INTRODUCTION

Glutamate is the main excitatory neurotransmitter in the cerebral cortex. As glutamate is released into the synaptic cleft, a specific reuptake system present on both neuronal and glial cells adjusts the extracellular concentrations of this neurotransmitter between 0.2 µM and 200 µM (1). Activation of glutamatergic receptors results in both rapid changes of ionic currents and second messenger formation. Among the second messenger pathways stimulated by glutamate, the release of AA¹ has been shown in brain slices (2) and primary cultures of neurons or astrocytes (3, 4, 5). The glutamate-evoked release of AA could be of particular relevance to the physiological regulation of the excitatory signaling, since it has been suggested that AA itself could amplify glutamatergic neurotransmission. Indeed, AA enhances glutamate release from presynaptic terminals (6), inhibits glutamate reuptake (7), and enhances NMDA currents (8). Interestingly, certain components of long term potentiation appear to involve AA formation (9).

AA is an unsaturated fatty acid predominantly located in the sn-2 position of membrane phospholipids from where it can be released by various types of phospholipases, mainly by phospholipase A₂ (PLA₂) or by sn-2 diacylglycerol lipase (for review, see Ref. 10). In neurons, the glutamate-evoked release of AA appears to involve the activation of PLA₂ (3, 4, 11). The release of AA is tightly modulated by different mechanisms; in particular, PLA₂ activity is regulated by phosphorylation. Thus, both PLA₂ activity and the subsequent release of AA are enhanced by the cAMP/cAMP-dependent protein kinases (cAMP/PKA) pathway (12, 13).

In the present study, we addressed the question of whether activation of the cAMP/PKA pathway in neurons would modulate the glutamate-evoked release of AA. For this purpose, we investigated the effect of VIP and PACAP on the glutamate response, since both peptides are potent and efficient stimulators of cAMP formation in cortical neurons (14, 15, 16, 17). Both VIP and PACAP interact with the G_s protein-coupled PACAP I and PACAP II receptors. PACAP I receptors are activated by PACAP in the nanomolar range and by VIP in the micromolar range, whereas PACAP II receptors are activated in the nanomolar range by both peptides (18, 19).

Here we report that VIP and PACAP, by activating PACAP I receptors, potentiate the glutamate-evoked release of AA from primary cultures of cerebral cortical neurons. However, the cascade of events that leads to this potentiation is not mediated by G_s proteins, nor by the cAMP/PKA or the phospholipase C (PLC)/PKC pathway. Therefore, our data describe a potentiating effect mediated by the activation of PACAP I receptors, which does not involve second messenger pathways classically associated with the activation of these types of receptors.

EXPERIMENTAL PROCEDURES

Materials

Poly-L-ornithine (M_r 30,000-70,000), laminin, Dulbecco's modified Eagle's medium (DMEM; D-7777), bovine pancreas insulin, human apo-transferrin, putrescin, progesterone, fatty acid free bovine serum albumin (BSA), isobutylmethylxanthine (IBMX), 8-bromoadenosine 3:5-cyclic monophosphate (8-Br-cAMP), L-glutamate, NMDA, kainate, D-serine, phorbol 12-myristate 13-acetate (PMA), and lanthanum were obtained from Sigma; (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX) from Tocris Neuramin, Bristol, United Kingdom; 8-Br-R_p-cAMPS and 8-Br-S_p-cAMPS from Biolog Life Science Institute, Bremen, Germany; (5R,10S)-(+)-5methyl-10,11-dihydro-5-H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801) and nifedipine from Research Biochemicals Inc., Natick, MA; VIP, PACAP-38 (PACAP), and secretin from Bachem, Bubendorf, Switzerland; U-73122 from Biomolecules Research Laboratories, PA; adenosine deaminase (EC 3.5.44) and ionomycin from Boehringer, Mannheim, Germany; [³H]arachidonic acid ([³H]AA, 8.25 TBq; 200 Ci/mmol), [¹⁴C]arachidonic acid ([¹⁴C]AA, 2.15 GBq; 58 mCi/mmol), ¹²⁵I-cAMP (59 KBq; 1.6 µCi/mmol), and myo-[2-³H]inositol with PTG-271 (633 GBq; 17.1 Ci/mmol) from Amersham, Buckinghamshire, United Kingdom; INDO-1 acetoxymethyl ester (INDO-1 AM) from Molecular Probes, OR; Ro 31-8220 was a kind gift from Dr. G. Lawton, Roche Products Ltd (Herts, UK); PD98059 was a kind gift from Dr. A. R. Saltiel, Parke-Davis Parmaceutical Research Division (Ann Arbor, MI).

