Pituitary adenylate cyclase-activating polypeptide modifies the electrical activity of CA1 hippocampal neurons in the rat

Neuroscience Letters 337 (2003) 97–100 www.elsevier.com/locate/neulet

Pituitary adenylate cyclase-activating polypeptide modifies the electrical activity of CA1 hippocampal neurons in the rat M. Di Mauro a, S. Cavallaro b, L. Ciranna a,* a

Dipartimento di Scienze Fisiologiche, Universita` di Catania, 95125 Catania, Italy b Istituto di Istituto di Scienze Neurologiche - CNR, 95123 Catania, Italy Received 25 October 2002; accepted 7 November 2002

Abstract The effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on neuronal excitability in the CA1 region of rat hippocampus were studied using in vivo and in vitro electrophysiological techniques. Extracellularly recorded spontaneous firing of CA1 neurons was transiently (2–7 min) increased by PACAP (106 ^ 32% enhancement, mean ^ SEM, n ¼ 11). Using whole-cell patch clamp, PACAP was tested on the resting membrane current of CA1 pyramidal neurons: PACAP activated a slow-onset (20–30 s) and long-lasting (over 20 min) inward current with a mean amplitude of 99 ^ 34 pA (mean ^ SD, n ¼ 8). These results indicate that PACAP induces depolarizing effects on CA1 hippocampal neurons. PACAP-induced long-lasting facilitation in the CA1 region might modify neuronal excitability and/or modulate the effect of other neurotransmitters. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pituitary adenylate cyclase-activating polypeptide; Hippocampus; CA1; Pyramidal neurons; Excitatory; Electrophysiology

The peptide PACAP, isolated from ovine hypothalamus, is widely distributed in the brain and modulates various physiological functions [4,7]. The rat hippocampus contains considerable amounts of PACAP [1] and PACAP receptors are expressed in dentate gyrus and in CA1 and CA2 pyramidal neurons [12]. The function of PACAP in the hippocampus is not fully known: PACAP stimulates intracellular Ca 21 release [14], exerts a neuroprotective action [11,15] and modulates synaptic transmission [5,8,9]; besides, PACAP receptor distribution changes from embryonic stages to adulthood, suggesting a role in development [13]. At a single cell level, nothing is known about a possible action of PACAP on membrane currents and on the excitability of hippocampal neurons. We have studied the electrophysiological effects of PACAP in CA1 region, where the presence of PACAP receptors has been demonstrated, using two different approaches: (1) extracellular recording in vivo to test PACAP effect on neuronal firing; (2) patch-clamp on hippocampal slices to test PACAP action on membrane ion currents of CA1 pyramidal neurons. * Corresponding author. Tel. 139-095-738-4055; fax 139-095330645. E-mail address: [email protected] (L. Ciranna).

Extracellular recordings of neuronal activity were performed on adult male Wistar rats; adequate measures were taken to minimize pain to the animal, according to the NIH Guide for the Care and Use of Laboratory Animals. The rat was anesthetized by intraperitoneal injection of urethane (1.3 g/Kg) and fixed in a stereotaxic frame; a hole was drilled in the skull to introduce a three barrel glass micropipette. After the experiment, the brain was removed, fixed in formaline (10%) and cut in frontal sections (60 mm) with a freezing microtome. The slices were stained with Neutral red; electrode tracks in the hippocampus were identified under a stereomicroscope. CA1 neuronal firing was recorded through one barrel of the micropipette (impedance 10–30 MV): single neuron activity was discriminated depending on spike shape and amplitude (Spike2 software, CED, Cambridge, UK) and was considered as unitary when the spike amplitude was approximately four times larger than background activity. Data were stored on a PC through an interface 1401 plus (CED, Cambridge, UK); the number of spikes/s was graphically represented as continuous time histogram over 5 s. One micropipette barrel contained PACAP-38 (Tocris, 100 mM in distilled water, pH 6.5 with NaOH 100 mM), applied by microiontophoresis with a positive ejection current (10–40 nA). Ejection currents were automatically balanced by the microionto-

0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S03 04 -3 94 0( 02 ) 01 316 - 2

