Abstract

To understand the functions of the neocortex, it is essential to characterize the properties of neurons constituting cortical circuits. Here, we focused on a distinct group of GABAergic neurons that are defined by a specific colocalization of intense labeling for both neuronal nitric oxide synthase (nNOS) and substance P (SP) receptor [neurokinin 1 (NK1) receptors]. We investigated the mechanisms of the SP actions on these neurons in visual cortical slices obtained from young glutamate decarboxylase 67-green fluorescent protein knock-in mice. Bath application of SP induced a nonselective cation current leading to depolarization that was inhibited by the NK1 antagonists in nNOS-immunopositive neurons. Ruthenium red and La3+, transient receptor potential (TRP) channel blockers, suppressed the SP-induced current. The SP-induced current was mediated by G proteins and suppressed by D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), but not by inhibitors of phosphatidylinositol-specific PLC, adenylate cyclase or Src tyrosine kinases. Ca2+ imaging experiments under voltage clamp showed that SP induced a rise in intracellular Ca2+ that was abolished by removal of extracellular Ca2+ but not by depletion of intracellular Ca2+ stores. These results suggest that SP regulates nNOS neurons by activating TRP-like Ca2+-permeable nonselective cation channels through a PC-PLC-dependent signaling pathway.

Introduction

Inhibitory GABAergic interneurons in the neocortex have been classified into various types based on distinctive electrophysiological properties, morphologies and the expression of specific chemical markers (reviewed in Markram et al. 2004; Druga 2009). In order to understand the mechanisms of the information processing in the neocortical circuits, it is essential to know the way how the functions of a variety of interneurons are regulated.

In this study, we focused on neuronal nitric oxide synthase (nNOS)-positive GABAergic neurons, which also express the substance P (SP) receptor (NK1 receptor) (Vruwink et al. 2001; Kubota et al. 2011; Dittrich et al. 2012). There are 2 types of nNOS neurons, intensely labeled relatively large neurons (type I) and weakly labeled small neurons (type II) with antibodies against nNOS or in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry (Yan et al. 1996; Yan and Garey 1997; Smiley et al. 2000; Lee and Jeon 2005; Cho et al. 2010; Kubota et al. 2011; Perrenoud et al. 2012). The NK1-expressing nNOS neurons have been considered to correspond to type I nNOS neurons (Kubota et al. 2011; Dittrich et al. 2012). Type I nNOS neurons are a subpopulation of somatostatin- and neuropeptide Y-positive GABAergic neurons (Dawson et al. 1991; Smiley et al. 2000; Tomioka et al. 2005; Higo et al. 2007; Kubota et al. 2011; Perrenoud et al. 2012) and give rise to long-distance projections as well as local axonal arborizations (Tomioka et al. 2005; Higo et al. 2007, 2009; Tomioka and Rockland 2007). Recently, these neurons were identified as “sleep active neurons” because they are highly active during slow-wave sleep (Gerashchenko et al. 2008; Kilduff et al. 2011).These neurons, exerting GABAergic inhibition, would also release NO in certain situations to modulate the function of the neocortical circuit. Considering the wide range of actions NO plays in the central nervous system (reviewed in Garthwaite and Boulton 1995; Calabrese et al. 2007; Garthwaite 2008), it is of great interest to reveal the mechanisms of the regulation of these nNOS neurons.

The highly specific colocalization of the intense labeling for nNOS and NK1 implies that SP is a critical regulator of the nNOS neurons. However, the functions of SP in these neurons have not been investigated except for a recent study reporting that SP depolarizes these neurons (Dittrich et al. 2012). Although that study showed that the depolarizing action of SP was commonly found in multiple species, the underlying mechanisms were largely unexplored. In the present study, we investigated the mechanisms of the SP action in visual cortical slices of mice. Our results indicate that SP activates transient receptor potential (TRP)-like Ca2+-permeable channels in the nNOS neurons.

Materials and Methods

All experimental procedures were approved by the Experimental Animal Care Committee, Research Institute of Environmental Medicine, Nagoya University.

Slice Preparation

Visual cortical slices were prepared as described previously (Yoshimura et al. 2003; Funahashi et al. 2013) from heterozygotes of glutamate decarboxylase 67 (GAD67)-green fluorescent protein (GFP) knock-in mice (Tamamaki et al. 2003). Twenty- to thirty-day-old mice were deeply anesthetized with isoflurane. The depth of anesthesia was carefully confirmed by the absence of reflexes to toe pinches. Coronal slices of visual cortex (300 µm thick) were cut in ice-cold artificial cerebrospinal fluid (ACSF), which contained (mm): 126 NaCl, 3 KCl, 1.2 NaH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3 and 10 glucose (pH 7.4, bubbled with 95% O2 and 5% CO2). Slices were incubated in the ACSF at 33°C for >1 h before recording.

Electrophysiological Recording

Whole-cell patch clamp recordings were obtained from the soma of GFP-positive neurons by visual control of patch pipettes. Slices were mounted in a recording chamber on an upright microscope (BX51WI, Olympus) and continuously superfused with the ACSF. Patch pipettes were prepared from borosilicate glass capillaries. Two types of internal solutions were used. One contained (mm): 140 K-gluconate, 8 KCl, 0.2 EGTA, 10 HEPES, 3 MgATP and 0.5 Na2ATP, adjusted to pH 7.3 with KOH. The other contained (mm): 130 Cs-gluconate, 8 CsCl, 10 EGTA, 10 HEPES, 3 MgATP and 0.5 Na2ATP, adjusted to pH 7.3 with CsOH. Biocytin (5 mg/mL) (Sigma) was dissolved in the internal solutions to stain recorded neurons. The K+-based solution was used in most of the experiments, unless otherwise stated. The resistance of the electrodes was 3–7 MΩ in the ACSF. The actual membrane potential was corrected by a liquid junction potential of −10 mV. Current–voltage curves were obtained by subtracting current responses to voltage steps (50 ms) or ramp (−200 mV/s) before SP applications from those at the peak of the SP-induced current responses. Series resistance was compensated by approximately 70% in the voltage-clamp recordings to obtain current–voltage curves. All recordings were performed at room temperature (25–28°C). Data were recorded with MultiClamp 700B patch clamp amplifier and pClamp software (Molecular Devices).

Ca2+ Imaging

Cells were loaded with the fluorescent Ca2+ indicator rhod-2 (0.2 mm) (Invitrogen) from the recording patch pipette. Fluorescent images were acquired at 0.1 Hz with an electron multiplying CCD camera system (ImagEM, Hamamatsu Photonics).

Histological Procedures

After recording, the slices were fixed with 4% formaldehyde in 0.1 m phosphate buffer (pH 7.4) for more than a day at 4°C and then immersed in 0.01 m phosphate-buffered saline (PBS) containing 30% sucrose. The slices for immunohistochemical staining were resectioned (50 µm thick) with a freezing microtome (Yamato Kohki). The sections were washed in PBS containing 0.3% Triton X 100 (PBST) and incubated for 1 h in PBST containing 10% normal donkey serum to block nonspecific binding. After blocking, the sections were incubated overnight with sheep anti-nNOS antibody (Millipore) (1 : 1000 dilution with PBS containing 0.3% Triton X 100 and 1% normal donkey serum). After washing in PBS, the sections were incubated for 2 h with AlexaFluor 568-labeled anti-sheep IgG (Invitrogen) and AMCA-labeled streptavidin (Jackson ImmunoResaerch) (1 : 200 and 1 : 1000 with PBS, respectively). After washing, the sections were examined with a fluorescent microscope (BX53, Olympus). All of the procedures were performed at room temperature.

To examine the morphology of recorded neurons, the 300-μm-thick slices without resectioning were incubated in methanol containing 0.6% H2O2, then in PBST containing 1% avidin–biotin–peroxidase complex (Vector Laboratories) for 3 h, and the cells were visualized with nickel-intensified diaminobenzidine.

Electrophysiological Measurement and Data Analysis

The amplitude of the current response was determined by the difference between the base line current before SP application and the current averaged across a 1- to 3-s time window at the visually determined maximum of the response. The resting potential was measured immediately after the whole-cell configuration was obtained. In the case of neurons with spontaneous firing, the resting potential was determined as the membrane potential averaged over 10 s. Current pulses (800 ms, 20 pA increments) were injected into the recorded cells through patch pipettes to measure their electrophysiological parameters. The input resistance and the membrane time constant were calculated from the response to a hyperpolarizing current pulse (−20 pA). The parameters related to the action potential were measured for the first spike in response to the minimum current required to induce the firing of the cell. The amplitude of the action potential and the afterhyperpolarization (AHP) were measured with reference to the spike onset defined as the point at which the slope of the voltage trace exceeded 10 mV/ms. The duration of the action potential was measured at half amplitude. The latency of the AHP was measured as the latency of the AHP peak with reference to the spike onset. When the AHP had 2 components, the amplitude and the latency of the second component were also measured with reference to the spike onset.

