Galanin inhibits neural activity in the subfornical organ in rat slice preparation

Neuroscience 143 (2006) 769 –777

GALANIN INHIBITS NEURAL ACTIVITY IN THE SUBFORNICAL ORGAN IN RAT SLICE PREPARATION A. KAI,a,b K. ONO,a H. KAWANO,c E. HONDA,a O. NAKANISHIb AND K. INENAGAa*

Rada et al., 1998; Björkstrand et al., 1993), whereas it decreases water intake and the plasma concentration of vasopressin and oxytocin (Brewer et al., 2005; Kondo et al., 1993; Björkstrand et al., 1993). The actions of GAL are mediated by at least three GAL receptor subtypes, GalR1, GalR2 and GalR3. The former two subtypes were widely expressed in the CNS but GalR3 was more sparsely distributed (Wang et al., 1997; Waters and Krause, 2000). Although all the three receptor subtypes show high affinity for GAL, the affinity of GAL for GalR1 is relatively higher than other GalRs (Wang et al., 1997). In some electrophysiological studies, GAL has been reported to have both inhibitory and excitatory actions. For example, GAL inhibits the neural activity of neurons in the hypothalamic supraoptic nucleus (SON) and locus coeruleus (LC) (Papas and Bourque, 1997; Sevcik et al., 1993; Xu et al., 2005) whereas it excites cholinergic neurons in the diagonal band of Broca (DBB) and dorsal root ganglion (DRG) neurons (Jhamandas et al., 2002; Puttick et al., 1994; Kerekes et al., 2003). The diversity of the effects of GAL is thought to be due to the different responses of the different GalR subtypes, that is, inhibition through GalR1 and GalR3, and excitation through GalR2. The signaling pathways are also different; the activation of both GalR1 and GalR3 is coupled to Gi/o, whereas that of GalR2 is predominantly coupled to Gq/11 (Wang et al., 1998; Branchek et al., 2000). The subfornical organ (SFO) is a circumventricular structure that plays important roles in the control of water intake and vasopressin release. Because the organ lacks a blood– brain barrier, many blood-borne peptides can directly affect SFO neurons. Many previous in vivo studies have reported that angiotensin II (ANGII) injected into the SFO induces drinking behavior and vasopressin release (Mangiapane and Simpson, 1980; Iovino and Steardo, 1984), so that GAL may inhibit the activity of ANGII-sensitive SFO neurons. Since it has been recently reported that the SFO is a central target for circulating feeding signals, such as ghrelin and amylin (Pulman et al., 2006), it is not surprising that some cells in the SFO respond to GAL which is known to be a feeding-related peptide. Additionally, in situ hybridization studies have revealed that GalR3 mRNA is expressed in the SFO (Mennicken et al., 2002), although the mRNAs for other GalR subtype have not been studied. In the present study, we investigated whether the three known GalRs are present in the rat SFO using the reverse transcription–polymerase chain reaction (RT-PCR) technique, and whether GAL affects the electrophysiological behavior of SFO neurons using selective agonists for the GalR subtypes techniques. In addition, we investigated

a

Department of Biosciences, Kyushu Dental College, 2-6-1 Manazuru, Kokurakitaku, Kitakyushu, 803-8580, Japan

b

Department of Control of Physical Function, Kyushu Dental College, 2-6-1 Manazuru, Kokurakitaku, Kitakyushu, 803-8580, Japan

c

Department of Anatomy and Physiology, Saga University, Nabeshima, Saga, 849-8501, Japan