Methods

Cortical neurons devoid of glial cells were prepared as described previously (11). Briefly, the cerebral cortex of 16-day-old Swiss albino mice embryos was dissected by removing the olfactory bulbs, striatum, hippocampus, and meninges. Cells were dissociated mechanically and were plated (800,000 cells/ml) on 12-well Falcon culture dishes (1 ml/well) previously coated with 15 µg/ml polyornithine. For intracellular Ca²⁺ ([Ca²⁺]_i) imaging, cells were plated on glass coverslips also coated with 15 µg/ml polyornithine, followed by 2 µg/ml laminin. In all cases, coating was carried out in serum-free medium. The culture medium was composed of DMEM supplemented with 2 mM glutamine, 7.5 mM NaHCO₃, 5 mM HEPES buffer (pH 7.0), 100 µg/ml streptomycin, 60 µg/ml penicillin, 25 µg/ml insulin, 100 µg/ml transferrin, 60 µM putrescine, 20 nM progesterone, and 30 nM sodium selenite. Cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ for 5-7 days in vitro, a time point at which they reach maximum viability and exclusively express neuronal markers (11).

Measurement of [³H]AA release was carried out as described previously (11). Briefly, cortical neurons cultured in 12-well culture dishes were incubated for 16-20 h in the presence of [³H]AA (1 µCi/well) by adding 100 µl/well of DMEM containing [³H]AA and 0.01% BSA as a lipid carrier. After washing three times at 37 °C with a Locke-HEPES buffer (L-H buffer (in mM): NaCl, 145; KCl, 5.5; CaCl₂, 1.1; MgCl₂, 1.1; NaHCO₃, 3.6; glucose, 5.5; HEPES, 20; pH, 7.4) containing 0.2% BSA, cells were preincubated for 15 min in L-H buffer, supplemented with adenosine deaminase (1 IU/ml). This latter procedure was used to degrade endogenous adenosine, which has been previously shown to affect glutamate-mediated signal transduction (20). Unless otherwise stated, inhibitors or antagonists were also added during this preincubation period. Neurons were then exposed for 15 min at 37 °C to the various agents in a similar L-H buffer, lacking Mg²⁺ (a condition necessary to yield full activity of NMDA receptors; Refs. 21 and 22) and in the presence of adenosine deaminase (1 IU/ml). At the end of the stimulation, the incubation medium was collected, centrifuged at 200 × g for 5 min, and an aliquot of the supernatant was assayed for ³H by liquid scintillation counting. To avoid misestimation due to the presence of BSA, the protein content was determined in sister cultures by the method of Bradford (23).