All values are given as mean ± s.e.m. The unpaired t-test and the Tukey–Kramer test were used to assess the statistical significance. Differences were considered to be significant at P < 0.05.

Drugs

The drugs used were obtained from the following sources: SP, [Sar9, Met(O2)11]-SP, (S)-3-Methyl-2-phenyl-N-(1-phenylpropyl)-4-quinolinecarboxamide (SB 222200), 4-aminopyridine, nifedipine, ruthenium red, 2-Aminoethyl diphenylborinate (2-APB), and 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) from Sigma; TTX, tetraethylammonium and thapsigargin from Wako; (3aR,7aR)-Octahydro-2-[1-imino-2-(2-methoxyphenyl)ethyl]-7,7-diphenyl-4H-isoindol (RP 67580), 1-[2-[(3S)-3-(3,4-Dichlorophenyl)-1-[2-[3-(1-methylethoxy)phenyl]acetyl]-3-piperidinyl]ethyl]-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR 140333), and DL-2-Amino-5-phosphonopentanoic acid (DL-AP5) from Tocris; 2,3-Dihydroxy-6-nitro-7-sulphamoylbenzo(f)-quinoxaline (NBQX), 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536), and tricyclodecan-9-yl xanthogenate (D609) from Enzo Life Science; 1-(1,1-Dimethylethyl)-1-(4-methylph­enyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP 1) from Santa Cruz Biotechnology; 1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF 96365) and 10,10-Bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991) from Abcam; Men 10376 from LKT Laboratories.

Results

Whole-cell recordings were obtained from GFP-positive visual cortical neurons in slice preparations obtained from the GAD67-GFP knock-in mice (Tamamaki et al. 2003). Most of our recordings targeted GFP-positive neurons, which had a relatively large soma size (approximately >15 μm in major diameter), because previous studies have shown that type I nNOS neurons are medium-to-large GABAergic neurons (Dawson et al. 1991; Yan et al. 1996; Yan and Garey 1997; Smiley et al. 2000; Lee and Jeon 2005; Tomioka et al. 2005; Cho et al. 2010; Kubota et al. 2011; Perrenoud et al. 2012). In response to 250 nm of SP applied in the perfusion medium, a subset of the large GFP-positive neurons showed a marked depolarization (n = 10) in current clamp mode or an inward current response in voltage-clamp mode (−67.2 ± 2.5 pA, at −45 – −70 mV, n = 203). The depolarization was accompanied by action potentials in most of the neurons examined in current clamp mode (n = 8/10) (Fig. 1A,B). The whole data set included 213 neurons with depolarization or inward current responses and 24 neurons without noticeable responses to SP (Fig. 1C). Thirty SP-responsive and 4 SP-unresponsive neurons were recorded in the supragranular layer, 157 and 19 in the infragranular layer, and 16 and 1 in the white matter. We did not encounter neurons showing hyperpolarizing or outward current responses to SP except infrequent neurons in which the frequency of inhibitory postsynaptic currents increased.

Figure 1.

Examples of SP-responsive and SP-unresponsive neurons. (A) A SP-responsive and nNOS-immunopositive neuron in Layer 2/3. (Top left) Double staining of the recorded GFP-positive neuron (arrow heads) for nNOS and biocytin. (Top middle and top right) Responses to current pulses from 2 different membrane potentials, indicated on the left of the traces. Injected currents were 20, −20, −40, and −60 pA for the middle panel and 100 pA for the right panel. The slow ramp depolarization (arrows) and the delayed start of firing were visible when current pulses were injected from a hyperpolarized level (−85 mV). (Bottom) Depolarizing response to SP (250 nm) applied in the bath. (B) A SP-responsive and nNOS-positive neuron in Layer 6. The arrangement is the same as in (A). Injected currents were 20, 0, −20, −40, and −60 pA for the top middle traces, and 40 pA for the top right trace. This neuron also showed the ramp-shaped depolarization (arrows) and the delayed firing. (C) A SP-unresponsive and nNOS-negative neuron in Layer 6. The arrangement is the same as in (A). Injected currents were 40, 0, −20, −40, and −60 pA for the top middle traces, and 80 pA for the top right trace.

Figure 1.

Examples of SP-responsive and SP-unresponsive neurons. (A) A SP-responsive and nNOS-immunopositive neuron in Layer 2/3. (Top left) Double staining of the recorded GFP-positive neuron (arrow heads) for nNOS and biocytin. (Top middle and top right) Responses to current pulses from 2 different membrane potentials, indicated on the left of the traces. Injected currents were 20, −20, −40, and −60 pA for the middle panel and 100 pA for the right panel. The slow ramp depolarization (arrows) and the delayed start of firing were visible when current pulses were injected from a hyperpolarized level (−85 mV). (Bottom) Depolarizing response to SP (250 nm) applied in the bath. (B) A SP-responsive and nNOS-positive neuron in Layer 6. The arrangement is the same as in (A). Injected currents were 20, 0, −20, −40, and −60 pA for the top middle traces, and 40 pA for the top right trace. This neuron also showed the ramp-shaped depolarization (arrows) and the delayed firing. (C) A SP-unresponsive and nNOS-negative neuron in Layer 6. The arrangement is the same as in (A). Injected currents were 40, 0, −20, −40, and −60 pA for the top middle traces, and 80 pA for the top right trace.

Identification of nNOS Neurons

We first examined nNOS expression in both SP-responsive and SP-unresponsive neurons. Thirty SP-responsive neurons and 24 unresponsive neurons were examined by immunohistochemical and biocytin staining after electrophysiological recordings. Most (n = 25/30, 83%) of the SP-responsive neurons were positive for nNOS (Fig. 1A,B). On the other hand, none (n = 0/24, 0%) of the SP-unresponsive neurons were positive for nNOS (Fig. 1C).

We noticed that SP-responsive neurons often showed a characteristic firing pattern in response to depolarizing pulses. These neurons showed varying degrees of adapting or irregular train of action potentials (upper middle and upper right traces in Fig. 1A,B) except for 1 nNOS-negative neuron that showed non-adapting high-frequency firing that is characteristic of fast-spiking neurons (Kawaguchi 1995; Kawaguchi and Kubota 1997). At the onset of the current pulse, 12 of 22 SP-responsive neurons examined showed a characteristic shoulder-shaped voltage trajectory, followed by a ramp depolarization (arrows in upper right traces, Fig. 1A,B) and delayed firing. The ramp depolarization and the delayed firing were seen only when current pulses were injected from hyperpolarized membrane potentials. All of the neurons showing the delayed firing were confirmed as nNOS positive. The remaining neurons (10/22) did not show the delayed firing, and 5 of those were confirmed as nNOS positive. In addition, SP-responsive neurons often showed inward rectification and a voltage sag in the responses evoked by hyperpolarizing current pulses. Inward rectification was found in 11 of 18 SP-responsive/nNOS-positive neurons examined (Fig. 1A,B), and a clear voltage sag was found in 8 of these neurons (Fig. 1A). Out of 5 SP-responsive/nNOS-negative neurons, 2 showed inward rectification and 1 showed a voltage sag.

We quantified electrophysiological parameters related to passive property and action potential waveform of the neurons tested for both SP responsiveness and nNOS immunoreactivity and compared them between the 3 groups, SP-responsive/nNOS-positive, SP-responsive/nNOS-negative, and SP-unresponsive/nNOS-negative neuron groups (Table 1). The SP-responsive/nNOS-positive neurons showed a significantly more depolarized resting membrane potential, a larger membrane time constant, a longer duration of action potentials, and a larger amplitude of the first component of AHP, compared with either 1 or both of the nNOS-negative groups. These results suggest that the SP-responsive/nNOS-positive neurons comprise an electrophysiologically distinct subtype of GABAergic neurons. Previous studies have reported similar differences in the comparison of the electrophysiological properties between putative type I nNOS neurons and putative type II nNOS neurons (Perrenoud et al. 2012) or NPY neurons without responses to SP (Dittrich et al. 2012), supporting the concept that SP-responsive/nNOS-positive neurons can be regarded as type I nNOS neurons.