Abstract—The activation of the subfornical organ (SFO), a circumventricular organ, induces water intake and vasopressin release. Since central administrations of galanin (GAL) suppress water intake and vasopressin release, GAL may inhibit the neural activity of SFO neurons. In the present study, we investigated effects of GAL on the SFO using molecular biological, electrophysiological and anatomical techniques. Reverse transcription–polymerase chain reaction analysis demonstrated the presence in the SFO of rats of the mRNAs for each of the three known GAL receptor subtypes (GalR1, GalR2 and GalR3). In extracellular recordings in SFO slice preparations, GAL dose-dependently inhibited the neural activity of cells from a number of recording sites. Many GAL-sensitive SFO neurons showed excitatory responses to angiotensin II (ANGII). The GalR1 agonist M617 inhibited the activity of SFO neurons, whereas the GalR2 and GalR3 agonist GAL(2–11) had almost no effect. In patch-clamp recordings, GAL induced an outward current in SFO neurons without influencing synaptic currents. An immunoelectron microscopic study revealed the existence of GAL-containing synaptic vesicles in the SFO. These results suggest that the SFO has neural inputs involving GAL. The response to GAL is inhibitory, mediated at least in part by GalR1 and provides a plausible explanation for the opposite effects of ANGII and GAL seen in vivo on water intake and vasopressin release. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: subfornical organ, galanin, immunoelectron microscopy, RT-PCR, extracellular recording.

Galanin (GAL), a 29 amino acid peptide first isolated from pig intestine (Tatemoto et al., 1983), is a widely distributed neurotransmitter in the nervous system (Melander et al., 1986), and is thought to play important roles in many physiological functions. Peripheral or central administration of GAL increases food intake and the concentration of cholecystokinin in plasma (Kyrkouli et al., 1986, 1990; *Corresponding author. Tel: ⫹81-93-582-1131; fax: ⫹81-93-582-8288. E-mail address: [email protected] (K. Inenaga). Abbreviations: ANGII, angiotensin II; DBB, diagonal band of Broca; DRG, dorsal root ganglion; GAL, galanin; LC, locus coeruleus; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT-PCR, reverse transcription–polymerase chain reaction; SFO, subfornical organ; SON, supraoptic nucleus; TTX, tetrodotoxin.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.08.043

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existences of GAL-immunoreactive nerve terminals in the SFO by using electron microscopy.

The care and handling of the animals used in these experiments was consistent with all the U.S. National Institutes of Health recommendations for the humane use of animals. All experimental procedures were reviewed and approved by the appropriate Animal Experiment Committees of the Kyushu Dental College where the experiments were performed. The number of animals used was kept to the minimum necessary for a meaningful interpretation of the data and animal discomfort was kept to the minimum.

than conventional extracellular recording using single glass micropipettes. Frequently a continuous recording could be obtained for 4 –5 h. The data were stored on a computer hard disk drive through Power Laboratory system (sampling rate: 10 –20 kHz). In offline analysis, amplitude ranges were set to discriminate neural activity as single- or multi-units using a window discriminator and with amplitude and interspike interval histograms (Spike Histogram, ADInstruments, Sydney, NSW, Australia). The interspike intervals of all discriminated single-units were over 50 ms throughout. The averaged discharge rate for 300 s before drug administration was used as control. The averaged discharge rate for 60 s was compared with control to evaluating the responses. When the averaged discharge rate was greater or smaller than the control value by 20% or more, the site was considered sensitive to the applied chemicals.

Conventional RT-PCR

Whole-cell patch-clamp recordings

The total RNA from the SFO in male Wistar rats was analyzed with a protocol similar to that reported previously (Honda et al., 2003; Ono et al., 2005). After making thick SFO slices by the same method as that used for electrophysiological experiments (see below), SFO tissues were dissected away from the hippocampal commissure and corpus callosum under the microscope. Total RNA was extracted using RNeasy Mini Kits (Qiagen, Hilden, Germany). Reverse transcription of the total RNA (40 ng) was performed in a final volume of 20 ␮l using oligo-dT12–18 primer (0.5 ␮g/␮l) and RNasin (10 units; TaKaRa, Otsu, Japan) with sensiscript RT kit (Qiagen). Polymerase chain reaction (PCR) was performed with a thermal cycler (PCR Thermal Cycler Dice; TaKaRa). Specific primers for GalR1, GalR2 and GalR3 were used in published sequences (Seth et al., 2004). PCR was performed with a PCR buffer containing 10 pmol primers, 2.5 U TaqDNA polymerase (Hot Start Version; TaKaRa) and each transcribed cDNA, in a final volume of 50 ␮l. Single-stranded cDNA products were denatured and subjected to PCR amplification (40 cycles). Each PCR cycle consisted of denaturation at 94 °C for 20 s, annealing at 63– 69 °C for 30 s and finally extension at 70 °C for 35 s. Genomic DNA (50 ng) and total RNA (10 ng) were respectively used as positive and negative controls. The PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. All positive amplicons were sequenced with a dye termination procedure and these sequences were found to match published sequences closely.