Characterization by thin layer chromatography (TLC) of the ³H-labeled product(s) released from cortical neurons was carried out as described previously (11). This procedure allows complete separation of AA from its metabolites including prostaglandins and most lipoxygenase metabolites, as well as from other classes of lipids such as phospholipids and mono- and diacylglycerols.

cAMP levels were determined by radioimmunoassay under conditions similar to those used for the [³H]AA release measurement. Briefly, cortical neurons cultured in 12-well dishes were washed three times with L-H buffer at 37 °C and subsequently preincubated for 15 min in L-H buffer supplemented with IBMX (1 mM) and adenosine deaminase (1 IU/ml). Neurons were then exposed for 15 min at 37 °C to the agents in a L-H buffer lacking Mg²⁺, supplemented with IBMX (1 mM), adenosine deaminase (1 IU/ml), and glutamate (100 µM). Glutamate was added in these experiments because cortical neurons possess metabotropic glutamate receptors, which are negatively coupled to the stimulation of adenylyl cyclase (24). Indeed, as cortical neurons were incubated in presence of 100 µM glutamate, the accumulation of cAMP evoked by 1 µM VIP and 100 nM PACAP was partially inhibited (data not shown). Therefore, we set out to determine whether agents that are known to stimulate cAMP formation were still efficient in the presence of 100 µM glutamate (see ``Results''). The reaction was stopped by replacement of the incubation buffer with ice-cold L-H buffer and subsequent sonication. The suspension was boiled for 10 min and centrifuged for 2 min at 9980 × g, and an aliquot of the supernatant was taken to assess cAMP levels (using ¹²⁵I-cAMP as a tracer; Ref. 25).

Formation of [³H]inositol phosphates ([³H]IPs) was determined under conditions similar to those used for the [³H]AA release measurement (11). Briefly, cells were plated in the presence of myo-[2-³H]inositol (4 µCi/ml) (in 12-well culture dishes). After 5-7 days in vitro, neurons were washed three times with L-H buffer (1 ml/well) at 37 °C, then preincubated for 15 min in L-H buffer supplemented with lithium (10 mM) and adenosine deaminase (1 IU/ml), and finally exposed for 15 min to the agents in a L-H buffer lacking Mg²⁺, supplemented with lithium (10 mM) and adenosine deaminase (1 IU/ml). Isolation of [³H]IPs by ion-exchange chromatography (Dowex AG 1-X8) was performed as described previously (see Ref. 11).

Determination of [Ca²⁺]_i was carried out as described previously (11). Briefly, cortical neurons grown on glass coverslips were studied with dual emission microfluorimetry. Cells were loaded with 12 µM INDO-1 AM and then exposed to various agents (in L-H buffer) using a multichannel cell superfusion device. All responses were measured in the absence of Mg²⁺ and in the presence of D-serine (100 µM). D-Serine was used instead of glycine because it yields full activation of NMDA receptor on perfused neurons and does not activate glycine-strychnine-sensitive chloride channels (26). The concentration of [Ca²⁺]_i (nM) was calculated according to the equation described by Grynkiewicz et al. (27). Under those conditions, 97% of neurons tested responded to 100 µM glutamate.

Results are expressed as mean ± S.E. of n independent determinations. Data were statistically analyzed using InStat, GraphPad Software, San Diego, CA.

RESULTS

As shown previously, glutamate (used at a maximally effective concentration of 100 µM) evoked the release of [³H]AA from cortical neurons in primary culture (Fig. 1, and see Ref. 11). Although neither VIP (1 µM) nor PACAP (10 nM) significantly affected the basal release of [³H]AA, they both potentiated the glutamate response by, respectively, 2- and 3-fold (Fig. 1). EC₅₀ values for these potentiating effects were 1 µM for VIP and 0.7 nM for PACAP (Fig. 2), suggesting that these peptides might exert their effect by activating PACAP I receptors. In agreement with this hypothesis, the release of [³H]AA evoked by the concomitant application of VIP (1 µM), PACAP (10 nM), and glutamate was similar to the response obtained with PACAP (10 nM) and glutamate (n = 9; data not shown). Therefore, in the following experiments, potentiation of the glutamate-evoked release of [³H]AA was investigated using 10 nM PACAP.

The effect of secretin was also studied, because this peptide shares a high degree of sequence homology with VIP and PACAP (18, 19). However, secretin (1 µM) had no effect on either basal [³H]AA release or on the glutamate response (Fig. 1).

