapticnAChRs located on afferents from the MD. This facilitatory effectlikely involves the activation of alpha4/beta2 nAChRs as this effectis blocked by DHBE, and alpha4/beta2 mRNAs are expressed in MD(Wadaet al., 1989).

However, this conclusion is necessarily tentative, as nAChRs arestructurally and pharmacologically heterogeneous and few subtypescan be identified with existing radioligands. In addition, the involve-ment of multiple nAChR subtypes cannot be ruled out, particularlyas their is evidence of heterogeneity among presynaptic nAChRseven within a single anatomical pathway (Kulaket al., 1997). Amongpossible candidates, alpha7-containing nAChRs suggest themselves,as they have been shown to enhance glutamate release in subcorticalbrain areas (Grayet al., 1996; Alkondonet al., 1996). Moreover, Gilet al. (1997) have recently shown that in rat brain slices containingthe ventrobasal thalamus and somatosensory cortex, the nicotine-induced facilitation of thalamocortical EPSPs was blocked by MLA,albeit at higher concentrations than typically required to block alpha7-type nAChRs. Against this, alpha7 subunit mRNA in MD appears tobe expressed at only low levels in the thalamus (Seguelaet al., 1993).


FIG. 9. Blockade by CNQX of the MD evoked long-latency excitatory response and of its facilitation by nicotine in a PrL cell. Post-stimulus time histogramsare shown, each comprising 50 cumulative sweeps. The first bar of each histogram corresponds to the stimulus artefact. Control: long-latency excitatory responseevoked by MD stimulation (5 Hz). Nicotine: iontophoretic application of nicotine (70 nA, 2 min) increased the excitatory response by 130%. Recovery: returnto the predrug response 5 min after cessation of nicotine application. CNQX: iontophoretic application of CNQX (70 nA, 2.5 min) blocked the excitatoryresponse. CNQX1 nicotine: nicotine (70 nA, 2 min) applied 3 min after the onset of CNQX application (70 nA), no longer induced any facilitation of theexcitatory response. Control: excitatory response 9 min after the cessation of CNQX and nicotine application. Numbers in parentheses indicate the number of spikes.

The long-latency responses evoked by MD stimulation are likelydue to the activation of recurrent collaterals of PFC pyramidal neuronsprojecting to the MD. The facilitation of these responses by nicotinicagonists could be due to involvement of presynaptic nAChRs locatedon these collaterals. In the rat, pyramidal cells are reported to expressalpha3 and alpha4 mRNA (Nakayamaet al., 1995; Lobronet al.,1995) and beta2-like immunoreactivity (Bravo & Karten, 1992; Hillet al., 1993); these cells may also express alpha7 subunits, as alpha7message is present in the cerebral cortex (Seguelaet al., 1993) andhas been detected in pyramidal cells in the human PFC (Weverset al., 1995). The facilitation by nicotinic agonists of these long-latency excitatory responses, probably due to the activation ofrecurrent collaterals of pyramidal cells, suggests that a pool of nAChRscould be present on the terminals of these recurrent collaterals. Thispool could represent, in part, the remaining nAChRs observed inlayer III of the PrL after lesion of the MD, as well as nAChRs presentin deeper layers.

In some cases, the local application of the nicotinic antagonistsinduced an increase or a decrease of the excitatory responses evokedby MD stimulation. Although, this finding is not in itself conclusive,it can be proposed that these effects could be due to blockade of atonic action of acetylcholine on the presynaptic nAChRs located onglutamatergic or on inhibitory GABAergic terminals. Blocking thistonic action would thus induce, respectively, an inhibitory or afacilitatory effect at the postsynaptic level.

Taken together, the present results suggest a modulation of thecortical glutamatergic transmission by the nicotinic agonists. Indeed,


as previously shown, the short- and long-latency responses evokedby MD stimulation were blocked by the AMPA receptor antagonistCNQX (Pirot et al., 1994, 1995). Furthermore, the facilitation ofthese responses by nicotine application was no more observedunder CNQX.

Microdialysis experiments

The present results show that nicotine, either infused locally orinjected intraperitoneally, increases the extracellular level of glutamatein the prelimbic area of the PFC. This effect appears to be mediatedby nAChRs as shown by the inhibitory action of the nicotinicantagonist DHBE. This result is in line within vitro and in vivostudies showing that in a number of brain areas, nicotine increasesthe release of various neurotransmitters, including glutamate, GABA,dopamine and noradrenaline (for reviews see Role & Berg, 1996;Wonnacott, 1997).