Whole-cell recordings were made as described previously (Honda et al., 2003). Perfusion flow rate was set to 1.5 ml/min at 33 °C. The pipette solution used in the microelectrodes contained (in mM): K gluconate 140, MgCl2 1, CaCl2 1, EGTA 5, Hepes 10, Na2ATP 4 (pH 7.2 adjusted with KOH). The electrodes were double-pulled (P-87, Sutter Instrument Co., Novato, CA, USA) from borosilicate thin-glass capillaries (i.d. 1.2 mm and o.d. 1.5 mm), and had a final resistance of 5–10 M⍀ when filled with the pipette solution. The series resistances (⬍25 M⍀) and membrane capacitance were compensated and checked regularly during the recording. The transmembrane current was recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA, USA). Potential values were corrected for the junction potential (⫺11 mV). An event-detection threshold of more than 5 pA was used, to discern from baseline noise. Signals were digitized at 4 kHz with an analog-digital converter (PowerLab/8, ADI, Sydney, Australia).

EXPERIMENTAL PROCEDURES Experimental animals

Slice preparations Male Wistar rats weighing 150 –200 g were deeply anesthetized with ketamine (250 mg/kg, s.c.) and decapitated. The brain was blocked at an angle of approximately 45° between the coronal and horizontal planes from the ventral to the rostrodorsal surfaces of the brain. Slices of 300-␮m thickness were prepared in a cold slice-cutting solution (in mM; sucrose 211, KCl 3, NaHCO3 26, glucose 10, MgSO4 1.3, KH2PO4 1.24, CaCl2 2.1), and preincubated in the bathing and perfusion solution (in mM; NaCl 124, KCl 3, NaHCO3 26, glucose 10, MgSO4 1.3, KH2PO4 1.24, CaCl2 0.75 (extracellular recording) or 2.1 (patch-clamp recording), which was oxygenated with 95% O2 and 5% CO2) at room temperature for at least 1 h before recording.

Extracellular recordings In extracellular recordings (MEA Multichannel Systems, Reutlingen, Germany), one slice was submerged in a recording chamber containing an array of 60 chevron-type microelectrodes with a volume of 1 ml at a room temperature (20 –23 °C). Perfusion flow rate was set to 1 ml/min. Often, spontaneous electrical activities from more than two sites were simultaneously measured. The recording system gives stable long-term recording and is simpler

Drug applications When required, a low Ca2⫹ (0.5 mM) and high Mg2⫹ (9 mM) solution was used to block synaptic transmission in extracellular recordings as reported in previous studies (Okuya et al., 1987). Tetrodotoxin (TTX) at 0.5 ␮M was applied to SFO slices to confirm whether or not the recorded signals represented true neural activity. In whole-cell patch-clamp recordings, TTX at 0.3 ␮M was routinely included in a perfusion solution to block TTX-sensitive neural activation. Drugs were applied to the slices for 2 min (extracellular recordings) or 1.5 min (whole-cell patch-clamp recordings) by perfusing from separate storage bottles containing medium to which they had been added. The drugs were purchased from the following pharmaceutical companies; TTX from Wako (Japan), GAL and ANGII from the Peptide Institute (Japan) and GAL(2–11) from Sigma (St. Louis, MO, USA). The GalR1 selective agonist M617 was kindly gift from Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden (Lundström et al., 2005).

Immunohistochemical electron microscopy Male Sprague–Dawley rats (7 weeks⫾1 day old) were used. After anesthesia with an i.p. injection of pentobarbital sodium (50 mg/ kg) the animals were perfused through the ascending aorta with physiological saline, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). The brains were dissected out and a brain block containing the SFO was immersed in the same fixative for 2–24 h. After cryoprotectant treatment with 20% sucrose in phosphate-buffered saline (PBS), 30 ␮m thick frontal sections throughout the SFO were cut serially using a freezing microtome, collected in PBS, and soaked in 0.3% H2O2 in PBS for 10 min to block intrinsic peroxidase activities before immunohistochemistry. The sections were incubated first with 2% normal

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Jose, CA, USA) was used to make minor adjustments of brightness and contrast of digitally captured electron microscopic images.