Thin layer chromatography (TLC) was used to verify that glutamate alone or in the presence of PACAP evoked the release of [³H]AA exclusively. Indeed, in three separate experiments, the increase in ³H-labeled product(s) evoked by glutamate either alone or in the presence of PACAP was exclusively recovered in the AA fraction. No significant radioactivity was recovered in the fraction containing AA metabolites.

PACAP I receptors are coupled to G_s proteins and have been shown to activate adenylyl cyclase (18). Indeed, PACAP (in the presence of glutamate) induced a strong accumulation of cAMP in cortical neurons (Fig. 3A). A similar accumulation of cAMP was obtained by either direct activation of the G_s proteins with cholera toxin (CTx) or by direct stimulation of adenylyl cyclase with forskolin (Fig. 3A). However, the glutamate-evoked release of [³H]AA was not affected in neurons similarly treated with either CTx or forskolin (Fig. 3B), indicating that intracellular increases in cAMP are not sufficient to potentiate the glutamate response.

The involvement of PKA is also unlikely. A specific cell membrane permeant inhibitor of PKA activity, i.e. 8-Br-R_p-cAMPS (100 µM) did not affect the release of [³H]AA evoked by either glutamate alone or by the co-application of glutamate plus PACAP (Fig. 4). Furthermore, application of cell membrane permeant PKA activators i.e. 8-Br-cAMP (100 µM) and 8-Br-S_p-cAMPS (100 µM) did not affect the release of [³H]AA evoked by glutamate (1.1 ± 0.19- and 0.98 ± 0.05-fold of the glutamate response, respectively; n = 9).

A few reports have indicated that PACAP can also activate the PLC pathway (28). Indeed, PACAP induced a significant accumulation of [³H]IPs in cortical neurons, this response being additive with the glutamate-induced accumulation of [³H]IPs (Table I). To assess the possible involvement of the PLC pathway in AA release, we used a potent inhibitor of several types of PLC, i.e. U73122 (29, 30). When added to cortical neurons, U73122 (3 µM) inhibited the PACAP plus glutamate-induced formation of [³H]IPs by 84% (Table I), indicating that this inhibitor was active on the PLC activity present in cortical neurons. However, PLC inhibition by U73122 (3 µM) did not affect the [³H]AA release evoked by the co-application of glutamate and PACAP (10 nM) (Fig. 4), indicating that PLC activation is not necessary to potentiate the glutamate response.

Table I. Glutamate- and PACAP-induced [3H]IP accumulation Cortical neurons were incubated for 15 min in the presence of agonists and 3H-IPs accumulation was determined as described under ``Experimental Procedures.'' U73122 was preincubated 15 min and maintained throughout the incubation. Results are the mean ± S.E. of 6-9 separate determinations. Statistical analysis: **, p < 0.01 significantly different from basal (ANOVA followed by Dunnett's test). Agonists added Amount added [3H]IP accumulation µM % of basal Basal 100  ± 10 PACAP 0.01 209  ± 7** Glutamate 100 159  ± 7** Glutamate + PACAP 100 + 0.01 258  ± 7** U73122 3 85  ± 5 Glutamate + PACAP + U73122 100 + 0.01 + 3 110  ± 4

The involvement of PKC is also unlikely. Indeed, we tested a potent inhibitor of PKC activity, i.e. Ro 31-8220, which has a specific effect between 1 and 10 µM on intact cells (31). Ro 31-8220 (10 µM) did not affect the release of [³H]AA evoked by either glutamate alone or by the co-application of glutamate plus PACAP (10 nM) (Fig. 4). Furthermore, activation of PKC by application of the phorbol ester PMA (0.1 µM) did not affect the glutamate-evoked release of [³H]AA (1.03 ± 0.16-fold of the glutamate response, n = 6).