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Fig. 1. (A) The firing activity of a CA1 hippocampal neuron, recorded extracellularly, was represented with continuous time histograms over 5 s epochs. A microiontophoretic application of PACAP-38 (PACAP, 10 nA, 30 s) increased the rate of firing; complete recovery was observed after the end of PACAP application. The shaded box includes the interval of PACAP effect (duration 100 s). (B) Histograms represent the mean firing rate (mean ^ SEM, n ¼ 11) in control conditions (ctrl), at maximal PACAP effect (PACAP max) and over the whole interval of PACAP-induced firing increment (PACAP interval). (C) Microphotograph of the pyramidal cell layer in the CA1 region of a freshly cut hippocampal slice (400 £ magnification). One of the pyramidal neurons studied in our experiments is visible in the middle (dotted circle) being impaled by the patch-clamp pipette (recording electrode). (D) Whole-cell spontaneous membrane current recorded from a CA1 pyramidal neuron at a holding potential (HP) of 270 mV. Bath application of PACAP-38 (PACAP, 100 nM, 5 min) induced a slow-onset inward current of about 150 pA amplitude, which appeared 1 min after the beginning of application and lasted longer than 9 min.

phoresis system through the third micropipette barrel containing 3 M NaCl. For each neuron studied we calculated the mean firing rate, standard deviation (SD) and standard error of the mean (SEM) during at least 2 min before drug application; PACAP was tested only if the SD did not exceed 20% of the mean firing rate value. PACAP effect was defined as a change in the mean firing rate by at least 2 SD from the mean background activity and lasting more than 20 s. Unpaired Student’s t-test was performed on both responsive and unresponsive neurons to compare firing rate value in control conditions and after PACAP application (Graph Pad Prism). We have recorded the electrical activity of 23 neurons (12 animals) located in the CA1 region, as confirmed by histological reconstruction of electrode tracks after each experiment. CA1 neurons fired spontaneously at a mean rate of 5.3 ^ 3 spikes/s (mean ^ SD, n ¼ 23, range 2–12 spikes/s). Application of PACAP-38 (10–40 nA, during 30 s) increased the firing rate in 11 out of 23 neurons studied

(47%); PACAP had no effect on the remaining 12 neurons tested. PACAP-induced excitation was fully reversible (Fig. 1A): neuronal firing was remarkably increased by PACAP (10 nA, 30 s), excitation appeared 40–50 s after the beginning of PACAP application and complete recovery was observed within 2 min. The amount of PACAP effect was calculated both as maximal firing rate increment after PACAP application (PACAP max) and as the increment over the whole interval (2–7 min in distinct neurons) of PACAP effect (PACAP interval, included in the shaded box in Fig. 1A). As shown in Fig. 1B, the mean spontaneous firing (5.4 ^ 1 spikes/s, n ¼ 11) was raised to 17.1 ^ 4.8 spikes/s at maximal PACAP effect, yielding an increment of 226 ^ 76% (mean ^ SEM, n ¼ 11); for each neuron, the firing rate increase was statistically significant (P , 0:03). When considering the whole interval of PACAP effect, firing rate reached 9.8 ^ 1.9 spikes/s (mean ^ SEM, n ¼ 11), with a mean enhancement of 99 ^ 33% (n ¼ 11). In unresponsive neurons (n ¼ 12), the mean firing rate after PACAP application (5.0 ^ 1.2 spikes/s, mean ^ SD)

