Potentiation of NMDA Currents by Pituitary Adenylate Cyclase Activating Polypeptide in Neonatal Rat Sympathetic Preganglionic Neurons

S. Y. Wu and N. J. Dun

Department of Anatomy and Neurobiology, Medical College of Ohio, Toledo, Ohio 43614

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Wu, S. Y. and N. J. Dun. Potentiation of NMDA currents by pituitary adenylate cyclase activating polypeptide in neonatal rat sympathetic preganglionic neurons. J. Neurophysiol. 78: 1175-1179, 1997. Whole cell patch-clamp recordings were made from sympathetic preganglionic neurons (SPNs) in the intermediolateral cell column of thoracolumbar spinal cord slices of 12- to 16-day-old rats, and the effects of pituitary adenylate cyclase activating polypeptide (PACAP)-38 on N-methyl-D-aspartate (NMDA)- and kainate (KA)-induced inward currents were examined. PACAP, in concentrations (10-30 nM) that caused no significant change of holding currents, reversibly increased NMDA-induced currents but not KA-induced currents. At higher concentrations (>30 nM), the peptide produced a sustained inward current. The potentiating effect of PACAP was nullified by prior incubation of the slices with the adenylate cyclase inhibitor MDL-12,330A (25 µM). Further, superfusing the slices with the membrane-permeable cyclic AMP analogue N6,2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (100-300 µM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (700 µM) increased the NMDA currents. This result suggests that PACAP selectively increases NMDA-receptor-mediated responses in the rat SPNs, probably via a cyclic-AMP-dependent mechanism, providing evidence that the peptide may be involved in synaptic plasticity.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Pituitary adenylate cyclase activating polypeptide(PACAP), a relatively new peptide first isolated from the ovine hypothalamus, occurs as a 38-amino-acid or a truncated 27-amino-acid peptide, the former being the prevailing form in mammalian tissues (Arimura 1992). The amino acid sequence of PACAP-38 is well preserved in different species and is identical in sheep, rat, and human (Arimura 1992). More interestingly, PACAP is homologous to a peptide encoded by the Drosophila memory gene amnesiac, which is thought to be involved in memory storage in the fruit fly (Feany and Quinn 1995). The possibility that PACAP may also be involved in synaptic plasticity in the mammalian nervous system has yet to be explored (Kandel and Abel 1995).

Biochemical studies have revealed two seven-transmembrane-domain PACAP receptors that are coupled to different intracellular pathways (Shivers et al. 1991). Type I receptors are positively coupled to adenylate cyclase and phospholipase C, whereas type II receptors are linked to adenylate cyclase only (Spengler et al. 1993). Activation of intracellular adenylate cyclase cascade has been shown to underlie long-lasting potentiation of glutamate responses in central synapses, which may represent a form of synaptic plasticity (Greengard et al. 1991; Siegelbaum and Kandel 1991; Wang et al. 1991).

The present study was undertaken to test the hypothesis that PACAP acting via the adenylate cyclase pathway may modulate glutamate responses in rat sympathetic preganglionic neurons (SPNs). SPNs appear to be a suitable model system because nerve terminals containing PACAP-like immunoreactivity have been shown to make synaptic-like contacts with retrogradely labeled SPNs in the rat intermediolateral cell column (Chiba et al. 1996; Dun et al. 1996b).