Table 1

Electrophysiological properties

 SP+/nNOS+ (n = 18) SP+/nNOS− (n = 5) SP−/nNOS− (n = 24) 
Resting membrane potential (mV) −55.3 ± 1.2**a, b −68.0 ± 4.7 −63.7 ± 1.4 
Input resistance (MΩ) 584 ± 46 335 ± 95 519 ± 91 
Membrane time constant (ms) 50.9 ± 5.5**b 30.8 ± 9.9 28.7 ± 3.8 
Action potential threshold (mV) −45.3 ± 0.7 −50.6 ± 1.3 −45.4 ± 1.1 
Action potential amplitude (mV) 77.4 ± 2.3 75.5 ± 1.7 72.4 ± 1.7 
Action potential duration (ms) 1.24 ± 0.06*a 0.85 ± 0.12 1.09 ± 0.07 
AHP first component amplitude (mV) −15.4 ± 0.7*b −21.4 ± 1.3 −19.8 ± 1.3 
AHP first component latency (ms) 5.7 ± 1.3 6.5 ± 2.7 8.3 ± 1.3 
AHP second component amplitude (mV) −16.3 ± 1.0 (n = 13) −18.8 (n = 1) −15.8 ± 2.1 (n = 7) 
AHP second component latency (ms) 27.4 ± 4.1 (n = 13) 15.4 (n = 1) 21.1 ± 2.5 (n = 7) 
 SP+/nNOS+ (n = 18) SP+/nNOS− (n = 5) SP−/nNOS− (n = 24) 
Resting membrane potential (mV) −55.3 ± 1.2**a, b −68.0 ± 4.7 −63.7 ± 1.4 
Input resistance (MΩ) 584 ± 46 335 ± 95 519 ± 91 
Membrane time constant (ms) 50.9 ± 5.5**b 30.8 ± 9.9 28.7 ± 3.8 
Action potential threshold (mV) −45.3 ± 0.7 −50.6 ± 1.3 −45.4 ± 1.1 
Action potential amplitude (mV) 77.4 ± 2.3 75.5 ± 1.7 72.4 ± 1.7 
Action potential duration (ms) 1.24 ± 0.06*a 0.85 ± 0.12 1.09 ± 0.07 
AHP first component amplitude (mV) −15.4 ± 0.7*b −21.4 ± 1.3 −19.8 ± 1.3 
AHP first component latency (ms) 5.7 ± 1.3 6.5 ± 2.7 8.3 ± 1.3 
AHP second component amplitude (mV) −16.3 ± 1.0 (n = 13) −18.8 (n = 1) −15.8 ± 2.1 (n = 7) 
AHP second component latency (ms) 27.4 ± 4.1 (n = 13) 15.4 (n = 1) 21.1 ± 2.5 (n = 7) 

Note: Statistical significance was assessed by Tukey–Kramer test.

aComparison to SP+/nNOS−.

bComparison to SP−/nNOS−.

*P < 0.05; **P < 0.01.

Figure 2 shows a plot of the input resistance against the resting membrane potential of the neurons examined for SP responsiveness and nNOS immunoreactivity. The distributions of the SP-responsive/nNOS-positive and the SP-responsive/nNOS-negative neurons were clearly different, but they partially overlapped each other (shown as red and blue symbols in Fig. 2, respectively). Three of the SP-responsive/nNOS-negative neurons, indicated by blue filled circles, seemed to be clearly separated from all of the other SP-responsive neurons. Two of these 3 neurons, with the lowest input resistance and the most hyperpolarized resting potential, were the only neurons that showed depolarizing responses without accompanying action potentials in response to SP in current clamp mode. One of these 2 neurons was the only 1 neuron that showed fast-spiking-like firing in the SP-responsive neuron groups. In regard to these 2 neurons, only 2 or 4 parameters of the electrophysiological measurements were within the ranges observed in SP-responsive/nNOS-positive neurons (Cells 1 and 2 in Supplementary Table 1). Thus, these 2 neurons seemed distinct from the nNOS-positive neurons. In contrast, in the 2 SP-responsive/nNOS-negative neurons indicated by the blue open circles in Figure 2, almost all of the parameters were within the ranges shown by the SP-responsive/nNOS-positive neurons (Cells 4 and 5 in Supplementary Table 1). These 2 neurons may have been false negatives in immunohistochemical staining after whole-cell recording, in which the cytosolic contents were replaced with the patch pipette solution. Based on these considerations, data were collected from SP-responsive neurons with input resistances of >250 MΩ and resting membrane potentials more depolarized than −70 mV (dashed lines in Fig. 2) in the following experiments, except in the case of whole-cell recordings using Cs+-based intracellular solution.

Figure 2.

Relationship between the resting membrane potential and the input resistance in SP-responsive and -unresponsive cells. The input resistance was plotted against the resting potential. Data were from SP-responsive/nNOS-positive (red circles), SP-responsive/nNOS-negative (blue circles), and SP-unresponsive/nNOS-negative (green circles) neurons. Open and filled circles indicate neurons with and without spontaneous firing, respectively. The horizontal and vertical dashed lines indicate 250 MΩ and −70 mV, respectively.

Figure 2.

Relationship between the resting membrane potential and the input resistance in SP-responsive and -unresponsive cells. The input resistance was plotted against the resting potential. Data were from SP-responsive/nNOS-positive (red circles), SP-responsive/nNOS-negative (blue circles), and SP-unresponsive/nNOS-negative (green circles) neurons. Open and filled circles indicate neurons with and without spontaneous firing, respectively. The horizontal and vertical dashed lines indicate 250 MΩ and −70 mV, respectively.

Morphological Properties of the SP-Responsive Neurons

We examined morphological properties of SP-responsive neurons stained with biocytin applied intracellularly from the recording pipette. Figure 3 shows representative examples of the SP-responsive neurons in Layers 2/3 (Fig. 3A), 5 (Fig. 3B), and 6 (Fig. 3C,D). Substance P-responsive neurons were multipolar neurons with sparsely spiny or aspiny dendrites. Their dendrites intruded the adjacent cortical layers. We could reconstruct substantial axonal arborizations in 51 neurons. Substance P-responsive neurons typically had dense axonal arborizations that covered an area approximately 200–300 μm in diameter surrounding their soma, and some axonal branches extended further vertically or horizontally (Fig. 3A1,B1,C1,D1). Thirty-two neurons showed axons that extended into all of the cortical layers. In the horizontal direction, we could trace at least 1 axonal branch over 600 μm long for 25 neurons, and the longest branch reached 1.2 mm from the soma (Fig. 3D). Seven neurons had axons running a substantial distance horizontally in the white matter. Eighteen neurons had axons that entered the adjacent cortical areas. Sixteen neurons had axons that entered the medial and/or lateral secondary visual cortex (Fig. 3B2,C2,D2), and 5 neurons had axons that reached the retrosplenial cortex (Fig. 3C2). One neuron had an axonal branch that crossed the underneath white matter and extended to and ramified in the subiculum (Fig. 3B2). Considering that these morphological analyses were performed using the 300-μm-thick slice preparations used for electrophysiological examinations, these neurons may have had long axonal braches more frequently. The presence of long horizontal axons is consistent with previous studies showing that most of the retrogradely labeled GABAergic projection neurons in the neocortex are nNOS positive and considered to be type I nNOS neurons (Tomioka et al. 2005; Higo et al. 2007, 2009; Tomioka and Rockland 2007), which further supports our identification of nNOS neurons.

Figure 3.

Morphology of the SP-responsive neurons. Camera lucida drawings of the SP-responsive neurons stained with intracellularly applied biocytin. The detailed (A1D1) and the corresponding wide area (A2D2) drawings of 4 representative SP-responsive neurons. Soma and dendrites are indicated in black and axons are in red. Dashed lines indicate the border of cortical layers in the detailed and the border of cortical areas in the wide area drawings, respectively. V1, primary visual cortex; V2, secondary visual cortex; RS, retrosplenial cortex; S, subiculum; W, white matter.

Figure 3.

Morphology of the SP-responsive neurons. Camera lucida drawings of the SP-responsive neurons stained with intracellularly applied biocytin. The detailed (A1D1) and the corresponding wide area (A2D2) drawings of 4 representative SP-responsive neurons. Soma and dendrites are indicated in black and axons are in red. Dashed lines indicate the border of cortical layers in the detailed and the border of cortical areas in the wide area drawings, respectively. V1, primary visual cortex; V2, secondary visual cortex; RS, retrosplenial cortex; S, subiculum; W, white matter.

Finally, we did not encounter cells with neurogliaform-like shape, which is the typical morphology of type II nNOS neurons (Smiley et al. 2000).

Characterization of the SP-Induced Current

In order to characterize the SP-induced current, we performed voltage-clamp recordings from SP-responsive neurons. Cells were held at −55 mV, which is close to the resting potential in the SP-responsive/nNOS-positive neurons, unless otherwise stated. In principle, our preparations did not allow for prior identification of nNOS neurons. Therefore, when we examined the effects of drugs on the SP-induced current responses, we applied SP twice and the second application was challenged by the test drugs. Most of the drugs were applied in the bath solution starting 5–25 min before the second SP application. When the test drug was applied intracellularly, the first SP application was started within 60 s after whole-cell configuration was established. In order to compensate for possible desensitization of the SP responses, the proportion of the second SP response to the first in the test experiments was compared with that in the control experiments in which SP was applied twice without test drugs.