The facilitatory effect of nicotine was observed after either systemicadministration or after local infusion via the dialysis probe. Theintraprobe concentrations of nicotine found to promote detectableglutamate release were 100 and 1000µM. These concentrations,which are lower than reported in comparable studies of otherneurotransmitters, would likely result in submicromolar values in theextracellular space (Nisellet al., 1994; Marshallet al., 1997). Theobserved inhibition by the selective nicotinic antagonist DHBE furthersupports mediation by nAChRs.

The blockade of the nicotine action by TTX or Ca21 removal isindicative of an effect involving sodium and calcium dependent

28 Y. Gioanniet al.

FIG. 10. Effects of nicotine on extracellular concentrations of endogenousglutamate, as measured by microdialysis in the prefrontal cortex of freelymoving rats. Dialysate samples were collected over periods of 20 min. Nicotinewas applied through the dialysis probe (bars) for 20-min periods. Results aremeans6 SEM (n 5 3 rats) of endogenous glutamate expressed as a percentageof basal values. (A) Dose-dependent effect of nicotine (20, 100, 1000µM).(B) Tetrodotoxin 10µM (TTX) applied together with nicotine 1 mM (N 1 TTX)through the dialysis probe prevented the effect of nicotine alone (N). (C)Calcium-free artificial CSF blocked the effect of nicotine on glutamate.*P , 0.05 (ANOVA).

mechanisms of neuronal origin. At this stage the precise mechanismfor the increase of glutamate release by nicotine remains speculative.If one follows the common view of presynaptic receptors locatedclose to the sites of exocytotic release, then the sensitivity to TTXsuggests the involvement of local excitatory circuits. However, theTTX sensitivity does not necessarily exclude a presynaptic controlof transmitter release. Indeed, there is good evidence for the presenceof nicotinic receptors located on the preterminal domain of nerveendings, which stimulate neurotransmitter release through a TTX


FIG. 11. The blockade by dihydro-beta-erythroidine (DHBE) of glutamaterelease evoked by nicotine in the prefrontal cortex of freely moving rats. (A)Nicotine 1 mM (N) applied through the dialysis probe produced a significantincrease in the level of extracellular glutamate. This effect was prevented bythe nicotinic antagonist DHBE (10 mM) applied in the perfusate. The nicotineeffect recovered following DHBE washout. (B) Nicotine (0.2 mg/kg) givenby intraperitoneal injection (N) produced a significant increase in glutamaterelease. An i.p. injection of saline (S) had no effect. DHBE applied throughthe dialysis probe prevented the effect of systemic nicotine.

sensitive mechanism (Le´naet al., 1993). The Ca21 sensitivity is alsoin agreement with the hypothesis of a presynaptic mechanism.

Our two functional approaches, electrophysiology and microdia-lysis, both suggest that nicotine can facilitate glutamatergic synapsesin the PrL. Further investigation will be needed to determine theextent to which glutamate release, as measured by dialysis, representsthalamocortical activity.

Conclusions

As a whole, the present data suggest that nicotinic agonists facilitatethe glutamatergic transmission in the PFC, by acting on presynapticnAChRs located on MD afferents and probably also on recurrentcollaterals of pyramidal cells. These observations may be relevant tobehavioural changes that have been observed following the localblockade of nAChRs in the PFC. Marked deficits are observed inrats trained to working memory tasks requiring high attentionaldemand (Granonet al., 1995). Interestingly, several studies in humans,monkeys and rats suggest that the MD nucleus and the PFC are bothinvolved in attentional processes (Isseroffet al., 1982; Markowitch,1982; Stokes & Best, 1990; Pardoet al., 1991). Thus, the control ofthe thalamocortical input by nAChRs could be involved in some ofthe cognitive effects of nicotine. Taken together, these data reinforcethe view that the PFC is an important target for nicotine in facilitatingattentional functions.


Acknowledgements

Supported by INSERM and by the Medical Research Council of Canada (toPBSC). PBSC is a Chercheur Boursier of the FRSQ. We thank MelanieReuben for preparation of figures and Anne-Marie Godeheu and MoniqueSaffroy for histological assistance.

Abbreviations

AI, agranular insular cortex; AMPA, (6)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-proprionic acid; Cg1, cingulate cortex area 1; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DHBE, dihydro-beta-erythroidine; DMPP, 1,1-dimethyl-4-phenylpiperazinium; EPSP, excitatory postsynaptic potential; Fr2,frontal cortex area 2; Fr3, frontal cortex area 3; LO, lateral orbital cortex; MD,mediodorsal thalamic nucleus; MLA, methyllycaconitine; nAChR, nicotiniccholinoceptor; NMDA, N-methyl-D-aspartate; PrL, prelimbic; TTX, tetro-dotoxin.

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nicotinic receptors in the rat prefrontal cortex: increase in glutamate release and facilitation of...

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