Some of the results have appeared as an abstract (Wu and Dun 1996a).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Procedures used in obtaining 500-µm transverse thoracolumbar spinal cord slices from 12- to 16-day-old Sprague-Dawley rats have been described (Shen and Dun 1990; Wu and Dun 1996b). Spinal slices were superfused with a Krebs solution of the following composition (in mM): 117 NaCl, 2.0 KCl, 1.2 KH2PO4, 2.3 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose. The solution was saturated with 95% O2-5% CO2. In preparing Mg2+-free Krebs solution, MgCl2 was omitted and replaced with an isomolar amount of NaCl. Whole cell recordings were made from SPNs under voltage-clamp mode with the use of an Axoclamp 2A (Wu and Dun 1996b). When filled with the solution containing (in mM) 130 K+ gluconate, 1 MgCl2, 2 CaCl2, 4 ATP, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, patch electrodes had a resistance of 3-5 MOmega . With the exception of glutamate agonists, pharmacological agents were dissolved in Krebs solution in known concentrations and applied to the slices by superfusion. N-methyl-D-aspartate (NMDA, 1 mM) or kainate (KA, 1 mM) was pressure ejected to the recording neuron from a glass pipette with the use of a Picospritzer (General Valve). A bipolar concentric electrode was placed close to the ventral rootlets for antidromic identification of SPNs (Shen and Dun 1990). Experiments were carried out at room temperature (21 ± 1°C). Results are expressed as means ± SD and analyzed statistically with the use of Student's t-test.

NMDA, KA, D-2-amino-phosphonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Cookson (St. Louis, MO); PACAP-38 was purchased from Peninsula Laboratories (Belmont, CA); MDL-12,330A and tetrodotoxin were purchased from RBI (Natick, MA); and other chemicals were from Sigma (St. Louis, MO).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

NMDA- and KA-induced currents

SPNs in the intermediolateral cell column of spinal slices were identified by their location and by the appearance of an antidromic spike following stimulation of ventral rootlets (Shen and Dun 1990). These neurons had a mean resting potential, input resistance, and time constant of -61 ± 7 mV, 530 ± 68 MOmega , and 43 ± 6 ms (n = 32), which were comparable with those reported earlier (Wu and Dun 1996b).

A brief (duration 10-20 ms) puff of NMDA or KA by pressure to SPNs voltage clamped to -60 mV, which is close to the resting potential of these neurons, evoked an inward current. Similar to what occurred in other central neurons (Collingridge and Lester 1989), NMDA currents and KA currents evoked in SPNs differed with respect to their electrophysiological and pharmacological properties. For example, changing the Krebs solution to a Mg2+-free Krebs solution markedly increased the NMDA currents (Fig. 1) but not the KA currents; the peak NMDA currents were increased by an average of 120 ± 10% (n = 5, P < 0.05) in Mg2+-free solution as compared with control Krebs solution.


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FIG. 1. N-methyl-D-aspartate (NMDA) currents potentiated by pituitary adenylate cyclase activating polypeptide(PACAP) and blocked by D-2-amino-phosphonopentanoic acid (AP5) in neonatal rat sympathetic preganglionic neuron (SPN). Pressure application of NMDA (1 mM, pulse duration 10 ms, indicated by black-down-triangle ) from micropipette positioned above recording neuron voltage clamped to -60 mV evoked inward current that was potentiated in Mg2+-free Krebs solution (black-triangle indicates change to Mg2+-free Krebs solution for remaining experiment). Note that fast transient immediately before inward current is pressure ejection artifact, which was used as marker of NMDA application in this and subsequent traces. Addition of PACAP (30 nM) to Mg2+-free solution, as indicated by arrow, further increased NMDA currents. Last, the NMDA receptor antagonist AP5 (10 µM) applied between the 2 arrows, reversibly eliminated NMDA currents.