When SP was applied with a 20-min interval, the amplitude of the second response was 81.0 ± 12.2% of the first (n = 8) (Fig. 4A,C). With this application interval, we first verified that the SP-induced current was elicited by the direct action of SP on the NK1 receptors expressed in the recorded neurons. The cocktail of the Na+ channel blocker TTX (1 μm), the non-NMDA receptor antagonist NBQX (10 μm), and the NMDA receptor antagonist DL-AP5 (100 μm) did not affect the SP-induced current (73.5 ± 11.5% of the first, unpaired t-test, P = 0.67, n = 6) (Fig. 4B,C), indicating that the SP-induced current was not mediated by the enhanced synaptic inputs from surrounding neurons. To confirm that this current was mediated by the activation of NK1 receptors, we examined the effect of tachykinin receptor antagonists on the SP-induced current. The SP-induced current was significantly suppressed by the selective NK1 antagonists SR 140333 (1 μm, 22.7 ± 5.9% of the first, unpaired t-test, P < 0.01, n = 5) (Fig. 5A,F) and RP 67580 (10 μm, 32.0 ± 3.8% of the first, unpaired t-test, P < 0.01, n = 8) (Fig. 5B,F). On the other hand, the NK2 antagonist Men 10376 (2 μm) and the NK3 antagonist SB 222200 (10 μm) were ineffective against the SP-induced current (79.5 ± 6.5% of the first, unpaired t-test, P = 0.93, n = 5 for Men 10376 and 73.6 ± 12.5% of the first, unpaired t-test, P = 0.68, n = 7 for SB 222200) (Fig. 5C,D,F). We further confirmed that [Sar9, Met(O2)11]-SP (250 nm), a selective NK1 agonist, readily induced an inward current in the SP-responsive neurons (181.1 ± 34.2 pA, n = 3) as did the endogenous NK1 agonist SP (Fig. 5E). These results indicate that the SP-induced current was mediated by the activation of NK1 receptors expressed in nNOS neurons.

Figure 4.

The SP-induced current is not the enhanced synaptic inputs from surrounding neurons. (A) An example of current responses to the first and the second SP application under control recording conditions. (B) An example of recordings in which the second SP application was performed in the presence of TTX, NBQX, and AP5. (C) Summary graph showing that TTX, NBQX, and AP5 did not show detectable effects on the SP-induced current. The percentage ratio (mean ± s.e.m) of the second to the first response amplitude was plotted.

Figure 4.

The SP-induced current is not the enhanced synaptic inputs from surrounding neurons. (A) An example of current responses to the first and the second SP application under control recording conditions. (B) An example of recordings in which the second SP application was performed in the presence of TTX, NBQX, and AP5. (C) Summary graph showing that TTX, NBQX, and AP5 did not show detectable effects on the SP-induced current. The percentage ratio (mean ± s.e.m) of the second to the first response amplitude was plotted.

Figure 5.

The response to SP is mediated by NK1 receptors. (AD) Examples of recordings in which the second SP application was performed in the presence of SR 140333 (A), RP 67580 (B), Men 10376 (C), and SB 222200 (D). (E) An example of current response to [Sar9, Met(O2)11]-SP in the SP-responsive neuron. [Sar9, Met(O2)11]-SP induced an inward current (right) when it was applied 20 min after the SP application (left). (F) Summary graph of the effects of the antagonists of tachykinin receptors. **P < 0.01.

Figure 5.

The response to SP is mediated by NK1 receptors. (AD) Examples of recordings in which the second SP application was performed in the presence of SR 140333 (A), RP 67580 (B), Men 10376 (C), and SB 222200 (D). (E) An example of current response to [Sar9, Met(O2)11]-SP in the SP-responsive neuron. [Sar9, Met(O2)11]-SP induced an inward current (right) when it was applied 20 min after the SP application (left). (F) Summary graph of the effects of the antagonists of tachykinin receptors. **P < 0.01.

We next examined the ionic nature of the SP-induced current. In this and the following experiments, SP was applied with a 30-min interval. The amplitude of the second SP response was 95.6 ± 7.7% of the first in control recordings (n = 11) (Fig. 6B). Bath application of a cocktail of the blockers of K+ channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (3 mm CsCl, 5 mm 4-aminopyridine, and 20 mm tetraethylammonium replacing equimolar NaCl) in the presence of 0.5 μm TTX did not significantly affect the SP-induced current (102.4 ± 17.5% of the first, unpaired t-test, P = 0.73, n = 12) (Fig. 6A,B), indicating that K+ and HCN channels do not contribute to the SP-induced current at least at the resting membrane potential level.

Figure 6.

Substance P induces a nonselective cation current. (A) An example of recordings in which the second SP application was performed in the ACSF containing K+ and HCN channel blockers (CsCl, 4-aminopyridine, and tetraethylammonium). Recordings were obtained in the presence of TTX. (B) Summary graph of the effects of K+ and HCN channel blockers, showing no significant effect on the SP-induced current. (C) An example of measurement of the reversal potential of SP-induced current. (C1) The SP-induced current at various voltages (lower traces) were obtained by subtracting the responses to 50-ms voltage steps applied before SP application (upper left traces) from those during SP application (upper right traces). The holding potential was −45 m,V and the test voltage was varied from −125 to −5 mV with 20-mV increments. Recordings were obtained in the ACSF containing 0-Ca2+, EGTA, nifedipine, TTX, and the blockers of K+ and HCN channels. (C2) The current–voltage curves constructed from the traces in C1. The reversal potential of the SP-induced current was −16 mV.

Figure 6.

Substance P induces a nonselective cation current. (A) An example of recordings in which the second SP application was performed in the ACSF containing K+ and HCN channel blockers (CsCl, 4-aminopyridine, and tetraethylammonium). Recordings were obtained in the presence of TTX. (B) Summary graph of the effects of K+ and HCN channel blockers, showing no significant effect on the SP-induced current. (C) An example of measurement of the reversal potential of SP-induced current. (C1) The SP-induced current at various voltages (lower traces) were obtained by subtracting the responses to 50-ms voltage steps applied before SP application (upper left traces) from those during SP application (upper right traces). The holding potential was −45 m,V and the test voltage was varied from −125 to −5 mV with 20-mV increments. Recordings were obtained in the ACSF containing 0-Ca2+, EGTA, nifedipine, TTX, and the blockers of K+ and HCN channels. (C2) The current–voltage curves constructed from the traces in C1. The reversal potential of the SP-induced current was −16 mV.

We then measured the reversal potential of the SP-induced current. In the presence of TTX and the K+ and HCN channel blockers, Ca2+ was omitted from the ACSF and EGTA (0.5 mm) and nifedipine (50 μm) were included in the ACSF to abolish the current through voltage-dependent Ca2+ channels. The current–voltage curves were obtained by subtracting the current responses to voltage steps (from −45 mV, 50 ms duration) before SP application from those during the peak of the SP-induced current (Fig. 6C1,C2). The mean reversal potential of the SP-induced current was −17.4 ± 1.9 mV (n = 4) (Fig. 6C2). These results indicate that SP activates nonselective cation channels expressed in nNOS neurons.

As a candidate for nonselective cation channels activated following NK1 receptor activation, we focused on TRP channels. We examined the effects of some broad-spectrum inhibitors of TRP channels: 2-aminoethyl diphenylborinate (2-APB) and SKF 96365, both of which preferentially inhibit the canonical subfamily of TRP channels (TRPCs), ruthenium red, which inhibits the vanilloid subfamily of TRP and some other TRP channels but not TRPCs, and La3+, which enhances TRPC4 and TRPC5 but inhibits many other TRP channels (Inoue 2005; Clapham 2007). Bath applied 2-APB (100 μm) and SKF 96365 (100 μm) seemed slightly reduced the SP-induced current; however, the reduction was not statistically significant (73.9 ± 4.9% of the first, unpaired t-test, P = 0.054, n = 7 for 2-APB and 80.1 ± 7.7% of the first, unpaired t-test, P = 0.21, n = 8 for SKF 96365) (Fig. 7A,B,E). On the other hand, ruthenium red (20 μm) consistently suppressed the SP-induced current (39.3 ± 6.4% of the first, unpaired t-test, P < 0.0001, n = 7) (Fig. 7C,E). La3+ (100 μm) also suppressed the SP-induced current (54.6 ± 7.0% of the first, unpaired t-test, P < 0.01, n = 8) (Fig. 7D,E). Thus, it seems plausible that the SP-induced current in the nNOS neurons is mediated by TRP channels other than TRPCs.

Figure 7.