The glutamate-evoked release of [³H]AA in primary cultures of cortical neurons is mediated by two receptor subtypes: AMPA/kainate and NMDA (11). Activation of AMPA/kainate receptors with either kainate (100 µM) alone or glutamate plus MK801 (1 µM to block NMDA receptors) evoked the release of [³H]AA; both of these reponses were similarly potentiated by PACAP (Fig. 5). Activation of NMDA receptors with either NMDA (100 µM) alone or glutamate plus DNQX (10 µM to block AMPA/kainate receptors) evoked the release of [³H]AA; both reponses were also potentiated by PACAP (Fig. 5).

Finally, although metabotropic receptors are not involved in the glutamate-evoked release of AA from cortical neurons in primary cultures (11), we considered the possibility that PACAP could reveal such a coupling. This hypothesis can, however, be ruled out since (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 mM) in the presence of PACAP did not evoke a significant release of [³H]AA (n = 9; data not shown).

The glutamate-evoked release of [³H]AA is dependent on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) (3, 32, 33). Since activation of glutamate receptors is known to induce Ca²⁺ influx (either through the ionotropic receptors themselves or by opening voltage-sensitive Ca²⁺ channels (VSCC)), an amplification of these influxes by PACAP could account for its potentiating effect on the glutamate-evoked release of [³H]AA.

In a first step, we studied the overall elevations in [Ca²⁺]_i in INDO-1-loaded cortical neurons. Application of PACAP did not affect basal [Ca²⁺]_i, nor did it significantly alter the elevation in [Ca²⁺]_i elicited by glutamate (Table II). Since modifications in [Ca²⁺]_i microdomains might not be detected by fluorescent imaging techniques (but could nevertheless be involved in regulation of enzymatic activities; Ref. 34), we considered the possibility that activation of PACAP I receptors could enhance Ca²⁺ influx due to the opening of Ca²⁺ channels. This hypothesis can be ruled out, since in the presence of a broad spectrum VSCC blocker, i.e. lanthanum (2 µM), PACAP similarly potentiated the glutamate-evoked release of [³H]AA (2.7 ± 0.24-fold enhancement of the glutamate response; n = 9). Confirming these results, a specific L-type VSCC blocker, i.e. nifedipine (10 µM) did not significantly affect the release of [³H]AA evoked by glutamate and PACAP (2.65 ± 0.11-fold enhancement of the glutamate response; n = 9).

Table II. Increases in [Ca2+]i evoked by glutamate and PACAP alone or in combination Increases in [Ca2+]i were measured in INDO-1-loaded cortical neurons exposed to agents for a period of 2 min in an incubation medium lacking Mg2+. Data represent [Ca2+]i calculated as previously described (11), corresponding to n individual neurons analysed for each condition and chosen at random from 5 independent coverslips. No significant difference was found when glutamate and glutamate plus PACAP responses were compared (two-tailed unpaired Student's t test; p > 0.05). Agents added Amount added [Ca2+i (nM) number of cells µM Basal 41  ± 1 327 PACAP 0.01 44  ± 4 52 Glutamate 100 715  ± 51 143 Glutamate + PACAP 100 + 0.01 855  ± 56 132

Taken together, these results suggest that the potentiating mechanism mediated by the activation of PACAP I receptors could be operative at a level further downstream of the glutamate induced Ca²⁺ influx. To strengthen this hypothesis, we examined whether PACAP would affect the release of [³H]AA evoked by a receptor-independent pathway, such as a large increase in [Ca²⁺]_i evoked by the Ca²⁺ ionophore ionomycin. Indeed, the release of [³H]AA induced by ionomycin (10 µM) was potentiated by PACAP (Fig. 5).

DISCUSSION

In the present study, we show that VIP and PACAP potentiate, in a concentration-dependent manner, the glutamate-evoked release of AA from primary cultures of cortical neurons. Considering the EC₅₀ values of this potentiating effect (1 µM for VIP and 0.7 nM for PACAP; see Fig. 2), it can be postulated that both peptides potentiate the glutamate response by interacting with PACAP I receptors (18, 19). Further strengthening this view is the result showing that secretin, which interacts with PACAP II, but not with PACAP I receptors (18, 19), had no effect on the glutamate-evoked AA release.