M. Di Mauro et al. / Neuroscience Letters 337 (2003) 97–100

did not significantly differ (P ¼ 0:9) from mean spontaneous firing (5.2 ^ 1.1, mean ^ SD). No relationship was found between the level of neuronal firing and responsiveness to PACAP: spontaneous firing rate was 5.4 ^ 1 (mean ^ SD, n ¼ 11) and 5.2 ^ 1.1 (mean ^ SD, n ¼ 12) spikes/s for responsive and unresponsive neurons respectively, and was not significantly different in the two groups (P ¼ 0:8). Neurons responding to PACAP with an increase of firing rate did not appear to be segregated to any particular CA1 zone, since both responsive and unresponsive neurons were scattered through the CA1 region (not shown). These results indicate that PACAP exerts excitatory effects on a subset of CA1 hippocampal neurons. To identify morphologically the type of neuron responsive to PACAP, we turned to freshly-cut slices where CA1 neurons could be visualized with infrared microscopy, and we investigated the effect of PACAP on the resting membrane current of pyramidal neurons using whole-cell patch-clamp. Young rats (9–20 days) were decapitated under ether anaesthesia; the brain was removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) bubbled with a mixture of O2 95% and CO2 5%. ACSF composition was (in mM): NaCl, 124; KCl, 3.0; NaH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.0; NaHCO3, 26; d-glucose, 10. Using an Oxford Vibratome, the hippocampus was cut into slices (300–400 mm), kept in oxygenated ACSF at room temperature (21–22 8C) during at least 1 h prior to recording. At the beginning of the experiment, one slice was transferred to the recording chamber (volume 2 ml), continually perfused with oxygenated ACSF at a rate of 1 ml/min and viewed under an infrared/differential interference contrast upright microscope (Leica DMLFS). The recording electrode was a micro-hematocrite glass pipette (resistance between 1.5 and 3 mV), filled with a solution containing (in mM): KCl, 170; HEPES, 10; MgCl2, 2; NaCl, 10; EGTA, 0.2; Mg-ATP, 3.5; Na-GTP, 1 (pH 7.3 with KOH). PACAP-38 was dissolved in ACSF. Data were acquired and analysed using both EPC and Signal softwares from Cambridge Electronic Instruments (CED, UK). CA1 pyramidal neurons were visually identified by their location and by their typical elongated shape and large dimension (Fig. 1C). The membrane potential of the neuron studied was held at 270 mV and spontaneous membrane current was recorded for at least 3 min after seal establishment. PACAP-38 was applied by bath perfusion only if baseline current remained stable, i.e. if SD did not exceed 20% of the mean value. In all the cells tested (n ¼ 12), application of PACAP induced a slow-onset inward current. The effect of PACAP was dose-dependent: the amplitude (mean ^ SD) of PACAP-induced inward current was 33 ^ 12 pA (n ¼ 4), 71 ^ 22 pA (n ¼ 4) and 99 ^ 59 pA (n ¼ 8) at PACAP concentrations of 1, 10 and 100 nM respectively. PACAP-induced current appeared 20–30 s after the beginning of PACAP application and persisted during at least 20 min after washout (Fig. 1D). In control experiments, no variation of spontaneous current was

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observed after application of ACSF solution containing either no PACAP (n ¼ 4) or PACAP 100 nM inactivated by previous heating at 56 8C during 20 min (n ¼ 5). All CA1 pyramidal neurons in vitro responded to PACAP with an inward current, whereas in extracellular recordings, where the cell type could not be identified, PACAP increased the firing rate in only a fraction (47%) of CA1 neurons. Conventionally, an inward current corresponds to positive charges entering the membrane and, whatever the ionic basis (either cation influx, anion efflux or closure of resting potassium channels), leads to membrane depolarization. PACAP-induced inward current in CA1 pyramidal neurons went in the same direction of PACAP-induced increase of CA1 neuronal firing in vivo, and thus might represent the same effect observed with a different technique. Further investigations will clarify whether the two effects share common mechanisms or diverge at some level. PACAP-induced inward current had a relatively long duration (.20 min) which cannot be attributed to the perfusion system used: the recording chamber in fact contained 2 ml of solution, thus at a flow rate of 1 ml/min washout of drugs was complete after 2 min. The time course of PACAPinduced current that we observed was similar to that described in neurons from rat supraoptic nucleus, where PACAP activated a long-lasting cation inward current leading to membrane depolarization, increase of firing and hormone release [10]. Other examples exist in literature: PACAP induced a slow inward current and membrane depolarization in rat pineal cells [3] and stimulated the firing of rat paraventricular [16] and sympathetic preganglionic neurons [6]. PACAP-induced inward current is likely to modify neuronal responsiveness to weak excitatory stimuli, which might cause further depolarization and cell firing. Neuronal depolarization is also crucial for long-term potentiation (LTP) induction in the CA3–CA1 synapse, removing the blockade of N-methyl-d-aspartate receptors by Mg 21 ions [2]. From our results, PACAP depolarized CA1 pyramidal neurons over long periods of time and two distinct articles have reported a modulatory action of PACAP on the CA3– CA1 synapse, respectively showing a reduction [5] and a facilitation of transmission [8]. Further studies will clarify if PACAP might interfere with LTP induction and modify the cellular basis for learning and memory. We are grateful to Prof. Francesca Santangelo, Flora Licata and Guido Li Volsi for helpful suggestions and critical reading of the manuscript. The present work was supported by a grant from the Biotech project n. BIO4-980517: “Physiological functions of PACAP/VIP receptors in the nervous system”. [1] Arimura, A., Somogyvari-Vigh, A., Miyata, A., Mizuno, K., Coy, D.H. and Kitada, C., Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes, Endocrinology, 129 (1991) 2787–2789.

