Measurement of Orexin (Hypocretin) and Substance P Effects on Constitutively Active Inward Rectifier K+ Channels in Brain Neurons

C H A P T E R

T H I R T Y

Measurement of Orexin (Hypocretin) and Substance P Effects on Constitutively Active Inward Rectifier Kþ Channels in Brain Neurons Yasuko Nakajima* and Shigehiro Nakajima† Contents 1. Introduction 2. Dissociated Culture of Cholinergic Neurons in the Basal Forebrain 2.1. Culture materials 2.2. Obtaining slices of the basal forebrain 2.3. Dissociation of nucleus basalis neurons 2.4. Plating dissociated neurons in culture dishes 2.5. Culture medium and maintenance of culture 2.6. Selection of cholinergic neurons 3. Effects of Orexin (Hypocretin) and Substance P on Constitutively Active Inward Rectifier Kþ (KirNB) Channels 3.1. Equipment 3.2. Effects of orexin and substance P on KirNB channels: whole-cell recordings 3.3. Orexin and substance P effects on KirNB channels: single-channel recordings 4. Signal Transduction of Substance P and Orexin Effects on KirNB Channels 4.1. PKC inhibitor 4.2. Pseudosubstrate of PKC 4.3. Phosphatase inhibitor Acknowledgments References

614 615 616 616 616 617 619 619 620 620 620 622 625 626 627 628 628 629

* Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

{

Methods in Enzymology, Volume 484 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)84030-3

#

2010 Elsevier Inc. All rights reserved.

613

614

Yasuko Nakajima and Shigehiro Nakajima

Abstract Electrophysiological experiments in our laboratory have led to the discovery that the cholinergic neurons in the nucleus basalis in the rat forebrain possess constitutively active inward rectifier Kþ channels. Unlike cloned inward rectifier Kþ channels, these constitutively active inward rectifier Kþ channels were found to have unique properties, and thus were named “KirNB” (inward rectifier Kþ channels in the nucleus basalis). We found that slow excitatory transmitters, such as orexin (hypocretin) and substance P, suppress the KirNB channel, resulting in neuronal excitation. Furthermore, it was discovered that suppression of KirNB channels by these transmitters is through protein kinase C (PKC). This chapter describes detailed electrophysiological techniques for investigating the effects of orexin and substance P on constitutively active KirNB channels. For this purpose, we also present a method for culturing nucleus basalis cholinergic neurons in which KirNB channels exist. Then, we describe the procedures through which PKC has been determined to mediate inhibition of KirNB channels by orexin and substance P. There are probably many other transmitters which may produce effects on KirNB channels. This chapter will enable researchers to investigate the effects of such transmitters on KirNB channels and their roles in neuronal functions.

1. Introduction In 1985, we discovered that unique constitutively active inward rectifier Kþ (Kir) currents exist in cholinergic neurons in the basal forebrain (Stanfield et al., 1985). These cholinergic neurons exist in the nucleus basalis, the diagonal band, and the medial septal nucleus. These neurons have been recognized to be important for memory and cognition (Deutsch, 1971; Perry et al., 1999; Riekkinen et al., 1990). It has also been found that these cholinergic neurons selectively degenerate in Alzheimer’s disease in humans (Coyle et al., 1983). We investigated the properties of this constitutively active inward rectifier Kþ channel in cholinergic neurons in the nucleus basalis by using the on-cell as well as the inside-out single-channel recordings. The constitutively active Kir channels in these neurons open and close spontaneously near the resting membrane potential. We designated such constitutively active Kir channels as “KirNB” channels (Bajic et al., 2002). When the recording method was changed from the on-cell recording to the inside-out recording, the activity of the KirNB channels remained intact with their constitutive activity unchanged. The mean open time of KirNB channels was 1 ms and their unitary conductance was 23 pS (155 mM [Kþ]0). These characteristic features of KirNB channels are different from those of cloned Kir channels (Bajic et al., 2002). At present, these KirNB channels have not been genetically determined.