The effects of TRP channel inhibitors on the SP-induced current. (AC) Examples of recordings showing the effects of 2-APB (A), SKF 96365 (B), ruthenium red (C), and La3+ (D) on the SP-induced current. (E) Summary graph of the effects of the inhibitors of TRP channels. **P < 0.01; ****P < 0.0001.

Figure 7.

The effects of TRP channel inhibitors on the SP-induced current. (AC) Examples of recordings showing the effects of 2-APB (A), SKF 96365 (B), ruthenium red (C), and La3+ (D) on the SP-induced current. (E) Summary graph of the effects of the inhibitors of TRP channels. **P < 0.01; ****P < 0.0001.

Signaling Pathway for the Activation of the SP-Induced Current

The SP-induced current was remarkably suppressed by intracellularly applied GTPγS (1 mm) (15.1 ± 4.9% of the first, unpaired t-test, P < 0.001, n = 4) (Fig. 8A,E) and GDPβS (1 mm) (44.9 ± 11.9% of the first, unpaired t-test, P < 0.01, n = 6) (Fig. 8E), indicating that the activation of the SP-induced current is dependent on G protein signaling. It is known that NK1 receptors can stimulate phosphatidylinositol hydrolysis or cyclic AMP signaling pathways (Nakajima et al. 1992). Therefore, we examined whether the activation of the SP-induced current is dependent on these signaling pathways. Bath application of U-73122 (5 μm), an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC) which cleaves phosphatidylinositol 4,5-bisphosphate, did not suppress the SP-induced current (101.3 ± 17.5% of the first, unpaired t-test, P = 0.76, n = 10) (Fig. 8B,E). SQ 22536 (300 μm), an inhibitor of adenylate cyclase which mediates the formation of cyclic AMP from ATP, also did not suppress the SP-induced current (109.3 ± 9.6% of the first, unpaired t-test, P = 0.28, n = 10) (Fig. 8C,E). These results suggest that the activation of the SP-induced current in nNOS neurons is not dependent on either phosphatidylinositol hydrolysis or cyclic AMP.

Figure 8.

The activation of SP-induced current is dependent on G protein, but not on PI-PLC, AC, or Src tyrosine kinases. (A) An example of recordings in the presence of GTPγS intracellularly. The first SP application was started within 60 s after whole-cell configuration was established. (BD) Examples of recordings in which the second SP application was performed in the presence of U-73122 (B), SQ 22536 (C), and PP 1 (D). (E) Summary graph of the effects of the inhibitors of intracellular signaling pathways. **P < 0.01; ***P < 0.001.

Figure 8.

The activation of SP-induced current is dependent on G protein, but not on PI-PLC, AC, or Src tyrosine kinases. (A) An example of recordings in the presence of GTPγS intracellularly. The first SP application was started within 60 s after whole-cell configuration was established. (BD) Examples of recordings in which the second SP application was performed in the presence of U-73122 (B), SQ 22536 (C), and PP 1 (D). (E) Summary graph of the effects of the inhibitors of intracellular signaling pathways. **P < 0.01; ***P < 0.001.

In hippocampal and ventral tegmental area neurons, it was shown that SP activated the Na+-leak channel (NALCN), a nonselective cation channel, through a Src family tyrosine kinase-dependent mechanism, which, however, was G protein independent (Lu et al. 2009). We examined the effects of PP 1, an inhibitor of Src, on the SP-induced current. However, instead of inhibition, PP 1 (20 μm) produced facilitation of the SP-induced current to some degree, but the effect was not statistically significant (138.2 ± 19.4% of the first, unpaired t-test, P = 0.1, n = 7) (Fig. 8D,E).

Finally, we tested the effect of D609, an inhibitor of phosphatidylcholine-specific PLC (PC-PLC), on the SP-induced current. PC-PLC is an enzyme that hydrolyzes phosphatidylcholine to produce diacylglycerol and phosphocholine. Unexpectedly, D609 (100 μm) itself induced an outward current that lasted as long as D609 was present (82.4 ± 14.9 pA, n = 5) (Fig. 9A). Substance P applied on top of D609 induced an inward current (−53.7 ± 20.0 pA, n = 5) (Fig. 9A). The reversal potentials of the outward and the inward current, measured with voltage ramp in a SP-responsive neuron, were both close to the equilibrium potential of potassium (−93 and −90 mV, respectively), suggesting that they are K+ currents but not nonselective cation currents. We therefore performed recordings using the Cs+-based intracellular solution. Substance P readily induced inward current responses with this solution (−58.1 ± 6.3 pA, n = 14), whereas D609 no longer induced outward currents (Fig. 9B). In control recordings with a 30-min interval, the response to the second SP application was 66.6 ± 5.1% of the first (Fig. 9C, n = 8). Under these recording conditions, D609 significantly suppressed the SP-induced current (26.6 ± 5.6% of the first, n = 6, unpaired t-test, P < 0.001) (Fig. 9B,C). These results suggest that SP induces the nonselective cation current through PC-PLC-dependent signaling.

Figure 9.

Effects of the PC-PLC inhibitor D609 on the SP-induced current. (A) An example of recordings using the K+-based intracellular solution. D609 induced an outward current (middle trace), and the subsequent coapplication of SP induced an inward current (right trace). (B) An example of recordings using Cs+-based intracellular solution. D609 did not induce the outward current and suppressed the SP-induced current significantly. (C) Summary graph of the effect of D609 on the SP-induced current when the Cs+-based intracellular solution was used. ***P < 0.001.

Figure 9.

Effects of the PC-PLC inhibitor D609 on the SP-induced current. (A) An example of recordings using the K+-based intracellular solution. D609 induced an outward current (middle trace), and the subsequent coapplication of SP induced an inward current (right trace). (B) An example of recordings using Cs+-based intracellular solution. D609 did not induce the outward current and suppressed the SP-induced current significantly. (C) Summary graph of the effect of D609 on the SP-induced current when the Cs+-based intracellular solution was used. ***P < 0.001.

These results also suggest that SP can modulate the K+ current that was unmasked in the presence of D609. It is known that SP can modulate M-current (Adams et al. 1983). However, it is likely that the current is mediated by some K+ channels other than the M-type K+ channel current, because the current appeared in the presence of D609 was not affected by the M-current inhibitor XE-991 (Supplementary Fig. 1).

SP-Activated Nonselective Cation Channels Are Ca2+ Permeable

To examine whether SP can increase intracellular Ca2+ concentration in nNOS neurons without the participation of voltage-dependent Ca2+ channels, neurons were loaded with the Ca2+-sensitive fluorescent dye rhod-2 from the recording pipette. In response to SP, the fluorescence intensity recorded from the soma was increased in parallel with the SP-induced current responses recorded at −55 mV (ΔF/F = 11.8 ± 1.2%, n = 11) (Fig. 10A,D), indicating that the activation of NK1 receptors is coupled to a raise in the intracellular Ca2+ concentration in nNOS neurons. After preincubation with thapsigargin (1–10 μm, 20–60 min) to deplete intracellular Ca2+ stores, the Ca2+ increase tended to be larger than that in control solution, but the difference was not statistically significant (17.7 ± 5.3%, n = 5, unpaired t-test, P = 0.18) (Fig. 10B,D). In contrast, the SP-induced Ca2+ rise was significantly smaller when Ca2+ was omitted from and EGTA (0.5 mm) was added to the extracellular solution (3.4 ± 0.9%, n = 12, unpaired t-test, P < 0.001) (Fig. 10C,D). The amplitude of the SP-induced current in the presence of thapsigargin (−55.4 ± 8.5 pA) and in the Ca2+-free condition (−60.1 ± 11.5 pA) was not significantly different from that in the control recordings (−53.8 ± 4.3 pA) (unpaired t-test, P = 0.86 and P = 0.62, respectively). These results suggest that SP induces a Ca2+ influx from the extracellular space through the nonselective cation channels, but not Ca2+ release from internal stores.

Figure 10.

Substance P induces an increase in the intracellular Ca2+ concentration under voltage-clamp recording. (AC) Examples of recordings in the control ACSF (A), in the presence of thapsigargin (B) and in the Ca2+-free condition (C). Top traces show the change in the fluorescence intensity (ΔF/F) of the Ca2+ indicator rhod-2 loaded from the recording pipette, and bottom traces show simultaneously recorded current responses in each panels. (D) Summary graph of the effect of eliminating different Ca2+ sources. ***P < 0.001.

Figure 10.

Substance P induces an increase in the intracellular Ca2+ concentration under voltage-clamp recording. (AC) Examples of recordings in the control ACSF (A), in the presence of thapsigargin (B) and in the Ca2+-free condition (C). Top traces show the change in the fluorescence intensity (ΔF/F) of the Ca2+ indicator rhod-2 loaded from the recording pipette, and bottom traces show simultaneously recorded current responses in each panels. (D) Summary graph of the effect of eliminating different Ca2+ sources. ***P < 0.001.