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[2] Bliss, T.V. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31–39. [3] Darvish, N. and Russell, J.T., Neurotransmitter-induced novel modulation of a nonselective cation channel by a cAMP-dependent mechanism in rat pineal cells, J. Neurophysiol., 79 (1998) 2546–2556. [4] Harmar, A.J., Arimura, A., Gozes, I., Journot, L., Laburthe, M., Pisegna, J.R., Rawlings, S.R., Robberecht, P., Said, S.I., Sreedharan, S.P., Wank, S.A. and Waschek, J.A., International Union of Pharmacology, XVIII: nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase- activating polypeptide, Pharmacol. Rev., 50 (1998) 265–270. [5] Kondo, T., Tominaga, T., Ichikawa, M. and Iijima, T., Differential alteration of hippocampal synaptic strength induced by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), Neurosci. Lett., 221 (1997) 189–192. [6] Lai, C.C., Wu, S.Y., Lin, H.H. and Dun, N.J., Excitatory action of pituitary adenylate cyclase activating polypeptide on rat sympathetic preganglionic neurons in vivo and in vitro, Brain Res., 748 (1997) 189–194. [7] Rawlings, S.R., PACAP PACAP receptors, and intracellular signalling, Mol. Cell Endocrinol., 101 (1994) C5–C9. [8] Roberto, M. and Brunelli, M., PACAP-38 enhances excitatory synaptic transmission in the rat hippocampal CA1 region, Learn. Mem., 7 (2000) 303–311. [9] Roberto, M., Scuri, R. and Brunelli, M., Differential effects of PACAP-38 on synaptic responses in rat hippocampal CA1 region, Learn. Mem., 8 (2001) 265–271. [10] Shibuya, I., Kabashima, N., Tanaka, K., Setiadji, V.S., Noguchi, J., Harayama, N., Ueta, Y. and Yamashita, H., Patch-

[11]

[12]

[13]

[14]

[15]

[16]

clamp analysis of the mechanism of PACAP-induced excitation in rat supraoptic neurones, J. Neuroendocrinol., 10 (1998) 759–768. Shioda, S., Ozawa, H., Dohi, K., Mizushima, H., Matsumoto, K., Nakajo, S., Takaki, A., Zhou, C.J., Nakai, Y. and Arimura, A., PACAP protects hippocampal neurons against apoptosis: involvement of JNK/SAPK signaling pathway, Ann. N.Y. Acad. Sci., 865 (1998) 111–117. Shioda, S., Shuto, Y., Somogyvari-Vigh, A., Legradi, G., Onda, H., Coy, D.H., Nakajo, S. and Arimura, A., Localization and gene expression of the receptor for pituitary adenylate cyclase-activating polypeptide in the rat brain, Neurosci. Res., 28 (1997) 345–354. Skoglosa, Y., Takei, N. and Lindholm, D., Distribution of pituitary adenylate cyclase activating polypeptide mRNA in the developing rat brain, Brain Res. Mol. Brain Res., 65 (1999) 1–13. Tatsuno, I., Yada, T., Vigh, S., Hidaka, H. and Arimura, A., Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons, Endocrinology, 131 (1992) 73–81. Uchida, D., Arimura, A., Somogyvari-Vigh, A., Shioda, S. and Banks, W.A., Prevention of ischemia-induced death of hippocampal neurons by pituitary adenylate cyclase activating polypeptide, Brain Res., 736 (1996) 280–286. Uchimura, D., Katafuchi, T., Hori, T. and Yanaihara, N., Facilitatory effects of pituitary adenylate cyclase activating polypeptide (PACAP) on neurons in the magnocellular portion of the rat hypothalamic paraventricular nucleus (PVN) in vitro, J. Neuroendocrinol., 8 (1996) 137–143.