Orexin and Substance P Effects on CA Kir Channels

615

Further characteristics of the KirNB channel were discovered. When a slow excitatory transmitter, such as substance P or neurotensin, is applied, KirNB channels are suppressed, and the cell produces neuronal excitation (Bajic et al., 2002; Farkas et al., 1994; Stanfield et al. 1985; Yamaguchi et al., 1990). We then investigated the effects of orexin on KirNB channels. Orexins (also named “hypocretins”) are recently discovered neuropeptides, consisting of orexin A and orexin B, and are implicated in the sleep disorder, narcolepsy (Chemelli et al., 1999; de Lecea et al., 1998; Nishino et al., 2000; Sakurai et al., 1998; Thannickal et al., 2000). In our laboratory, orexin A was found to inhibit KirNB channels, resulting in neuronal excitation (Hoang et al., 2004). We further investigated the signal transduction mechanisms of substance P and orexin effects on KirNB channels (Hoang et al., 2004; Takano et al., 1995). It was observed through single-channel recordings that substance P effects are induced by a diffusible messenger. It was found that staurosporine (a protein kinase C (PKC) inhibitor) and PKC pseudosubstrate PKC (19–36) suppressed substance P effects on KirNB (Takano et al., 1995). In addition, it was noticed that substance P irreversibly suppressed KirNB channels in neurons treated with okadaic acid (a phosphatase inhibitor), suggesting that substance P effects on KirNB channels are mediated by PKC phosphorylation of KirNB channels (Takano et al., 1995). We have also shown that the orexin A-induced suppression of KirNB channels is mediated by a pertussis toxin (PTX)-insensitive G protein (such as Gq/11). The recovery from this suppression is performed by dephosphorylation (Hoang et al., 2004), suggesting that the effects of orexin are mediated through PKC. KirNB channels seem to always be active in the nucleus basalis cholinergic neurons. We have not encountered such a unique characteristics of KirNB channels in other types of neurons we have investigated, such as noradrenergic neurons in the locus coeruleus (Grigg et al., 1996; Nakajima et al., 1996). Thus, for KirNB channel investigations, we have been using the dissociated culture of cholinergic neurons (developed by Nakajima et al., 1985) from the basal forebrain. In this chapter, we first explain our method of culturing cholinergic neurons. We then present electrophysiological techniques for investigating the orexin or substance P effects on the constitutively active KirNB channel.

2. Dissociated Culture of Cholinergic Neurons in the Basal Forebrain Dissociated culture of cholinergic neurons in the basal forebrain was developed in our laboratory (Nakajima et al., 1985). In this culture, we discovered the existence of constitutively active Kir channels (Stanfield

616

Yasuko Nakajima and Shigehiro Nakajima

et al., 1985); we later named them KirNB channels (Bajic et al., 2002). This unique channel is so far encountered only in the basal forebrain cholinergic neurons. We used the cultured nucleus basalis cholinergic neurons for our studies on the effects of orexin or substance P on KirNB channels. First, we describe the protocol of making dissociated culture of nucleus basalis cholinergic neurons. Since the initial reports of our culture methods (Nakajima and Masuko, 1996; Nakajima et al., 1985), steady improvements have been made.

2.1. Culture materials Culture neurons are obtained from young new born rats (2–5-day-old rats). After anesthesia with isoflurane, the forebrain region is rapidly removed, and the rats are quickly sacrificed by decapitation.

2.2. Obtaining slices of the basal forebrain Four to six pieces of the forebrain are placed in a 3.5 cm petri dish with the anterior surface of the forebrain facing down. The forebrain is embedded in warm (45  C) 3.5% agar dissolved in a balanced salt solution; a buffer (pH 7.4) consisting of 130 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 33 mM glucose, and 5 mM PIPES (piperazine-N,N0 -bis[2-ethanesulfonic acid]). The petri dish is then immediately placed over ice-cold water for 5 min to solidify the agar. To prepare for sectioning, the hardened agar block is trimmed and affixed to a cutting dish (a sterile disposable square petri dish, 10  10  1.5 cm) using instant glue (Alpha cyanoacrylate). The cutting dish is mounted on a Vibratome machine (Lancer 1000) (Fig. 30.1). The Vibratome is placed in a laminar flow cabinet. The cutting dish is then filled with oxygenated cold BBS solution (Fig. 30.1). The forebrain coronal slices of 400 mm thickness are aseptically sectioned by positioning the blade holder at a 16 angle with slow cutting speed and large amplitude vibration.

2.3. Dissociation of nucleus basalis neurons From the forebrain sections, the area of the nucleus basalis is dissected under a microscope, using a pair of micro-knives (30 gauge hypodermic needles attached to 1 ml tuberculin syringes). Figure 30.2A shows an example of the nucleus basalis containing cholinergic neurons histochemically stained for acetylcholinestease (AChE). After training with stained materials, we successfully dissected the nucleus basalis without staining. The dissected pieces are collected in a conical 15 ml centrifuge tube and treated with oxygenated papain solution (1.5 ml) for 15 min at 37  C. Additional fresh oxygenated papain solution ( 1 ml) is then added for another 15 min at 37  C.

617

Orexin and Substance P Effects on CA Kir Channels

O2

A

B

Vibratome C

Figure 30.1 Schematic diagram showing brain slice sectioning. An agar block containing forebrains (A) is attached to the bottom of a square petri dish (10  10  1.5 cm) (B), and this cutting dish is mounted on a Vibratome by the plastic pedestal (C). The agar block is immersed in a balanced salt solution which is continuously oxygenated (Nakajima and Masuko, 1996).

After these treatments, the pieces of the nucleus basalis are rinsed three times with culture medium and then dissociated by gentle trituration using a firepolished Pasteur glass pipette. Papain solution is made by dissolving 12 units/ml papain (Worthington Biochemical Co.) in L-15 culture medium (1.5 ml) containing 0.2 mg/ml DL-cysteine and 0.2 mg/ml bovine serum albumin (pH 7.3). When the papain solution is made, the solution is initially cloudy, but this turbidity disappears within 15 min. Then, the papain solution is sterilized by filtering.