Discussion

The main findings in the present study were as follows. First, SP activates NK1 receptors in nNOS neurons and induces a TRP-like nonselective cation current. Second, the activation of the nonselective cation channels is probably dependent on PC-PLC. Third, SP induces extracellular Ca2+ influx through the nonselective cation channels, consequently raising the intracellular Ca2+ concentration.

Selective Activation of Type I nNOS Neurons by SP

Recently, Dittrich et al. (2012) showed that SP depolarizes type I nNOS neurons in multiple species. In the present study, the majority (25/30) of SP-responsive neurons were positive for nNOS, whereas none of the SP-unresponsive neurons were positive for nNOS. Previous studies have reported selective colocalization of intense immunoreactivities for nNOS and NK1 (Vruwink et al. 2001; Kubota et al. 2011; Dittrich et al. 2012). A small number of SP-responsive/nNOS-negative neurons found in the present study might correspond to another population that are weakly positive for NK1 (Nakaya et al. 1994; Vruwink et al. 2001; Dittrich et al. 2012). However, we must consider the possibility that some of them were judged as nNOS negative due to the washout of intracellular contents during whole-cell recordings. Furthermore, it has been demonstrated that there are a considerable number of NK1-positive/nNOS-negative neurons in rats and monkeys, but only a very limited number in mice (Dittrich et al. 2012).

While previous studies have consistently demonstrated that intense NK1 labeling is highly specific to type I nNOS neurons, at present there is no definitive description regarding whether some of the type II nNOS neurons express NK1 or not. Type II nNOS neurons have been defined as small neurons labeled weakly for nNOS or NADPHd (Yan et al. 1996; Yan and Garey 1997; Smiley et al. 2000; Lee and Jeon 2005; Cho et al. 2010; Kubota et al. 2011; Perrenoud et al. 2012) and have neurogliaform morphology (Smiley et al. 2000). In the present experiment, our recordings were mostly targeted to large neurons among GFP-labeled neurons in the slice preparations. In addition, we have never encountered neurogliaform-like neurons among our samples of intracellularly stained cells.

Taken all together, we conclude that it is safe to assume that the vast majority of the SP-responsive neurons in the present study were type I nNOS neurons, although we cannot entirely rule out the possibility of some contaminations of other types of neurons. It is likely that SP selectively depolarizes type I nNOS neurons in the mouse visual cortex.

SP Activates TRP-like Ca2+-Permeable Nonselective Cation Channels in Type I nNOS Neurons

Blockade of voltage-dependent K+ channels and HCN channels produced no significant effect, indicating a negligible contribution of these channels to the SP-induced current. On the other hand, the reversal potential of the SP-induced current indicates that SP activates nonselective cation channels. The SP-activated channels are permeable to Ca2+, because the removal of extracellular Ca2+ abolished the SP-induced increase in the intracellular Ca2+ concentration under voltage-clamp recording conditions whereas the depletion of the intracellular Ca2+ stores did not.

Transient receptor potential channels are a superfamily of nonselective cation channels permeable to Ca2+ and modulated in various ways, including G protein-coupled receptor activation. It has been reported that the activation of NK1 receptors induces TRP-like currents in noradrenergic A7 neurons (Min et al. 2009). In the present study, 2 TRPC inhibitors had only a minor effect, if any, on the SP-induced current, whereas other TRP inhibitors ruthenium red and La3+ markedly suppressed the SP-induced current. These results suggest that the SP-induced current in the nNOS neurons is mediated by some TRP channels other than TRPCs.

It has been demonstrated that SP activates NALCN channels in hippocampal and ventral tegmental area neurons (Lu et al. 2009). Therefore, NALCN is another possible candidate for the SP-activated channels, although the inhibition of the signaling pathway for the activation of NALCN failed to suppress the SP-induced current in the present study (see below). The lack of specific inhibitor of NALCN prevented further examination.

Signaling Pathway for the Activation of SP-Induced Currents

It is widely accepted that NK1 receptors are coupled to Gq/11 protein, which in turn activates PI-PLC. The PI-PLC-dependent signaling is a well-established mechanism for the modulation of TRP channels (Ramsey et al. 2006). Previous studies have reported that NK1 agonists activate nonselective cation currents in striatal cholinergic neurons (Bell et al. 1998) and TRP-like currents in noradrenergic A7 neurons (Min et al. 2009) through a PI-PLC-dependent pathway. NK1 can also activate cyclic AMP-dependent signaling cascades (Nakajima et al. 1992). In the present study, however, the inhibition of PI-PLC or adenylate cyclase failed to suppress the SP-induced current. The inhibition of Src family tyrosine kinase, which is involved in the SP-induced activation of NALCN (Lu et al. 2009), did not suppress the SP-induced current either.

On the other hand, we found that the PC-PLC inhibitor D609 itself elicited an outward current and the subsequent coapplication of SP induced an inward current. The reversal potentials of these currents and the blockade with the Cs+-based intracellular solution suggested that these responses were mediated by K+ channels. Although the details remain unclear, it is likely that D609 increases the current mediated by some K+ channels, which are in a suppressed state in normal conditions, and SP decreases the current. Under the blockade of these K+ currents by Cs+, D609 remarkably suppressed the SP-induced current, suggesting that SP activates the TRP-like channels through a PC-PLC-dependent signaling pathway (Fig. 11).

Figure 11.

Signaling pathway for the SP-induced currents in nNOS neurons. Schematic drawing of the hypothetical signaling pathway for the activation of SP-induced currents. Plus and minus signs indicate activation and suppression, respectively, and dashed lines depict possible intermediate cascades, which were not revealed in the present study.

Figure 11.

Signaling pathway for the SP-induced currents in nNOS neurons. Schematic drawing of the hypothetical signaling pathway for the activation of SP-induced currents. Plus and minus signs indicate activation and suppression, respectively, and dashed lines depict possible intermediate cascades, which were not revealed in the present study.

Although PC-PLC is involved in various cellular functions, the PC-PLC-dependent regulation of ion channels has been relatively unexplored. As for TRP channels, a previous study has associated the P2Y receptor-mediated elevation of intracellular Ca2+ with PC-PLC and TRP channel in a renal cell line (Turvey et al. 2010). In the central nervous system, it has been reported that D609 suppresses the regulation of ion channels by ghrelin in pedunculopontine tegmental neurons (Kim et al. 2009) and by orexin-A/hypocretin-1 in pedunculopontine tegmental neurons (Kim et al. 2009), ventral tegmental dopamine neurons (Uramura et al. 2001), nucleus tractus solitarius neurons (Yang et al. 2003), and prefrontal cortical pyramidal neurons(Song et al. 2005; Xia et al. 2005, 2009). The present results provide another example of the PC-PLC-dependent activation of ion channels and may imply that it is not an unusual mechanism in the central nervous system.

In addition to the PC-PLC-dependent activation of the TRP-like channels, the present results suggest the presence of the PC-PLC-dependent and -independent inhibition of K+ channels (Fig. 11). The latter inhibition seems usually masked by the former. The switching between these 2 signaling pathways may be implicated in the modulation of the functions of nNOS neurons.

The Source of SP

Previous immunohistochemical studies have demonstrated the presence of SP-immunoreactive fibers and cell bodies in the neocortex (Ljungdahl et al. 1978; Inagaki et al. 1982; Penny et al. 1986; Jones et al. 1988; Iritani et al. 1989; Conti et al. 1992; Zhang and Harlan 1994; Vruwink et al. 2001). Substance P-expressing cell bodies in the neocortex are also immunopositive for glutamate decarboxylase (GAD) and account for approximately 20% of GAD-positive neurons (Penny et al. 1986). It has also been reported that the majority (71%) of the SP-expressing neurons are parvalbumin (PV)-immunopositive neurons (Vruwink et al. 2001), which are practically equivalent to fast-spiking GABAergic interneurons (Kawaguchi and Kubota 1997, 1998). Another possible source may be the ascending projection from the laterodorsal tegmentum, which provides dense SP-positive fibers in the medial frontal cortex (Sakanaka et al. 1983; Vincent et al. 1983). However, it is uncertain currently whether these fibers also project to other cortical areas. In any case, it seems conceivable that the GABAergic, especially PV-positive, interneurons are a major source of SP in the cerebral cortex.