Statistical analysis Student’s t-test was used in comparisons of two groups. The numerical data are given as mean⫾S.E.M., and n represents the number of neurons tested. Fig. 1. Identification of GAL receptor subtypes, GalR1, GalR2 and GalR3 in rat SFO tissues. The panel shows the results of agarose gel electrophoresis of fragments amplified by RT-PCR using subtypespecific primers. The PCR products of all three receptor types (GalR1, GalR2 and GalR3) were found in SFO tissue. (⫺), No reverse transcriptase present (as negative control); (⫹), cDNA of SFO after reverse transcriptase; (g), rat genome DNA (as positive control); M, molecular markers from 200 to 900 bp (in 100 bp increments).

swine serum in PBS (also used as the diluent for all antisera used) for 1 h, then with rabbit polyclonal rat anti-GAL (Peninsula, Belmont, CA, USA; Cat. No. T4334, diluted 1:4000) overnight at 4 °C, biotinylated donkey anti-rabbit IgG (Jackson, West Grove, PA, USA; diluted 1:200) for 1.5–2 h at room temperature and avidinbiotinylated peroxidase complex (Vector, Burlingame, CA, USA; diluted 1:200) for 1.5–2 h at room temperature. Thereafter, the sections were reacted with 0.02% diaminobenzidine-tetrahydrochloride (Dotite, Kumamoto, Japan) in 0.05 M Tris-buffered saline (pH 7.6) containing 0.005% H2O2 for 10 –15 min. After immunohistochemistry, the sections were postfixed with a mixture of 1% osmium tetroxide and 1.5% potassium ferrocyanide, dehydrated, and embedded in Spurr’s resin (Nisshin EM, Tokyo, Japan). Ultrathin sections of the SFO were prepared on a Sorvall MT2-B Ultra Microtome (Ivan Sorvall, Newtown, CT, USA), observed under a JEOL 100CX electron microscope (Jeol, Tokyo, Japan) without metal staining, and photographed on negative films. Digital images were captured using a film scanner and an image processing software (Adobe Photoshop, Adobe Systems, San

RESULTS GAL receptor subtypes in the SFO To determine which GAL receptor subtypes were expressed in the SFO, RT-PCR was performed from SFO tissues with GalR1, GalR2 and GalR3 specific primers. PCR products with the expected length of all GAL receptor subtypes were detected in SFO tissues (Fig. 1). Nucleotide sequence analysis of all PCR products revealed that these bands were made from fragments of the respective GAL receptor subtypes. These results suggest that the SFO contains the mRNAs of all three known GAL receptor subtypes. Here, it should be noted that the band for GalR3 from cDNA of SFO was thinner than others. Analysis of extracellular recordings from SFO slice preparations We obtained 135 multi-unit recordings in SFO slice preparation. From the 135 multi-unit recordings, 25 single-units were discriminated, as shown as Fig. 2A and B. The maximal amplitude of spikes of the single-units ranged from 44 to 170 ␮V and the mean firing rate of spontaneously activities was 1.5⫾0.4 Hz. At the end of recording, TTX at

Fig. 2. Signals and noise in extracellular recording. (A) TTX application (indicated by horizontal bar) suppressed spontaneous electrical activity. The two panels on the right are enlarged voltage traces before (1) and after the application of TTX at 0.5 ␮M (2). (B) Rate meter record of two different single units (white and black squares). Single unit activity completely disappeared after the application of TTX. (C) Amplitude histogram of two different single units (white and black squares). The discrimination analysis was performed from the data for 1 min (indicated 3 in A) before the application of TTX.