2.4. Plating dissociated neurons in culture dishes We plate dissociated nucleus basalis neurons in a central small well ( 1.2 cm in diameter) inside a 3.5-cm culture dish (Fig. 30.3). The well was made by drilling a hole at the center of a culture dish. A plastic Aclar film or a cover glass is adhered to the bottom of the culture dish using inert glue (Dow Corning, 3140 RTV) (Fig. 30.3). This small well is necessary to efficiently utilize the limited number of cells obtained from small pieces of the nucleus basalis. The bottom of the well is coated with rat tail collagen and glial feeder layer cells (Fig. 30.3; Nakajima and Masuko, 1996). Glial feeder layer cells are obtained by culturing cerebral cortex cells of newborn rats in a 10-cm culture dish. After a few weeks of culture, the cells start to divide. These cells are replated into another culture dish. As the procedure is repeated, eventually neurons die and glial cells remain. These glial feeder layer cells are plated in a well inside a culture dish 2–3 days

618

Yasuko Nakajima and Shigehiro Nakajima

A

GP

500 mm B

50 mm

Figure 30.2 (A) Coronal vibratome slice of the forebrain of a 2-day-old newborn rat. The slice was stained for acetylcholinesterase. The dark areas are positive to acetylcholinesterase stain (arrowheads) and are located at the medial aspect of the globus pallidus (GP). They are neurons homologous to those in the nucleus basalis of Meynert in humans. (B) A cultured neuron from the nucleus basalis, showing intense staining for acetylcholinesterase, indicating that the neuron is cholinergic. Cultured for 21 days. Modified from Nakajima et al. (1985).

Culture dish Well

Neuron Glial cell

Collagen Coverglass or aclar film

Figure 30.3 Schematic diagram showing a center well (1.2 cm in diameter) in a culture dish (3.5 cm in diameter). A well is made in the center of the culture dish. The bottom of the well is covered by a plastic Aclar film or a cover glass. The well is first covered by collagen and then by glial feeder layer cells. Dissociated neurons are cultured on the glial feeder layer which has been prepared beforehand (Nakajima and Masuko, 1996).

Orexin and Substance P Effects on CA Kir Channels

619

before culturing the neurons of the nucleus basalis. One day after the feeder layer cells are plated, antimitotic chemicals [50 fluoro-20 deoxyuridine (15 mg/ml) and uridine (35 mg/ml)] are applied to the feeder layer to suppress the overgrowth of feeder layer cells. This method of using the glial feeder layer promotes the survival of cultured neurons. After the dissociation of dissected nucleus basalis pieces, 0.15 ml of the cell suspension is placed in a feeder layer-covered well at a cell density of 2–5  104 cm 2 (Fig. 30.3). The culture dishes are kept in an incubator with 10% CO2 at 37  C. After 2–3 h of incubation, 2.5 ml of culture medium is added to each culture dish.

2.5. Culture medium and maintenance of culture The culture medium is a minimum essential medium with Earle’s salts, modified with 0.292 mg/ml L-glutamine, 3.7 mg/ml NaHCO3, 5 mg/ml D-glucose, 2% rat serum (prepared in our laboratory) and 10% horse serum, 10 mg/ml L-ascorbic acid, 50 units/ml penicillin, and 50 mg/ml streptomycin. A conditioned culture medium is always used, that is, a medium which has been kept in a culture dish with glial feeder layer cells overnight. With this procedure, toxic glutamate contained in fresh culture medium can be absorbed by glial cells (Baughman et al., 1991). The culture medium is usually exchanged with a new medium every 4 weeks. Most of our electrophysiological experiments are performed on 2- to 3-week-old cultures. In some experiments, we use up to 2- to 3-month-old cultures.

2.6. Selection of cholinergic neurons We found that cholinergic neurons in the basal forebrain tend to be quite large with a diameter range of 22–38 mm (Nakajima et al., 1985). After electrophysiological experiments, 13 cells were processed for AChE histocytochemistry. Twelve of these 13 (92%) cells produced AChE positivity (Nakajima et al., 1985), suggesting that they are cholinergic. According to Levey et al. (1983), all neurons with strong AChE positivity in the basal forebrain were choline acetyltransferase positive. Figure 30.2B shows a cultured cholinergic neuron stained with AChE histocytochemistry. KirNB channels are a particular type of ion channels located in cholinergic neurons in the basal forebrain. To investigate KirNB channels of nucleus basalis neurons, it is important to select large neurons (about 30 mm in diameter), which are most likely to be cholinergic. It is also recommended that at an early stage of experiments, a certain number of cells be treated with AChE histochemistry or choline acetyltransferase immunochemistry after electrophysiological data are obtained, to confirm that experimented cells are cholinergic.

620

Yasuko Nakajima and Shigehiro Nakajima

3. Effects of Orexin (Hypocretin) and Substance P þon Constitutively Active Inward Rectifier K (KirNB) Channels 3.1. Equipment A simple oscilloscope for observation during experiments; an inverted phase-contrast microscope (with DC power supply for the light bulb to avoid AC interference); a binocular microscope (Axiovert 135; Zeiss); a manipulator (to insert a glass pipette into the cell); a patch-clamp amplifier (Axopatch 200B; Axon Instruments); a digital data recorder (Instrutech; VB-10B); P-clamp software (Molecular Devices); a videocassette recorder (VCR, Sony SLV-N51); a digidata 1440A converter (Axon Instruments); a drug application device (OCTAFLOW system; ALA Scientific Instruments), a pipette puller (Sutter Instrument, Co.); a stimulator (Medical Systems Corp; Greenvale, NY); a pipette tip polisher (Narishige); a Faraday cage (a home-made cage); an air-cushioned table (TMC; MICRO-g); a chart recorder (Astro-med; DASH II).