Functional Implications

The present results showed that SP could depolarize nNOS neurons and hence increase their firing activities. In the neocortex, SP-positive synaptic terminals are seldom apposed to NK1-positive profiles and SP would act in a paracrine fashion (Vruwink et al. 2001). The response to SP is slow and long-lasting, as shown in the present and previous studies (Dittrich et al. 2012). Therefore, it is likely that SP acts in a long time scale rather than in a short time scale precisely timed to its release. Repetitive firing of PV neurons or ascending SP afferents, if present in the visual cortex, would cause a diffuse increase in the ambient SP concentration, inducing a tonic firing of the nNOS neurons to regulate the network functions.

However, it is also possible that the activity of PV neurons would exert strong GABAergic inhibition, which can counteract the depolarizing action of SP. Therefore, unlike the present experiments, in which SP was applied alone, the activation of the NK1 receptors may not always facilitate firing activities in physiological conditions. Even when the activation of NK1 receptors fails to initiate action potentials, Ca2+ entering through the SP-activated channels may play some functional roles in the somatodendritic domain. The long-lasting increase in the intracellular Ca2+ induced by SP, independent of the firing of cells, may contribute to the setting of the basal level of cytosolic Ca2+ concentration and modulate the action of the Ca2+ transient induced by other synaptic inputs and/or subsequent action potentials. Alternatively, the Ca2+ influx induced by SP by itself may directly regulate intracellular processes in a longer time scale than the phasic Ca2+ increase does. The firing activity of the nNOS neurons would be regulated by the balance of various synaptic inputs. We speculate that the SP-NK1 system regulates the function of nNOS neurons differentially, depending on whether the activation of NK1 receptors initiates action potentials in these neurons or not.

The SP-responsive visual cortical neurons investigated in the present study had extensive axonal arborizations around their soma and also had axonal branches passing through all of the cortical layers and extending a long distance horizontally into other cortical areas. Previous studies have shown that most of the GABAergic projection neurons are type I nNOS neurons (Tomioka et al. 2005; Higo et al. 2007, 2009; Tomioka and Rockland 2007), which probably correspond to the SP-responsive neurons in the present study. These axonal projections would enable the SP-responsive nNOS neurons to coordinate large-scale network activity, including the slow-wave activity during non-rapid eye movement sleep, as proposed recently (Kilduff et al. 2011), through releasing NO in addition to GABA. Because NADPHd was detected in the axon of type I nNOS neurons (Higo et al. 2009), it can be expected that NO is released in wide area when SP activates these neurons. On the other hand, because NK1 and nNOS are colocalized in the soma and dendrites (Vruwink et al. 2001; Kubota et al. 2011; Dittrich et al. 2012), Ca2+ entering through the SP-activated channels may directly modulate NO production in the somatodendritic domains. The increase in intracellular Ca2+ mediated by SP-activated channels would be long-lasting and may enhance phasic NO production induced by action potentials and/or regulate by itself tonic NO production, which acts locally in the areas surrounding the soma and dendrite of these neurons even in the absence of firing. NO produced by nNOS neurons may regulate various cellular processes, such as neuronal firing, synaptic plasticity, and local blood flow (Garthwaite and Boulton 1995; Calabrese et al. 2007; Garthwaite 2008). In the visual cortex, several studies have suggested that NO regulates visual responses (Cudeiro et al. 1997; Kara and Friedlander 1999) and excitation-inhibition balance (Le Roux et al. 2009), as well as long-term and short-term modification of synaptic transmission (Volgushev et al. 2000; Wei et al. 2002; Haghikia et al. 2007). It is important to clarify the mechanisms for NO production in SP-responsive nNOS neurons, in order to increase our understanding of NO-mediated regulation of these processes.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

This work was supported by grants from the Japan Society for the Promotion of Science (22700412 and 24500457 to T.E., 23300116 to Y.K.) and Grant-in-Aids for Scientific Research on Innovative Areas (23110004 to Y.K.) from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

Notes

Conflict of Interest: None declared.