Effects of PACAP on SPNs

PACAP, by superfusion to SPNs in slices, produced two conspicuous effects that were concentration dependent. At lower concentrations (10-30 nM), PACAP reversibly increased the NMDA currents in 28 of 42 SPNs examined; the holding currents were not significantly changed in any of these neurons. PACAP (10 nM) caused a small (12-16%) but statistically insignificant increase of NMDA currents (n = 4, P > 0.05), whereas PACAP (1 nM) produced no detectable increase in NMDA currents (n = 3). At higher concentrations (>30 nM), the peptide produced a sustained inward current in SPNs (Wu and Dun 1996a). BecausePACAP (30 nM) consistently potentiated NMDA currents without causing a significant change of holding currents, this concentration was used in all the experiments described below. The potentiation lasted 10-20 min and could be demonstrated in SPNs irrespective of the presence or absence of Mg2+ ions in the superfusing solution. Figure 1 shows that PACAP further increased the NMDA current in an SPN in which the NMDA current was first enhanced by superfusing the slice with an Mg2+-free solution. The mean increase of NMDA currents by PACAP (30 nM) in a Krebs solution with or without Mg2+ ion was 51 ± 6% (n = 17) and 47 ± 5.7% (n = 6) over the respective control responses (Fig. 2); the differences were statistically significant (P < 0.05). PACAP (30 nM) increased the NMDA currents by an average of 41 ± 4.5% (n = 4, P < 0.05) from spinal cord slices treated with tetrodotoxin (0.3 µM). Last, NMDA currents evoked in SPNs were effectively blocked by AP5 (10-20 µM) in all four neurons tested (Fig. 1). PACAP in the concentrations that potentiated NMDA currents had no significant effect on KA currents in any of the eight SPNs tested (Fig. 3). The mean amplitude of KA currents before and after PACAP (30 nM) was 83 ± 49 pA and 80 ± 47 pA (n = 8) and the difference was statistically insignificant (P > 0.05, Fig. 2). KA currents evoked in SPNs were reversibly blocked by CNQX (1 µM), as shown in Fig. 3.


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FIG. 2. Percent change of NMDA currents or kainate (KA) currents by PACAP and other pharmacological agents in rat SPNs. Mean values of NMDA or KA currents obtained in normal Krebs solution are assigned as 100% and responses obtained in presence of PACAP and/or other pharmacological agents are calculated relative to 100%, with the exception that NMDA/Mg2+-free/PACAP responses were compared with responses obtained in NMDA/Mg2+-free solution. PACAP (30 nM) was used in all experiments shown in this table; n, number of cells studied. Stars: statistically significant (P < 0.05). dbcAMP, N6,2'-0-dibutyryladenosine 3':5'-cyclic monophosphate; IBMX, 3-isobutyl-1-methylxanthine.


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FIG. 3. Lack of potentiation of KA currents by PACAP in an SPN. KA applied by pressure (1 mM, black-down-triangle ) evoked inward current, which was not enhanced by superfusing slice with PACAP (30 nM; between up-arrow and down-arrow ). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, 1 µM, horizontal bar) reversibly blocked KA currents. Chart recordings were interrupted by 3 periods of 3, 6, and 3 min.

Involvement of adenylate cyclase

The membrane-permeable cyclic AMP analogue N6,2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP, 300 µM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 700 µM) potentiated the NMDA currents (Fig. 4); the mean increase was 46 ± 9.7% (n = 5, Fig. 2). In the concentrations used here, dbcAMP or IBMX alone caused a small but statistically insignificant increase of NMDA currents in four neurons tested (P > 0.05). Incubation of the spinal cord slices with MDL-12,330A (25 µM), an adenylate cyclase inhibitor (Lippe and Ardizzone 1991), effectively blocked the potentiating effect of PACAP in all five neurons examined (Fig. 2). A representative experiment is shown in Fig. 4. Neither the holding current nor the amplitude of NMDA current was significantly changed by MDL-12,330A alone.