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Fig. 3. Inhibitory effects of GAL on spontaneous neural activities. Horizontal bars indicate drug application time, and the concentrations of GAL are indicated above the bars. (A) GAL inhibited the spontaneous electrical activity in a multi-unit recording (Aa) and a single-unit (Ab) that was discriminated from the multi-unit in Aa in dose-dependent manner. (B) The dose-response relationships of inhibitory effects of GAL (white circles; multi-units, n⫽14 and black circles; single-units, n⫽5). Each point represents the mean⫾S.E.M. (C) Persistence of inhibitory effect of GAL after synaptic blockade. The left and right panels show inhibitory responses to the application of GAL at 300 nM in a control perfusion medium (Ca) and a low Ca2⫹ and high Mg2⫹ medium (Cb).

0.5 ␮M was always applied to confirm neural activities over noise level (Fig. 2C). Inhibitory effects of GAL on SFO neurons To investigate effects of GAL on spontaneous electrical activity of SFO neurons, GAL was applied for 2 min at concentrations of 1, 10, 100 and 300 nM with 15– 40 min intervals between applications in 23 multi-unit recordings. Fig. 3Aa shows the rate-meter record of a multi-unit in a single recording site. From 23 multi-unit recordings, seven single-units were discriminated. Fig. 3Ab shows a ratemeter record in a single-unit that was discriminated from data of the multi-unit in Fig.3Aa. In 13 of 23 multi-units and five of seven single-units, GAL dose-dependently suppressed the spontaneous electrical activities (Fig. 3B). The threshold concentration of GAL was around 10 nM. It is unclear whether the responses were direct or indirect through other neurons located nearby. To resolve this problem, a low Ca2⫹ and high Mg2⫹ medium was used to block synaptic transmission (Okuya et al., 1987). Because the medium led to a decrease in the amplitude of the spike, it was sometimes impossible to discriminate singlefrom multi-units. Therefore, multi-unit analysis was only performed in some of the experiments (n⫽18). In the normal solution, eight of 18 multi-units were inhibited by GAL at 300 nM to 64.6⫾5.1% of the control (Fig. 3Ca). After blockade of synaptic transmission, six of the eight GALsensitive units were inhibited to 41.6⫾6.2% by the same concentration of GAL (Fig. 3Cb). The results indicate that SFO neurons are themselves sensitive to GAL. The re-

maining two GAL-sensitive units could not be analyzed because of smallness of the spikes in the low Ca2⫹ and high Mg2⫹ medium. In voltage-clamp patch-clamp recordings, GAL at 1 ␮M was applied to 20 SFO neurons in slice preparations in the presence of TTX at 0.3 ␮M. Holding potential was set to ⫺60 mV. In nine (45%) neurons, GAL induced long-lasting outward currents (15.0⫾3.7 pA, Fig. 4A). Time course of the responses was similar to that in extracellular recordings. In the experiments, mEPSCs and mIPSCs were often recorded (mIPSCs: n⫽18 and mEPSCs: n⫽12). Application of GAL did not affect the frequency and amplitude of either type of synaptic current (Fig. 4B, C and D). We have reported that the mEPSCs are mainly due to glutamatergic inputs and mIPSCs are mainly due to GABAergic ones (Honda et al., 2001). The present results suggest that GAL may be related to neither glutamatergic nor GABAergic spontaneous synaptic transmission in the rat SFO. GAL and ANGII responses in same SFO neurons To investigate subpopulations of GAL- and ANGII-sensitive neurons, the effects of both GAL and ANGII were analyzed on 21 discriminated single-units. GAL at either 300 nM or 1 ␮M was used to confirm the sensitivity to this compound. The concentration of ANGII (100 nM) was chosen because this was the concentration used in some previous studies (Okuya et al., 1987; Rauch et al., 1997). An inhibitory response to GAL was seen in 14 units (67%, Fig. 5A, left side), and a GAL-induced excitatory response was observed in only one unit (5%, Fig. 5B, left side).

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Fig. 4. GAL induces outward currents without influencing synaptic currents in the presence of TTX (0.3 ␮M). (A) Outward current by the application of GAL at 1 ␮M in a patch-clamp recording. Holding potential was ⫺60 mV. Horizontal bars indicate drug application time. (B) Expanded current records in control (a) and after the application of GAL (b) in A. (C) Cumulative probability plots of mIPSCs amplitude and inter-event intervals in the control conditions (Control) and after the application of GAL. The mean amplitude and number of mIPSCs were 21.6⫾0.2 pA and 553 events in the control (Control) and 21.4⫾0.2 pA and 492 events in GAL during a 60-s recording period. (D) Cumulative probability plots of mEPSCs amplitude and inter-event intervals in Control and after the application of GAL (GAL). The mean amplitude and number of mEPSCs were 21.8⫾1.1 pA and 76 events in Control and 23.5⫾1.1 pA and 55 events in GAL during a 60-s recording period.