References

Adams
PR
Brown
DA
Jones
SW
.
1983
.
Substance P inhibits the M-current in bullfrog sympathetic neurons
.
Br J Pharmacol
 .
79
:
330
333
.
Bell
MI
Richardson
PJ
Lee
K
.
1998
.
Characterization of the mechanism of action of tachykinins in rat striatal cholinergic interneurons
.
Neuroscience
 .
87
:
649
658
.
Calabrese
V
Mancuso
C
Calvani
M
Rizzarelli
E
Butterfield
DA
Stella
AM
.
2007
.
Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity
.
Nat Rev
 .
8
:
766
775
.
Cho
KH
Jang
JH
Jang
HJ
Kim
MJ
Yoon
SH
Fukuda
T
Tennigkeit
F
Singer
W
Rhie
DJ
.
2010
.
Subtype-specific dendritic Ca2+ dynamics of inhibitory interneurons in the rat visual cortex
.
J Neurophysiol
 .
104
:
840
853
.
Clapham
DE
.
2007
.
SnapShot: mammalian TRP channels
.
Cell
 .
129
:
220
.
Conti
F
De Biasi
S
Fabri
M
Abdullah
L
Manzoni
T
Petrusz
P
.
1992
.
Substance P-containing pyramidal neurons in the cat somatic sensory cortex
.
J Compar Neurol
 .
322
:
136
148
.
Cudeiro
J
Rivadulla
C
Rodriguez
R
Grieve
KL
Martinez-Conde
S
Acuna
C
.
1997
.
Actions of compounds manipulating the nitric oxide system in the cat primary visual cortex
.
J Physiol
 .
504
(Pt 2)
:
467
478
.
Dawson
TM
Bredt
DS
Fotuhi
M
Hwang
PM
Snyder
SH
.
1991
.
Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues
.
Proc Natl Acad Sci USA
 .
88
:
7797
7801
.
Dittrich
L
Heiss
JE
Warrier
DR
Perez
XA
Quik
M
Kilduff
TS
.
2012
.
Cortical nNOS neurons co-express the NK1 receptor and are depolarized by Substance P in multiple mammalian species
.
Front Neural Circuits
 .
6
:
31
.
Druga
R
.
2009
.
Neocortical inhibitory system
.
Folia Biologica
 .
55
:
201
217
.
Funahashi
R
Maruyama
T
Yoshimura
Y
Komatsu
Y
.
2013
.
Silent synapses persist into adulthood in layer 2/3 pyramidal neurons of visual cortex in dark-reared mice
.
J Neurophysiol
 .
109
:
2064
2076
.
Garthwaite
J
.
2008
.
Concepts of neural nitric oxide-mediated transmission
.
Eur J Neurosci
 .
27
:
2783
2802
.
Garthwaite
J
Boulton
CL
.
1995
.
Nitric oxide signaling in the central nervous system
.
Ann Rev Physiol
 .
57
:
683
706
.
Gerashchenko
D
Wisor
JP
Burns
D
Reh
RK
Shiromani
PJ
Sakurai
T
de la Iglesia
HO
Kilduff
TS
.
2008
.
Identification of a population of sleep-active cerebral cortex neurons
.
Proc Natl Acad Sci USA
 .
105
:
10227
10232
.
Haghikia
A
Mergia
E
Friebe
A
Eysel
UT
Koesling
D
Mittmann
T
.
2007
.
Long-term potentiation in the visual cortex requires both nitric oxide receptor guanylyl cyclases
.
J Neurosci
 .
27
:
818
823
.
Higo
S
Akashi
K
Sakimura
K
Tamamaki
N
.
2009
.
Subtypes of GABAergic neurons project axons in the neocortex
.
Front Neuroanat
 .
3
:
25
.
Higo
S
Udaka
N
Tamamaki
N
.
2007
.
Long-range GABAergic projection neurons in the cat neocortex
.
J Compar Neurol
 .
503
:
421
431
.
Inagaki
S
Sakanaka
M
Shiosaka
S
Senba
E
Takatsuki
K
Takagi
H
Kawai
Y
Minagawa
H
Tohyama
M
.
1982
.
Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis—I. Forebrain and upper brain stem
.
Neuroscience
 .
7
:
251
277
.
Inoue
R
.
2005
.
TRP channels as a newly emerging non-voltage-gated CA2+ entry channel superfamily
.
Curr Pharm Design
 .
11
:
1899
1914
.
Iritani
S
Fujii
M
Satoh
K
.
1989
.
The distribution of substance P in the cerebral cortex and hippocampal formation: an immunohistochemical study in the monkey and rat
.
Brain Res Bull
 .
22
:
295
303
.
Jones
EG
DeFelipe
J
Hendry
SH
Maggio
JE
.
1988
.
A study of tachykinin-immunoreactive neurons in monkey cerebral cortex
.
J Neurosci
 .
8
:
1206
1224
.
Kara
P
Friedlander
MJ
.
1999
.
Arginine analogs modify signal detection by neurons in the visual cortex
.
J Neurosci
 .
19
:
5528
5548
.
Kawaguchi
Y
.
1995
.
Physiological subgroups of nonpyramidal cells with specific morphological characteristics in layer II/III of rat frontal cortex
.
J Neurosci
 .
15
:
2638
2655
.
Kawaguchi
Y
Kubota
Y
.
1997
.
GABAergic cell subtypes and their synaptic connections in rat frontal cortex
.
Cereb Cortex
 .
7
:
476
486
.
Kawaguchi
Y
Kubota
Y
.
1998
.
Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex
.
Neuroscience
 .
85
:
677
701
.
Kilduff
TS
Cauli
B
Gerashchenko
D
.
2011
.
Activation of cortical interneurons during sleep: an anatomical link to homeostatic sleep regulation?
Trends Neurosci
 .
34
:
10
19
.
Kim
J
Nakajima
K
Oomura
Y
Wayner
MJ
Sasaki
K
.
2009
.
Orexin-A and ghrelin depolarize the same pedunculopontine tegmental neurons in rats: an in vitro study
.
Peptides
 .
30
:
1328
1335
.
Kubota
Y
Shigematsu
N
Karube
F
Sekigawa
A
Kato
S
Yamaguchi
N
Hirai
Y
Morishima
M
Kawaguchi
Y
.
2011
.
Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons
.
Cereb Cortex
 .
21
:
1803
1817
.
Lee
JE
Jeon
CJ
.
2005
.
Immunocytochemical localization of nitric oxide synthase-containing neurons in mouse and rabbit visual cortex and co-localization with calcium-binding proteins
.
Mol Cells
 .
19
:
408
417
.
Le Roux
N
Amar
M
Moreau
AW
Fossier
P
.
2009
.
Roles of nitric oxide in the homeostatic control of the excitation-inhibition balance in rat visual cortical networks
.
Neuroscience
 .
163
:
942
951
.
Ljungdahl
A
Hokfelt
T
Nilsson
G
.
1978
.
Distribution of substance P-like immunoreactivity in the central nervous system of the rat—I. Cell bodies and nerve terminals neuroscience
.
3
:
861
943
.
Lu
B
Su
Y
Das
S
Wang
H
Wang
Y
Liu
J
Ren
D
.
2009
.
Peptide neurotransmitters activate a cation channel complex of NALCN and UNC-80
.
Nature
 .
457
:
741
744
.
Markram
H
Toledo-Rodriguez
M
Wang
Y
Gupta
A
Silberberg
G
Wu
C
.
2004
.
Interneurons of the neocortical inhibitory system
.
Nat Rev
 .
5
:
793
807
.
Min
MY
Shih
PY
Wu
YW
Lu
HW
Lee
ML
Yang
HW
.
2009
.
Neurokinin 1 receptor activates transient receptor potential-like currents in noradrenergic A7 neurons in rats
.
Mol Cell Neurosci
 .
42
:
56
65
.
Nakajima
Y
Tsuchida
K
Negishi
M
Ito
S
Nakanishi
S
.
1992
.
Direct linkage of three tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected Chinese hamster ovary cells
.
J Biol Chem
 .
267
:
2437
2442
.
Nakaya
Y
Kaneko
T
Shigemoto
R
Nakanishi
S
Mizuno
N
.
1994
.
Immunohistochemical localization of substance P receptor in the central nervous system of the adult rat
.
J Compar Neurol
 .
347
:
249
274
.
Penny
GR
Afsharpour
S
Kitai
ST
.
1986
.
Substance P-immunoreactive neurons in the neocortex of the rat: a subset of the glutamic acid decarboxylase-immunoreactive neurons
.
Neurosci Lett
 .
65
:
53
59
.
Perrenoud
Q
Geoffroy
H
Gauthier
B
Rancillac
A
Alfonsi
F
Kessaris
N
Rossier
J
Vitalis
T
Gallopin
T
.
2012
.
Characterization of Type I and Type II nNOS-expressing interneurons in the barrel cortex of mouse
.
Front Neural Circuits
 .
6
:
36
.
Ramsey
IS
Delling
M
Clapham
DE
.
2006
.
An introduction to TRP channels
.
Ann Rev Physiol
 .
68
:
619
647
.
Sakanaka
M
Shiosaka
S
Takatsuki
K
Tohyama
M
.
1983
.
Evidence for the existence of a substance P-containing pathway from the nucleus laterodorsalis tegmenti (Castaldi) to the medial frontal cortex of the rat
.
Brain Res
 .
259
:
123
126
.
Smiley
JF
McGinnis
JP
Javitt
DC
.
2000
.
Nitric oxide synthase interneurons in the monkey cerebral cortex are subsets of the somatostatin, neuropeptide Y, and calbindin cells
.
Brain Res
 .
863
:
205
212
.
Song
CH
Xia
JX
Ye
JN
Chen
XW
Zhang
CQ
Gao
EQ
Hu
ZA
.
2005
.
Signaling pathways of hypocretin-1 actions on pyramidal neurons in the rat prefrontal cortex
.
Neuroreport
 .
16
:
1529
1533
.
Tamamaki
N
Yanagawa
Y
Tomioka
R
Miyazaki
J
Obata
K
Kaneko
T
.
2003
.
Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse
.
J Compar Neurol
 .
467
:
60
79
.
Tomioka
R
Okamoto
K
Furuta
T
Fujiyama
F
Iwasato
T
Yanagawa
Y
Obata
K
Kaneko
T
Tamamaki
N
.
2005
.
Demonstration of long-range GABAergic connections distributed throughout the mouse neocortex
.
Eur J Neurosc
 .
21
:
1587
1600
.
Tomioka
R
Rockland
KS
.
2007
.
Long-distance corticocortical GABAergic neurons in the adult monkey white and gray matter
.
J Compar Neurol
 .
505
:
526
538
.
Turvey
MR
Wang
Y
Gu
Y
.
2010
.
The effects of extracellular nucleotides on [Ca2+]i signalling in a human-derived renal proximal tubular cell line (HKC-8)
.
J Cell Biochem
 .
109
:
132
139
.
Uramura
K
Funahashi
H
Muroya
S
Shioda
S
Takigawa
M
Yada
T
.
2001
.
Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area
.
Neuroreport
 .
12
:
1885
1889
.
Vincent
SR
Satoh
K
Armstrong
DM
Fibiger
HC
.
1983
.
Substance P in the ascending cholinergic reticular system
.
Nature
 .
306
:
688
691
.
Volgushev
M
Balaban
P
Chistiakova
M
Eysel
UT
.
2000
.
Retrograde signalling with nitric oxide at neocortical synapses
.
Eur J Neurosci
 .
12
:
4255
4267
.
Vruwink
M
Schmidt
HH
Weinberg
RJ
Burette
A
.
2001
.
Substance P and nitric oxide signaling in cerebral cortex: anatomical evidence for reciprocal signaling between two classes of interneurons
.
J Compar Neurol
 .
441
:
288
301
.
Wei
JY
Jin
X
Cohen
ED
Daw
NW
Barnstable
CJ
.
2002
.
cGMP-induced presynaptic depression and postsynaptic facilitation at glutamatergic synapses in visual cortex
.
Brain Res
 .
927
:
42
54
.
Xia
J
Chen
X
Song
C
Ye
J
Yu
Z
Hu
Z
.
2005
.
Postsynaptic excitation of prefrontal cortical pyramidal neurons by hypocretin-1/orexin A through the inhibition of potassium currents
.
J Neurosci Res
 .
82
:
729
736
.
Xia
JX
Fan
SY
Yan
J
Chen
F
Li
Y
Yu
ZP
Hu
ZA
.
2009
.
Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels
.
J Physiol Biochem
 .
65
:
125
136
.
Yan
XX
Garey
LJ
.
1997
.
Morphological diversity of nitric oxide synthesising neurons in mammalian cerebral cortex
.
J fur Hirnforschung
 .
38
:
165
172
.
Yan
XX
Jen
LS
Garey
LJ
.
1996
.
NADPH-diaphorase-positive neurons in primate cerebral cortex colocalize with GABA and calcium-binding proteins
.
Cereb Cortex
 .
6
:
524
529
.
Yang
B
Samson
WK
Ferguson
AV
.
2003
.
Excitatory effects of orexin-A on nucleus tractus solitarius neurons are mediated by phospholipase C and protein kinase C
.
J Neurosci
 .
23
:
6215
6222
.
Yoshimura
Y
Ohmura
T
Komatsu
Y
.
2003
.
Two forms of synaptic plasticity with distinct dependence on age, experience, and NMDA receptor subtype in rat visual cortex
.
J Neurosci
 .
23
:
6557
6566
.
Zhang
L
Harlan
RE
.
1994
.
Ontogeny of the distribution of tachykinins in rat cerebral cortex: immunocytochemistry and in situ hybridization histochemistry
.
Brain Res Dev Brain Res
 .
77
:
23
36
.