It is well established that PACAP I receptors are coupled to G_s proteins (18). However, activation of G_s protein per se was not sufficient in potentiating the glutamate response, since the release of AA evoked by glutamate was not affected when the cells were treated with CTx (a toxin that activates G_s protein by ADP-ribosylation). It should be emphasized that cells responded to CTx, since a strong accumulation of cAMP was measured (see Fig. 3). This result suggests that the PACAP I receptors involved in this particular potentiating effect are not coupled to the G_s protein family. The family of G_i/o proteins can also be ruled out, since the potentiation of the glutamate-evoked release of AA by PACAP is not affected by treating the neurons with 1 µg/ml pertussis toxin for 24 h (data not shown).

In line with the non-involvement of G_s proteins, several arguments allow to rule out the cAMP-PKA pathway. Indeed, the glutamate-evoked release of AA was not affected by (i) treatments that induce an accumulation of cAMP to the same extent than that measured in the presence of PACAP (i.e. forskolin and CTx; Fig. 3) or (ii) application of cell membrane permeant cAMP (i.e. 8-Br-cAMP). Furthermore, the potentiating effect of PACAP was (i) not inhibited by a specific PKA inhibitor (i.e. 8-Br-R_p-cAMPS; Fig. 4) and (ii) not mimicked by a specific PKA activator (i.e. 8-Br-S_p-cAMPS).

In order to rule out the involvement of PLC in the glutamate-evoked release of AA, we used the specific inhibitor U73122. At a concentration shown here to dramatically inhibit PLC activity, this compound did not affect the release of AA evoked by glutamate alone or in the presence of PACAP (Fig. 4). This result confirms that PLC is not involved in the glutamate-evoked release of AA (11), nor in its potentiation by PACAP. Although a role of PKC was not expected, mainly in view of the non-involvement of PLC as discussed above, this possibility could be ruled out since: (i) the specific inhibitor of PKC Ro 31-8220 did not affect the AA release evoked by glutamate alone or in the presence of PACAP (Fig. 4) and (ii) the activation of the PKC by application of the phorbol ester PMA did not affect the glutamate-evoked release of AA (see ``Results'').

Recently, it has been shown that a PACAP-like neuropeptide present in Drosophila stimulates a distinct protein kinase pathway, which is independent of PKA or PKC, i.e. the mitogen-activated protein kinase (MAP kinase) cascade (35). Since MAP kinase directly phosphorylates cPLA₂ (36), an activation of this pathway could be considered for the potentiating effect of PACAP on the glutamate-evoked release of AA. However, the use of available inhibitors, such as PD098059 (37, 38), did not allow to prove the involvement of the MAP kinase pathway (data not shown). Furthermore, whether cPLA₂ is the enzyme involved in the glutamate-evoked release of AA from cortical neurons remains to be demonstrated. Therefore, at this point, we can only speculate on a possible involvement of the MAP kinase pathway in the potentiating effect of PACAP.