ANGII induced excitatory responses in 17 (81%) of 21 units. Of 17 ANGII-sensitive SFO neurons, 12 (71%) showed GAL-induced inhibition of activity (Fig. 5A) and one showed a GAL-induced excitatory response (Fig. 5B). The remaining four units were insensitive to GAL. Effects of GalR subtype selective agonists on spontaneous activity To investigate subtypes of the GAL-induced responses, the GalR1 specific agonist M617 and the GalR2/3 agonist

GAL(2–11), which is a GAL peptide chimera, at 1 ␮M were applied to 46 and 25 multi-units respectively,(Lundström et al., 2005; Lu et al., 2005a,b) and subsequently the same concentration of GAL was applied. In 20 of 24 multi-units that showed inhibitory response to GAL, M617 (Fig. 6A) inhibited the neural activity. The remaining four multi-units showed no responses to M617 (data not shown). The inhibitory response by M617 (62.8⫾4.2% of the control) was significantly weaker than that by GAL (51.8⫾3.9%, P⬍0.01). As shown in Fig. 6A,

Fig. 5. Responses to GAL and ANGII in two single-units. (A) Inhibitory effects of GAL at 300 nM and excitatory effects of ANGII at 100 nM on the spontaneous electrical activity of an SFO neuron. Of 14 neurons that were inhibited by GAL, 12 were excited by ANGII. (B) GAL at 300 nM showed an excitatory response and ANGII at 100 nM also showed an excitatory response.

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Fig. 6. Effects of the agonists selective for GalRs on spontaneous neural activities. (A and B) GalR1 selective agonist M617 at 1 ␮M as well as GAL inhibited spontaneous electrical activity in a multi-unit (A) and a single-unit recording (B). (C) GalR2 and GalR3 agonist GAL(2–11) at 1 ␮M either had no affect (a) or inhibited multi-unit recordings (b). Data in Ca and Cb were recorded at the same time. Note that the inhibitory effects of GAL(2–11) started more slowly and continued longer than the effects of GAL and M617.

M617-induced inhibitory responses were relatively shortlasting, compared with GAL-induced responses. In only one multi-unit recording, GAL showed an excitatory response, like the response of the single-unit in Fig. 5B, although M617 induced no response (data not shown). Seven single-units were discriminated from 46 multi-units that were tested with M617. Four single units showed inhibitory responses to both M617 and GAL, to 23.8⫾ 17.0% and 11.5⫾11.5%, of the control respectively (Fig. 6B). The remaining three neurons showed no response to either drug. On the other hand, GAL(2–11) did not affect neural activity in 12 of 14 multi-units that showed inhibitory responses by GAL (Fig. 6Ca). In the remaining two units, although GAL(2–11) induced inhibitory responses, the responses were slow and small (Fig. 6Cb), compared with the GAL- and M617-induced inhibitory responses. Fig. 6Ca and Cb show two multi-unit recordings observed during a single experiment. In just one multi-unit recording, GAL(2– 11) induced an excitatory response, but the following application of GAL showed no response (data not shown). Four single-units were discriminated from 25 multi-unit recordings that were tested with GAL(2–11). Although two of four single-units showed inhibitory responses to GAL, none showed any responses to GAL(2–11) (data not shown).

GAL-containing synaptic terminals in the SFO To confirm the existence of GAL-containing synaptic terminals in the SFO, an immunoelectron microscopic study was performed. There were a considerable number of GAL-immunoreactive axon varicosities in the SFO (Fig. 7). They were about 1.1 ␮m in diameter and were round, ovoid or ellipsoid in shape. Their axoplasm was filled with round small clear vesicles and some large-cored vesicles. Out of 31 immunoreactive axon varicosities observed in the SFO, 11 were detected that made synaptic contacts. All of them were symmetric synapses onto non-immunoreactive dendrites. Occasionally, single GAL-immunoreactive axon terminals were found that made independent synapses onto different dendrites. There were no GAL immunoreactivities in other neuronal elements, such as cell bodies and dendrites, or glial cells.