3.2. Effects of orexin and substance P on KirNB channels: whole-cell recordings 3.2.1. Introduction Orexins are excitatory transmitters consisting of orexin A and orexin B and implicated in sleep disorders and narcolepsy (Chemelli et al., 1999; de Lecea et al., 1998; Sakurai et al., 1998). The orexin receptors (orexin receptor type 1 and type 2) are present in certain brain nuclei, including the ascending arousal system. It is reported that the nucleus basalis contains both orexin receptor type 1 and type 2 (Marcus et al., 2001; Trivedi et al., 1998). The nucleus basalis is located in the basal forebrain and contains large cholinergic neurons. Degeneration of the cholinergic neurons in the nucleus basalis, diagonal band, and medial septal nuclei would cause memory loss and Alzheimer’s disease (Coyle et al., 1983). In order to obtain neurons containing orexin receptors, neurons from the nucleus basalis are cultured for 2–4 weeks. Large neurons ( 30 mm in diameter), which are mostly cholinergic (Nakajima et al., 1985), are used for the experiments. Application of orexin A or substance P inhibits a special type of channel, which we call KirNB channels, located in the basal forebrain cholinergic neurons. The KirNB channels seem to be one type of inward rectifying channels, causing neuronal inhibition (Bajic et al., 2002; Hoang et al., 2004; Stanfield et al., 1985; Yamaguchi et al., 1990). Application of orexin A to a nucleus basalis neuron reduces cell conductance through a PTX-insensitive G protein (Hoang et al., 2004).

Orexin and Substance P Effects on CA Kir Channels

621

The orexin-suppressed currents are inwardly rectifying with a reversal potential around the Kþ equilibrium potential (EK). Therefore, the orexin-induced inhibition of KirNB is mediated by a PTX-insensitive G protein (Gq/11). In many respects, the action of orexin is very similar to that of substance P. 3.2.2. Experiments using the whole-cell clamp (a) External and internal solutions for KirNB channels: The external solution—141 mM NaCl, 10 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 11 mM D-glucose, and 0.5 mM tetrodotoxin (pH 7.2). The patch pipette solution—141 mM K-D-gluconate, 10 mM NaCl, 5 mM HEPES–KOH, 0.5 mM EGTA–KOH, 0.1 mM CaCl2, 4 mM MgCl2, 3 mM Na2ATP, 0.2 mM GTP (pH 7.4). (b) Experimental procedures: Neurons, cultured for 2–4 weeks, were used. First, square-wave voltages are repetitively applied to the electrode. Then, let the electrode tip approach toward the cell surface. The square-wave current amplitude begins to decrease, indicating that the effective electrode resistance increases (because of the immediate presence of the cell membrane). Now, a negative pressure is applied to the electrode. This would suck up part of the cell membrane into the inside of the electrode tip. Addition of a larger negative pressure breaks a part of the membrane located inside the electrode. Thus, the electrode is now electrically continuous with the cell inside.

3.2.3. Effect of orexin A: experiments Both orexin A and substance P suppress the inward rectifier Kþ channels in cultured nucleus basalis neurons. Figure 30.4 shows one of our experiments (Hoang et al., 2004). The patch pipette contained 0.2 mM GTPgS together with the routine ingredients. The holding potential was 84 mV. In Fig. 30.4A, orexin A (3 mM) was applied. This resulted in a decrease of the membrane conductance. Each of the recurrent command pulses consisted of a square-wave depolarization (20 mV, 100 ms) and a hyperpolarization (50 mV, 100 ms). Figure 30.4B shows the current–voltage (I–V ) relation of the orexin A-suppressed current in a GTPgS-loaded NB neuron. First, a I–V relation was determined before (filled squares) and after (open circles) application of orexin. The difference between those two I–V curves would be the current amplitude that was suppressed by the orexin A application; this orexin A-suppressed current is plotted in Fig. 30.4C. It is clear from Fig. 30.4C that this current is inwardly rectifying Kþ channel since its reversal potential ( 74 mV) is near EK (71 mV).

622

Yasuko Nakajima and Shigehiro Nakajima

A

GTPg S

1 nA 40 s OXA C

B

0 pA -120

-80

-40 mV

OXA-suppressed current

-140

-100

-60 mV

0 pA

-350

-1000

-700

-2000

Figure 30.4 (A) Application of orexin A (3 mM) to a GTPgS-loaded nucleus basalis neuron resulted in an irreversible conductance decrease (closing of KirNB channels). (B) I–V relationship of whole-cell current before orexin A application (solid squares, solid line) and after orexin A application (hollow circles, dotted line). Comparison of the two curves indicates that the resting potential shifted from 64 mV (solid arrow) to  52 mV (hollow arrow) with about 12 mV depolarization by orexin A. (C) I–V relationship of the orexin A-suppressed (sensitive) current. Conductance showed an inward rectification and its reversal potential was close to EK ( 71 mV). Modified from Hoang et al. (2004).