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FIG. 4. Potentiation of NMDA currents by dbcAMP and by PACAP in neonatal rat SPNs. A: NMDA currents induced by pressure application of NMDA (1 mM, pulse duration 10 ms, marked by arrowhead) were increased by addition of dbcAMP (300 µM) and the phosphodiesterase inhibitor IBMX (700 µM), as indicated by the 2 arrows. This effect was reversible after period of wash with normal Krebs solution. Continuous chart recording was interrupted by 2 periods lasting 8 and 4 min. B: NMDA currents in this neuron were first increased by superfusing slice with PACAP (30 nM), applied between the 2 arrowheads. After NMDA responses had returned to control level, the adenylate cyclase inhibitor MDL-12,330A (25 µM) was added to Krebs solution. Addition of PACAP, as indicated by horizontal bar, caused no increase of NMDA currents in presence of MDL-12,330A. Both neurons were voltage clamped to -60 mV.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The peptide PACAP at concentrations of <= 30 nM selectively enhanced NMDA currents but not KA currents in immature rat SPNs. The potentiation occurred at concentrations that caused no significant change of holding currents. At higher concentrations, PACAP induced a long-lasting inward current (Wu and Dun 1996a). Further, the peptide increased the NMDA currents in Mg2+-containing and Mg2+-free solutions to about the same magnitude, implying that the potentiating action of PACAP is independent of Mg2+ ions. In this respect, protein kinase C appears to potentiate NMDA currents in isolated trigeminal neurons by reducing the Mg2+ block of NMDA receptor channels (Chen and Huang 1992). Moreover, PACAP potentiated NMDA currents in a tetrodotoxin-containing medium, indicating that the potentiation is not likely to be caused secondarily by a release of bioactive substances from the spinal cord slices.

The possibility that adenylate cyclase may be the intracellular messenger underlying the PACAP-mediated potentiation is suggested by the following observations. First, incubation of the spinal cord slices with the adenylate cyclase inhibitor MDL-12,3330A effectively prevented the potentiating effect of a second application of PACAP in SPNs in which an enhancement of NMDA currents could first be demonstrated by the peptide. Second, the membrane-permeable dbcAMP in the presence of a phosphodiesterase inhibitor mimicked the potentiating effect of PACAP. A potentiation of NMDA currents via a cyclic-AMP-dependent mechanism has been reported by several groups of investigators. For example, NMDA currents were enhanced by 8-bromo-cyclic AMP, or by intracellular application of adenosine 3',5'-cyclic monophosphate (cAMP) or of catalytic subunits of protein kinase A (Cerne et al. 1993). Similarly, activation of beta -adrenergic receptors potentiated NMDA responses via a cAMP-dependent protein kinase mechanism in basolateral amygdala and cultured hippocampal neurons (Huang et al. 1993; Raman et al. 1996), whereas activation of protein kinase C appears to be involved in the sustained potentiation of NMDA responses by the µ-opioid receptor agonist D-Ala2-MePhe4-gly-ol5-enkephalin (Chen and Huang 1991) and by the tachykinins substance P and neurokinin A (Rusin et al. 1993). In addition to neuropeptides, activation of metabotropic receptors has been shown to selectively potentiate NMDA but not alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid responses in hippocampal neurons (Aniksztejn et al. 1991; Harvey and Collingridge 1993). The mechanism underlying metabotropic-receptor-agonist-mediated potentiation of NMDA responses appears also to be cyclic AMP independent (Harvey and Collingridge 1993).

PACAP is homologous to a peptide that is thought to be involved in memory storage in Drosophila (Feaney and Quinn 1995). Glutamate-receptor-mediated long-term potentiation is implicated in certain forms of synaptic plasticity in the mammalian central synapse (Greengard et al. 1991; Siegelbaum and Kandel 1991). Viewed in this context,PACAP-mediated facilitation of NMDA responses may constitute a form of plasticity in certain central synapses. The distribution of PACAP-like immunoreactivity in the human spinal cord (Dun et al. 1996a) is similar to that of the rat and mouse spinal cord (Dun et al. 1996b; Narita et al. 1996), underscoring the potential importance of this peptide in synaptic plasticity of all mammals. Furthermore, our results support the concept that a common pathway may exist between neuropeptides and adenylate cyclase cascade on one hand and synaptic plasticity on the other (Kandel and Abel 1995).

    ACKNOWLEDGEMENTS

  This study was supported by National Institutes of Health Grants NS-18710 and HL-51314.

    FOOTNOTES

  Address for reprint requests: N. J. Dun, Dept. of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, PO Box 70577, Johnson City, TN 37614.

  Received 8 January 1997; accepted in final form 18 April 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society