DISCUSSION In the present study, the MEA system was used to investigate effects of GAL on neural activity of SFO neurons. Using the MEA system, extracellular electrical recordings were easily obtained from SFO slice preparations and in some cases recordings could be made from several recording sites in a single recording experiment. We selected and analyzed electrical activities in one or a few sites that showed a good signal to noise ratio in each recording

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Fig. 7. An electron micrograph of the SFO showing GAL-like immunoreactivity. Reaction products of diaminobenzidine are seen diffusely in the immunoreactive axoplasm. An immunoreactive axon terminal makes two symmetrical synapses (*) with different dendrites, and the one is enlarged in an inset.

experiment. In most cases electrical recordings were of the multi-unit type, reflecting the activity of several different neurons, because of relatively large surface area of the electrodes used by the MEA system. Twenty five singleunits were discriminated from over 100 multi-unit recordings in this study. Although in some ways, single-unit recordings give results that are easier to interpret in evaluating the drug responses, the present results show that multi-unit recordings may also be valuable for this purpose. This is the first report demonstrating electrophysiologically that the neuropeptide GAL has mainly inhibitory effects on the neural activity of SFO neurons. In extracellular recordings, GAL-induced inhibitory responses were observed during blockade of synaptic inputs. Furthermore, in whole-cell patch-clamp recordings, GAL-induced outward currents were observed in the presence of TTX where presynaptic effects are excluded. Further, the miniature PSCs were not affected by GAL. Thus, the GAL-induced responses are thought to be due to activation of GalR on the somata of SFO neurons. Both inhibitory and excitatory effects have been reported in earlier electrophysiological studies; inhibitory effects were observed in the SON and LC neurons (Papas and Bourque, 1997; Sevcik et al., 1993; Xu et al., 2005; Kozoriz et al., 2006), and excitatory effects in cholinergic neurons in DBB and DRG neurons (Jhamandas et al., 2002; Puttick et al., 1994; Kerekes et al., 2003). The diverse actions of GAL are thought to be due to the participation of different GAL receptor subtypes. In the LC, immunoreactive densities for GalR1 and GalR3 were stronger than those for GalR2 (Hawes and Picciotto, 2004). Similarly, the SON had much of GalR1 mRNA, but not GalR2 mRNA (O’Donnell et al., 1999; Gundlach et al., 2001). Thus, it seems that the GAL-induced inhibitory response is basically mediated through activations of GalR1 and/or GalR3. The cholinergic DBB neuron has been presumed to have only GalR2 (Jhamandas et al., 2002). Similarly, in DRG neurons showing GAL-induced excita-

tory responses, abundant expression of GalR2 mRNAs has been found (Kerekes et al., 2003; O’Donnell et al., 1999), suggesting that the effect is mediated through activations of GalR2. Since the RT-PCR study showed that the SFO has mRNAs of these three GalR subtypes, it was not unexpected that both excitatory responses and inhibitory responses were observed following the applications of GalR agonists. In almost all multi-units that showed an inhibitory response to GAL, the GalR1 selective agonist M617 induced inhibitory responses. However, GalR2 and GalR3 agonist GAL(2–11) induced inhibitory responses in only a small number of multi-unit recordings. These results suggest that GAL inhibits SFO neurons through mainly the GalR1. The duration of the response to M617 was obviously shorter than that to GAL, and the degree of inhibition following application of M617 was also smaller than that by GAL. A previous study has reported that the application of M617 that induces the activation of GalR1 shows a smaller decrease of intracellular cyclic AMP than GAL (Lundström et al., 2005). This may partly explain the weak inhibitory effect of M617 on the electrical activity of SFO neurons. It seems likely that GAL(2–11)-induced inhibitory responses observed in some multi-unit recordings may be mediated through the activation of GalR3, because the subtype as well as GalR1 is thought to be coupled with Gi/Go. On the other hand, GAL- and GAL(2–11)-induced excitatory responses that were observed in a few singleand multi-unit recordings might be mediated through activation of GalR2. In the RT-PCR study, the PCR product band for GalR3 from SFO cDNA was very much thinner than that from the genomic DNA (as positive control) and different from that of the other GalRs. Taken together with the electrophysiological results, it is reasonable to consider that GalR3-related inhibition may have only minor importance in the SFO. At the same time, PCR product band for GalR2 from SFO cDNA was obviously heavy like that for