3.3. Orexin and substance P effects on KirNB channels: single-channel recordings Almost all large nucleus basalis neurons are cholinergic. These neurons show spontaneously active KirNB channels at the single channel level (Fig. 30.5A1). The unitary conductance of KirNB channels was  23 pS at 155 mM [Kþ]0 (Fig. 30.5C; Hoang et al., 2004). The mean open time of KirNB channels was 1.1–1.3 ms (Fig. 30.5D) (Hoang et al., 2004). These single-channel characteristics of KirNB channels are quite different from any of the known inward rectifier Kþ channels. (Nucleus basalis cholinergic neurons also possess Kir3 (G protein-gated inward rectifier potassium) channels. These Kir3 channels have larger unitary conductance, namely, 30–40 pS).

623

Orexin and Substance P Effects on CA Kir Channels

Before OXA

A2

After OXA 2 pA

A1

10 ms B

0.8

NPo

0.6 0.4 0.2 OXA

0.0 0

100

200 Time (s)

300

400

D

C 100

80

80 N

N

60 60

40

40

20

20 0

0 0

1 2 3 Amplitude (pA)

4

2

8 4 6 Open-time (ms)

10

Figure 30.5 A1 and A2. On-cell single-channel events of KirNB were recorded from a nucleus basalis neuron before (A1) and after (A2) orexin A (OXA) (3 mM, 30 s) application. OXA closes channels in a nucleus basalis neuron. Patch pipette contained 155 mM KCl. Bath solution contained 5 mM KCl. Transmembrane potential was estimated to be  87 mV. (B) Application of OXA (3 mM, 30 s) resulted in a transient decline of NPo. Each circle represents the average of a 10 s interval. (C) Amplitude histogram of KirNB channels. The unitary conductance was calculated to be 23 pS. (D) Open time histogram of KirNB channels. The mean open time was 1.33 ms. Modified from Hoang et al. (2004).

Previously, we noticed that orexin A and substance P suppressed KirNB channels through PKC (Bajic et al., 2002; Hoang et al., 2004; Takano et al., 1995). In those experiments, we investigated the effects of orexin and

624

Yasuko Nakajima and Shigehiro Nakajima

substance P on KirNB channels using the cell-attached (on-cell) mode of single-channel recordings with the application of orexin A or substance P outside the patch pipette. The single-channel activity of KirNB channels was observed using both cell-attached mode as well as the inside-out mode of single-channel recordings as seen in Fig. 30.6A1 and A3 (Bajic et al., 2002). 3.3.1. Solutions for the single-channel recordings with cell-attached mode The external solution (5K Krebs solution): 5 mM KCl, 146 mM NaCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 11 mM D-glucose, 0.5 mM tetrodotoxin, and 5 mM HEPES–NaOH (pH 7.4). The patch pipette solution: 155 mM KCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 11 mM D-glucose, and 5 mM HEPES–NaOH (pH 7.4). 3.3.2. Experimental procedures First, the culture medium is exchanged to the 5Kþ Krebs solution. Then, a rather large neuron (30 mm in diameter) is selected. This is likely to be a cholinergic neuron. The cell is patched with a patch electrode with a resistance of  5 MO. When the gigaohm seal is obtained, we do not break the cell membrane. With this procedure, spontaneously active A Inside-out

Cell-attached A1

A3

A2 SP

SP

A1

A2

10 s

10 pA 20 ms

A3

5 pA

NPo

B 0.3 0.2 0.1 0

0 SP 50

100

150

200

250

300

Time (s)

Figure 30.6 Single-channel activity of KirNB channels from a nucleus basalis neuron. (A) A1 and A2: Single-channel currents with cell-attached mode. The external solution was the 5K Krebs solution. 0.5 mM substance P (SP) application induced a transient decrease in channel activity. The membrane potential of the patched region was 27 mV more hyperpolarized than resting potential. A3: KirNB channels recorded with the inside-out configuration. (B) SP application produced a transient decline of NPo. This graph was derived from the same patch as that in A. Modified from Bajic et al. (2002).

Orexin and Substance P Effects on CA Kir Channels

625

single-channel events (KirNB) will be observed as shown in Figs. 30.5A1 and 30.6A1. During the on-cell mode of single-channel recordings, the holding potential is about 27 mV more negative than the resting potential. The chance of the opening of KirNB channel in the patch is expressed as “NPo” (N ¼ the number of channels being recorded; Po ¼ probability of each channel to open) (Friedrich et al., 1988). The NPo of the spontaneous activity of KirNB channels in the Fig. 30.5A1 was 0.25 (Hoang et al., 2004). We also obtained NPo of 0.30 in another experiment (Bajic et al., 2002). After application of orexin A (3 mM) or substance P (0.3 mM) in the external solution, the opening of single-channel events drastically decreased as seen in Fig. 30.5A2 and B (Hoang et al., 2004) and in Figs. 30.6A2 and B (Bajic et al., 2002). Then, after washing out orexin or substance P, KirNB channel activity reappears as seen in Fig. 30.5B (Hoang et al., 2004) and in Fig. 30.6A3 and B (Bajic et al., 2002).