The Ca²⁺ influx mediated by glutamate receptor activation is a necessary step in the glutamate-evoked release of AA (3, 32, 33). Confirming these results, ionomycin induced a release of AA from cortical neurons in primary culture (Fig. 5). This is consistent with the fact that PLA₂ activities are enhanced by Ca²⁺ specifically interacting with a Ca²⁺-binding domain on cPLA₂ (39). Furthermore, Ca²⁺-regulated PLA₂ activities are present in primary cultures of cortical neurons (11). Thus, one possible step in the signal transduction cascade associated with the activation of PACAP I receptors would be the enhancement of the glutamate-induced Ca²⁺ influx. In primary cultures of cortical neurons, the glutamate-evoked release of AA is mediated by the activation of AMPA/kainate as well as NMDA receptor subtypes (11). Activation of glutamate receptors enhances [Ca²⁺]_i by several mechanisms: (i) a depolarization-induced gating of VSCC channels; (ii) in several neocortical neurons, which express specific AMPA receptors subunits, a Ca²⁺ influx that occurs directly through the ionotropic receptor channel (40), or (iii) through the NMDA receptor channel (41). In the present study, an involvement of VSCC could be ruled out, since potent blockers of these channels, such as nifedipine and lanthanum, did not reduce the AA release evoked by glutamate plus PACAP. Furthermore, since the kainate as well as the NMDA-evoked release of AA were similarly potentiated by PACAP (Fig. 5) a mechanism acting on a specific receptor subtype can also be excluded, suggesting a level of interaction that is downstream of the receptor-induced Ca²⁺ influx. Further strengthening this view is the fact that PACAP did not significantly affect the glutamate-induced enhancement in [Ca²⁺]_i (Table II). Even more striking is the observation that the release of AA evoked by ionomycin was also potentiated by PACAP (Fig. 5).

In summary, results reported here suggest that the substrate, whose activity is modulated following the activation of PACAP I receptors, is involved at a level downstream of the Ca²⁺ influx. Whether this substrate is PLA₂ itself remains to be demonstrated. This study also shows that activation of PACAP I receptors alone (by VIP or PACAP) is not sufficient to promote the release of AA, whereas it clearly potentiated a Ca²⁺-dependent release of AA evoked by either glutamate or ionomycin.

Is VIP a cell-specific neuromodulator of the glutamate signaling? The morphological characteristics and physiological properties of VIP containing neurons in the neocortex, the region from which the cultures used in this study were prepared, have been characterized in detail (for review, see Ref. 42). In particular, VIP is almost exclusively localized to a homogeneous population of bipolar, radially oriented neurons (43). The dendrites of these neurons extend from the pial surface to layer V-VI arborizing to a maximum of 100-120 µm in the horizontal plane. The axons of VIP-containing neurons are radially oriented and ramify within the radial domain of the dendritic arborization (43). These morphological characteristics imply that the actions of VIP released from VIP-containing neurons are confined within cortical columnar ensembles (for review, see Ref. 44). Since glutamate is released from activated afferent pathways to a given cortical area, VIP-containing bipolar neurons are ideally positioned to receive laminarly specified excitatory inputs from afferent pathways. Frequent asymmetrical (excitatory) synapses have been identified on VIP-containing neurons (45); furthermore, glutamate (in addition to other depolarizing stimuli) has been shown to evoke VIP release (46). Thus, the activation of excitatory afferents can result in synergistic interactions between VIP and glutamate on potential common target cells within discrete cortical domains.

From the data presented in this study, and from previous observations showing that VIP actually inhibits the glutamate-evoked release of AA from astrocytes in primary cultures (5), it follows that the concomitant release of VIP and glutamate within discrete radially oriented cortical domains will selectively enhance the release of AA within neuronal rather than glial populations of activated cortical columns. This cell type-specific modulation of the glutamate signaling by VIP could result in a transient sharpening of the information flow to target neurons resulting in the enhancement of the signal-to-noise ratio of AA-mediated cellular processes. Indeed, AA has been shown to regulate the efficacy of glutamatergic neurotransmission by several mechanisms. AA increases (i) NMDA currents (possibly by interacting with the ion channel protein itself) (8, 47) and (ii) the glutamate concentration in the synaptic cleft (possibly by enhancing synaptic release of glutamate and by inhibiting its reuptake) (48, 7). In line with these observations are the results obtained in the hippocampus showing that certain components of long term potentiation appear to require AA formation (9, 49). Therefore, data reported here suggest that VIP and PACAP could participate in the amplification of glutamate signaling, as already shown for the glutamate-induced c-fos expression (16).