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the GalR1 and this is inconsistent with the electrophysiological results. Such inconsistency for GalR2 between electrophysiological and molecular biological results has been also reported in LC neurons, suggesting that a downregulation occurs in protein-translation level (Hawes and Picciotto, 2004). Such down-regulation may also occur in SFO neurons. It is also possible to consider that GalR2 is expressed, but not externalized on the plasma membrane. As another possibility, GalR2 protein may be expressed specifically in axon terminals, rather than the neural soma or dendrites. If it were present, the activation of GalR2 in presynaptic terminals might have a little physiological significance because of the present evidence showing that GAL had no effect on synaptic currents. It is well-known that the excitation of SFO neurons by ANGII induces drinking behavior and vasopressin release. By contrast, it has been reported that drinking behavior and vasopressin release are inhibited by the central administration of GAL (Brewer et al., 2005; Kondo et al., 1993; Björkstrand et al., 1993). In the present study, many ANGII-sensitive SFO neurons showed a GAL-induced inhibitory response (n⫽12/17, 71%). This result suggests that the inhibitory response to the central administration of GAL on drinking behavior and vasopressin release is mediated through the SFO. The immunoelectron microscopy demonstrated that there were obvious GAL immunoreactive synaptic terminals in the SFO. This suggested the possibility that GAL is released from the synaptic terminals as a neurotransmitter or neuromodulator. Furthermore, the synapses showing GAL immunoreactivity were symmetric, generally indicating the inhibitory synapses. The feature is consistent with the electrophysiological evidence. GAL-immunoreactive neurons are reported to localize in several preoptic and hypothalamic areas, such as the medial preoptic nucleus and dorsomedial hypothalamic nucleus (Melander et al., 1986; Merchenthaler et al., 1993), some of which are known to project to the SFO (Lind et al., 1982). It is conceivable, therefore, that GAL in the SFO is derived from certain preoptic and hypothalamic areas. Since hypothalamic magnocellular neurons also express high levels of GAL (Melander et al., 1986), the GAL-induced inhibitory neural response in the SFO may contribute to negative feedback in the vasopressin- and oxytocin-releasing mechanisms. A humoral route of circulating GAL in plasma blood may provide another source, because the SFO lacks a blood– brain barrier. A study (Grenbäck et al., 2005) has reported that GAL-like immunoreactivity in human plasma was up to approximately 0.2 nM. We found in this study that the threshold concentration of inhibitory responses by GAL was around 10 nM (Fig. 3B). The concentration is 50 times higher than that in plasma. However, we cannot completely deny a possibility that the circulating GAL affects directly SFO neurons. As for ion channel mechanism, an increase of K⫹ conductance has been suggested to be involved in the GAL-induced inhibitory responses in LC and SON neurons, myenteric neurons of small intestine and mudpuppy parasympathetic neurons (Papas and Borque, 1997; Ren

et al., 2001; Parsons et al., 1998). GAL-induced inhibitory responses in SFO neurons may be mediated through activation of similar K⫹ channels since GAL elicited outward currents in the present study.

CONCLUSION In conclusion, the present study revealed that GAL inhibited the electrical activity of ANGII-sensitive SFO neurons that may be related to drinking behavior and vasopressin release. This observation offers a plausible explanation for the different effects of ANGII and GAL seen in vivo on water intake and vasopressin release. Furthermore, it suggests that the GAL-induced inhibitory response is mediated mainly through activation of GalR1, although the SFO contains cells that express the mRNAs of all three known GAL receptor subtypes. Acknowledgments—We thank L. Lundström Ph.D. and Prof. U. Langel for gift of M617. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to K.O. (17791327) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to K. I. (18592042).

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(Accepted 21 August 2006) (Available online 4 October 2006)