4. Signal Transduction of Substance P and Orexin Effects on KirNB Channels Both orexin and substance P inhibit KirNB channels, leading to neuronal excitation. We have investigated the signal transduction mechanisms of substance P and orexin effects on KirNB channels (Hoang et al., 2004; Nakajima et al., 1988; Takano et al., 1995, 1996). First we observed that the substance P effect on KirNB was mediated through a PTXinsensitive G protein (Nakajima et al., 1988). Next, we identified this G protein to be a Gq/11(Takano et al., 1996). We also noticed that the substance P effect is mediated through phospholipase C-b1 (PLC-b1) (Takano et al., 1996). In addition, we found that staurosporine (a PKC inhibitor) as well as PKC pseudosubstrate PKC (19–36) suppressed the substance P effects on KirNB (Fig. 30.7; Takano et al., 1995), suggesting that PKC is a signal transducer. In addition, we observed that substance P irreversibly suppressed KirNB channels in neurons treated with okadaic acid (a phosphatase inhibitor), suggesting that the substance P effects on KirNB channels are mediated by phosphorylation of KirNB channels by PKC. In orexin experiments, we observed that the orexin A-induced suppression of KirNB channels is mediated through a PTX-insensitive G protein (perhaps G q/11) (Hoang et al., 2004). It was also found that the pretreatment with okadaic acid prevented the recovery of this orexin A effect. This fact also suggests that the orexin A effects on KirNB channels are mediated through phosphorylation of the channels by the signal transducer, PKC (Fig. 30.8) (Hoang et al., 2004). Figure 30.9 is a schematic diagram of our proposed signal transduction pathways, through which KirNB channels are inhibited by orexin or substance P.

626

Yasuko Nakajima and Shigehiro Nakajima

1nA

A

SP

1 nA 1 nA

C

SP

SP

% 60

*

*

40 20 0

Control stau

PKI PKC (5–24) (19–36)

0.5 nA

D

SP-induced conductance inhibition

E

B

SP

10 s

Figure 30.7 PKC inhibitors (staurosporine and PKC(19–36)) suppressed the substance P (SP) effects on KirNB currents in nucleus basalis neurons: this could indicate that PKC is a signal transducer. (A) SP effects on a control neuron. (B) SP effects on a staurosporine (100 nM)-treated neuron. (C) SP effects on a neuron loaded with protein kinase A inhibitor, PKI (5–24) (20 mM). (D) SP effects on a neuron loaded with PKC inhibitor, PKC (19–36). (E) SP-induced inhibition of neurons treated with staurosporine (stau) or loaded with protein kinase inhibitors PKI (5–24) or PKC (19–36). Modified from Takano et al. (1995).

Below, we describe three possible tests, by which the PKC involvement in orexin and substance P effects on KirNB channels could be determined. These strategies will use (1) PKC inhibitor, (2) pseudosubstrate of PKC, or (3) phosphatase inhibitor.

4.1. PKC inhibitor Staurosporine, a broad spectrum protein kinase inhibitor (Tamaoki et al., 1986), was used in our experiments with the whole-cell clamp technique (Takano et al., 1995). Cultured nucleus basalis neurons are first treated with staurosporine (100 nM in 0.01% DMSO for 40 min), followed by the application of SP (0.3 mM) or orexin A (3 mM). For controls, the external solution containing 0.01% DMSO is used. Figure 30.7 shows our experimental results indicating that staurosporine suppressed the substance P effect on KirNB channels (Takano et al., 1995).

627

Orexin and Substance P Effects on CA Kir Channels

Control (DMSO, 0.01%)

Okadic acid-treated (100 nM)

B

1.0

1.0

0.8

0.8 NPo

NPo

A

0.6

0.6

0.4

0.4 OXA

OXA 0.2

0.2 0

120

60 Time (s)

0

60 Time (s)

120

Figure 30.8 Effects of okadaic acid pretreatment on the orexin A-induced inhibition of KirNB channels. The cell-attached mode of single-channel recordings was used. Patch pipette contained 155 mM KCl. Bath contained 5 mM Kþ. (A) Orexin A (OXA) (3 mM) application to control nucleus basalis cells (incubated in 0.01% DMSO in the external solution for 2–5 h) induced a transient decline in NPo. (B) OXA application to cells pretreated with okadaic acid (0.1 mM in the external solution containing 0.01% DMSO, 2–3.5 h) induced a long-lasting decline in NPo. Modified from Hoang et al. (2004).

OX

OX receptor

SP

SP receptor

PIP2 Gaq

PLCb DAG

PKC

Close

KirNB

Figure 30.9 Proposed signal transduction pathways of orexin (OX) and substance P (SP) effects on KirNB channels. PLCb (phospholipase Cb); PIP2 (phosphatidyl inositol4,5-bisphosphate); DAG (diacylglycerol).

4.2. Pseudosubstrate of PKC PKC (19–36), being a PKC pseudosubstrate, is a specific inhibitor of PKC since it competes with endogenous substrates of PKC (House and Kemp, 1987). PKC (19–36) is applied into the cell cytoplasm through a patch pipette. For this purpose, patch pipettes having a low resistance (2 MO) and filled with a patch pipette solution containing 30 mM PKC (19–36) are used. After patching a neuron and breaking the cell membrane, it is necessary to wait at least 5 min before starting to investigate the effects of orexin A (3 mM) or substance P (0.3 mM); waiting is necessary to ensure the introduction of PKC (19–36) into the cytoplasm. As a control for the PKC (19–36), the same concentration of a protein kinase A specific inhibitor,

628

Yasuko Nakajima and Shigehiro Nakajima

PKI (5–24), is used. Figure 30.7C, D, and E (summary) show that the effect of substance P on KirNB was reduced with the intracellular application of PKC (19–36).

4.3. Phosphatase inhibitor Inhibition of KirNB by orexin A and substance P recovers spontaneously (Hoang et al., 2004; Stanfield et al., 1985; Yamaguchi et al., 1990). We found that the recovery of these transmitters’ effects was suppressed by the pretreatment with okadaic acid (Hoang et al., 2004; Takano et al., 1995). Okadaic acid is an inhibitor of protein phosphatase type 1 and type 2A, which belong to serine/threonine protein phosphatase (Bialojan and Takai, 1988; Nairn and Shenolikar, 1992). These experiments suggest that the suppression of KirNB channels by orexin A and substance P is due to the phosphorylation of KirNB channels by PKC and that the recovery would be caused by dephosphorylation of the channels (Bajic et al., 2002; Hoang et al., 2004; Takano et al., 1995). In the experiments using okadaic acid, we employed on-cell singlechannel recordings of KirNB channels. In this experiment, KirNB channel activities are expressed in NPo (the possible numbers of channels multiplied by the open probability of the channel, namely, opening frequency of channels in the patch; see Section 3.3). For the okadaic acid experiments, 2–3-week-old nucleus basalis cultures are used. The culture medium is exchanged with 5K Krebs solution (5 mM KCl, 146 mM NaCl, 2.4 mM CaCl2, 1.3 mM MgCl2, 11 mM D-glucose, 0.5 mM tetrodotoxin, and 5 mM HEPES–NaOH, pH 7.4). Then, 5K Krebs solution is exchanged—5K Krebs solution containing 100 nM okadaic acid and 0.01% DMSO. The culture is incubated at 37  C for about 2.5 h, after which on-cell singlechannel experiments are performed, and orexin A (3 mM, 12 s) is applied. During the experiment, NPo is obtained at a 10 second interval. Control experiments are performed in 5K Krebs solution containing 0.01% DMSO. Figure 30.8 is an example of such experiments (Hoang et al., 2004), showing an irreversible decrease of NPo in okadaic acid treated neurons. In the control experiment, a decrease of NPo after the application of orexin A is seen; however, as expected, its values spontaneously recovered. The results of the above three types of experiments (use of staurosporine, use of PKC (19–36), and use of okadaic acid) all agree with the idea that PKC is involved in substance P- and orexin-induced inactivation of KirNB channels. Figure 30.9 is a schematic diagram showing this idea.

ACKNOWLEDGMENTS We deeply thank the investigators who contributed greatly to the success of the experiments. The research relevant to this chapter was supported by the National Institute of Health grants NS043239 and AG06093.

Orexin and Substance P Effects on CA Kir Channels

629

REFERENCES Bajic, D., Koike, M., Albsoul-Younes, A. M., Nakajima, S., and Nakajima, Y. (2002). Two different inward rectifier Kþ channels are effectors for transmitter-induced slow excitation in brain neurons. Proc. Natl. Acad. Sci. USA 99, 14494–14499. Baughman, R. W., Huettner, J. E., Jones, K. A., and Khan, A. A. (1991). Cell culture of neocortex and basal forebrain from postnatal rats. In “Culturing Nerve Cells,” (Gary Banker and Kimberly Goslin, eds.), pp. 227–249. M.I.T. Press, Cambridge. Bialojan, C., and Takai, A. (1988). Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Biochem. J. 256, 283–290. Chemelli, R. M., Willie, J. T., Sinton, C. M., Elmquist, J. K., Scammell, T., Lee, C., Richardson, J. A., Williams, S. C., Xiong, Y., Kisanuki, Y., Fitch, T. E., Nakazato, M., et al. (1999). Narcolepsy in orexin knockout mice: Molecular genetics of sleep regulation. Cell 98, 437–451. Coyle, J. T., Price, D. L., and DeLong, M. R. (1983). Alzheimer’s disease: A disorder of cortical cholinergic innervation. Science 219, 1184–1190. de Lecea, L., Kilduff, T. S., Peyron, C., Gao, X.-B., Foye, P. E., Danielson, P. E., Fukuhara, C., Battenburg, E. L. F., Gautvik, V. T., Bartlett, F. S., II, Frankel, W. N., Van Den Pol, A. N., et al. (1998). The hypocretins: Hypothalamus-specific peptides with neuroexcitatory activity. Proc. Natl. Acad. Sci. USA 95, 322–327. Deutsch, J. A. (1971). The cholinergic synapse and the site of memory. Science 174, 788–794. Farkas, R. H., Nakajima, S., and Nakajima, Y. (1994). Neurotensin excites basal forebrain cholinergic neurons: Ionic and signal-transduction mechanisms. Proc. Natl. Acad. Sci. USA 91, 2853–2857. Friedrich, F., Paulmichl, M., Kolb, H. A., and Lang, F. (1988). Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin. J. Membr. Biol. 106, 149–155. Grigg, J. J., Kozasa, T., Nakajima, Y., and Nakajima, S. (1996). Single-channel properties of a G-protein-coupled inward rectifier potassium channel in brain neurons. J. Neurophysiol. 75, 318–328. Hoang, Q. V., Zhao, P., Nakajima, S., and Nakajima, Y. (2004). Orexin (hypocretin) effects on constitutively active inward rectifier Kþ channels in cultured nucleus basalis neurons. J. Neurophysiol. 92, 3183–3191. House, C., and Kemp, B. E. (1987). Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 238, 1726–1728. Levey, A. I., Wainer, B. H., Mufson, E. J., and Mesulam, M. M. (1983). Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum. Neuroscience 9, 9–22. Marcus, J. N., Aschkenasi, C. J., Lee, C. E., Chemelli, R. M., Saper, C. B., Yanagisawa, M., and Elmquist, J. K. (2001). Differential expression of orexin receptors 1 and 2 in the rat brain. J. Comp. Neurol. 435, 6–25. Nairn, A. C., and Shenolikar, S. (1992). The role of protein phosphatases in synaptic transmission, plasticity and neuronal development. Curr. Opin. Neurobiol. 2, 296–301. Nakajima, Y., and Masuko, S. (1996). A technique for culturing brain nuclei from postnatal rats. Neurosci. Res. 26, 195–203. Nakajima, Y., Nakajima, S., Obata, K., Carlson, C. G., and Yamaguchi, K. (1985). Dissociated cell culture of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei. Proc. Natl. Acad. Sci. USA 82, 6325–6329. Nakajima, Y., Nakajima, S., and Inoue, M. (1988). Pertussis toxin-insensitive G protein mediates substance P-induced inhibition of potassium channels in brain neurons. Proc. Natl. Acad. Sci. USA 85, 3643–3647.

630

Yasuko Nakajima and Shigehiro Nakajima

Nakajima, Y., Nakajima, S., and Kozasa, T. (1996). Activation of G protein-coupled inward rectifier Kþ channels in brain neurons requires association of G protein beta gamma subunits with cell membrane. FEBS Lett. 390, 217–220. Nishino, S., Ripley, B., Overeem, S., Lammers, G. J., and Mignot, E. (2000). Hypocretin (orexin) deficiency in human narcolepsy. Lancet 355, 39–40. Perry, E., Walker, M., Grace, J., and Perry, R. (1999). Acetylcholine in mind: A neurotransmitter correlate of consciousness? Trends Neurosci. 22, 273–280. Riekkinen, P., Jr., Sirvio, J., and Riekkinen, P. (1990). Similar memory impairments found in medial septal-vertical diagonal band of Broca and nucleus basalis lesioned rats: Are memory defects induced by nucleus basalis lesions related to the degree of non-specific subcortical cell loss? Behav. Brain Res. 37, 81–88. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R. M., Tanaka, H., Williams, S. C., Richardson, J. A., Kozlowski, G. P., Wilson, S., Arch, J. R. S., Buckingham, R. E., et al. (1998). Orexins and Orexin receptors: A family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92, 573–585. Stanfield, P. R., Nakajima, Y., and Yamaguchi, K. (1985). Substance P raises neuronal membrane excitability by reducing inward rectification. Nature 315, 498–501. Takano, K., Stanfield, P. R., Nakajima, S., and Nakajima, Y. (1995). Protein kinase C-mediated inhibition of an inward rectifier potassium channel by substance P in nucleus basalis neurons. Neuron 14, 999–1008. Takano, K., Yasufuku-Takano, J., Kozasa, T., Singer, W. D., Nakajima, S., and Nakajima, Y. (1996). Gq/11 and PLC-beta 1 mediate the substance P-induced inhibition of an inward rectifier Kþ channel in brain neurons. J. Neurophysiol. 76, 2131–2136. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986). Staurosporine, a potent inhibitor of phospholipid/ Caþþ dependent protein kinase. Biochem. Biophys. Res. Commun. 135, 397–402. Thannickal, T. C., Moore, R. Y., Nienhuis, R., Ramanathan, L., Gulyani, S., Aldrich, M., Cornford, M., and Siegel, J. M. (2000). Reduced number of hypocretin neurons in human narcolepsy. Neuron 27, 469–474. Trivedi, P., Yu, H., MacNeil, D. J., Van der Ploeg, L. H., and Guan, X. M. (1998). Distribution of orexin receptor mRNA in the rat brain. FEBS Lett. 438, 71–75. Yamaguchi, K., Nakajima, Y., Nakajima, S., and Stanfield, P. R. (1990). Modulation of inwardly rectifying channels by substance P in cholinergic neurones from rat brain in culture. J. Physiol. 426, 499–520.