Localization and Function of Nk3 Subtype Tachykinin

Neuroscience 156 (2008) 987–994

LOCALIZATION AND FUNCTION OF3 SUBTYPENK TACHYKININ RECEPTORS OF LAYER V PYRAMIDAL NEURONS OF THE GUINEA-PIG MEDIAL PREFRONTAL CORTEX

M. A. SIMMONS,a,b* C. D. SOBOTKA-BRINERa bral cortex. In layer V pyramidal neurons of the medial pre- a AND A. M. MEDD frontal cortex, activation of the3 receptor NK system plays an aDepartment of Neuroscience Biology, AstraZeneca Pharmaceuticals, excitatory role in modulating synaptic transmission. © 2008 Wilmington, DE 19850, USA IBRO. Published by Elsevier Ltd. All rights reserved. b Department of Integrative Medical Sciences, Northeastern Ohio UniKey- words: neurokinin B, autoradiography, receptor binding, versities College of Medicine, 4209 State Route 44, P.O. Box electrophysiology95, . Rootstown, OH 44272-0095, USA

The tachykinins are a family of peptides that primarily act Abstract—The NK3 subtype of tachykinin receptor has been implicated as a modulator of synaptic transmission in severaas l neurotransmitters or neuromodulators in the CNS and brain regions, including the cerebral cortex. The localizatioperipheraln nervous system. The tachykinin family is de- and expression of 3 receptorsNK within the brain vary from fined characteristically by the conserved carboxy terminal species to species. In addition, the pharmacology3 of sequenceNK -Phe-Xaa-Gly-Leu-Met-NH2, where Xaa is a receptor-specific antagonists shows significant species vari- variable amino acid. The endogenous mammalian tachy- ability. Among commonly used animal models, the pharma- kinins are substance P (SP), neurokinin A (NKA) and cology of the guinea-pigreceptor NK most closely resem- 3 neurokinin B (NKB) (Beaujouan et al., 2004). bles that of the human3 receptor. NK Here, we provide anatomical localization studies, receptor binding studies, and These endogenous peptides act preferentially at three tachykinin receptors, termed the NK ,NK and NK sub- studies of the electrophysiological effects 3 receptoof NKr 1 2 3 ligands of guinea-pig cortex using two commercially availtype- tachykinin receptors. The NK1 receptor exhibits a able ligands, the 3NK receptor peptide analog agonist senk- higher affinity for the binding of SP than for NKA or NKB tide, and the quinolinecarboxamide receptorNK antagonist 3 (Krause et al., 1994). The NK2 receptor binds NKA with the SB-222,200. highest affinity (Buck and Shatzer, 1998). For the NK3 Saturation binding studies with membranes isolated from receptor, the order of agonist potency is NKBϾNKAϾSP guinea-pig cerebral cortex showed saturable binding consistent with a single high affinity site. Autoradiographic studie(Laufers et al., 1986); however, all three ligands are capable revealed dense specific binding in layers II/III and layer ofV activatingof the receptor. Like adrenergic, opioid, taste the cerebral cortex. For electrophysiological studies, brain and other G-protein-coupled receptors, the deduced amino slices were prepared from prefrontal cortex of 3- to 14-dayacid- sequence of the tachykinin receptors is consistent old guinea pigs. Whole cell recordings were made fromwith laye ther seven transmembrane domain structure proposed ؉ V pyramidal neurons. In current clamp mode -con-with a K for this family of receptors. taining pipette solution, senktide depolarized the pyramidal The pharmacology of tachykinin receptors has been neurons and led to repetitive firing of action potentials. In voltage clamp mode with ؉-containinga Cs pipette solution, well characterized in pain, and the roles of tachykinin senktide application produced an inward current and a receptorcon- ligands in lacrimation, salivation, and smooth centration-dependent enhancement of the amplitude and thmusclee contraction have been well documented (Regoli et frequency of spontaneous excitatory postsynaptic poten- al., 1994). Only recently has evidence emerged regarding tials. The glutamatergic nature of these events was demontheir- role in psychopharmacology. Interest in the role of strated by block by glutamate receptor antagonists. TheNK ef-receptors in the brain has been piqued by the devel- fects of senktide were blocked by SB-222,200,recep an- NK 3 3 opment of the brain permeant, potent, NK -selective com- tor antagonist. 3 Taken together, these results are consistent with a funcpounds- osanetant (Kamali, 2001) and talnetant (Evange- lista, 2005). tional role for 3NK receptors located on neurons in the cere- Studies of the function and pharmacology of NK re- *Correspondence to: M. A. Simmons, Department of Integrative Med- 3 ical Sciences, Northeastern Ohio Universities College of Medicineceptors, have proven to be complicated in several respects. 4209 State Route 44, P.O. Box 95, Rootstown, OH 44272-0095,One USA. of the difficulties is that the distribution of NKB ex- Tel: ϩ1-330-325-6525; fax:ϩ1-330-325-5912. pressing neurons and of NK3 receptors in the brain varies E-mail address: [email protected] (M. A. Simmons). among commonly used animal models (Massi et al., 2000; Abbreviations: ACSF, artificial cerebrospinal fluid;AP5, D-(Ϫ)-2-amino- Langlois et al., 2001) and between some of these models 5-phosphonopentanoic acid;B max, maximal binding; EPSC, excitatory postsynaptic current;Kd, dissociation constant of the radioli- and primates (Nagano et al., 2006). The pharmacology of gand; K , inhibition constant; NBQX, 2,3-dioxo-6-nitro-1,2,3,4-tet- i small molecule antagonists also shows interspecies vari- rahydrobenzo[f]quinoxaline-7-sulfonamide; NKA, neurokinin A; NKB, ation. In general, tachykinin receptor antagonists show neurokinin B; NK3,NK3 subtype tachykinin receptor; RT, room temperature; SP, substance P. selectivity for either rat and mouse or guinea-pig and gerbil 0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.08.037

987 988 M. A. Simmons et al. / Neuroscience 156 (2008) 987–994

NK3 receptors (Chung et al., 1995; Emonds-Alt et al., radioligand. The reaction was allowed to equilibrate for 1.5 h at RT 2002; Sarau et al., 1997, 2000). The pharmacology of and harvested onto GF/B filter plates presoaked in 0.5% BSA. Filters were washed with cold wash buffer (0.02 M Tris–HCl, pH 7, these compounds at the human NK3 receptor is more accurately replicated in guinea-pig and gerbil models than 0.02% BSA), dried and counted on a Topcount instrument (Perkin Elmer, Waltham, MA, USA). For competition binding experiments, in rat and mouse (Regoli et al., 1994). compounds were dissolved in DMSO and serially diluted 1:3. Two Given the relative receptor distribution, pharmacology microliters of each compound concentration was transferred to a and potential utility of the guinea pig as an animal model, 96-well 500 ␮l polypropylene U-bottom plate for final assay con- ␮ 125 7 we have examined the localization and function of NK3 centrations in the range of 10 M to 0.17 pM. The [ I]-MePhe - receptors in the cerebral cortex of the guinea pig. Here, we NKB ligand was used at concentrations of 0.02–0.07 nM. The provide anatomical localization studies, ligand binding remainder of the assay was as described above. studies, and studies of the physiological effects of NK Saturation binding data were analyzed by non-linear regres- 3 sion using GraphPad Prism software to yield K (dissociation receptor activation and blockade. d constant of the radioligand) and Bmax (maximal binding). For competition binding experiments, inhibition constants (Ki) were calculated using the Cheng-Prusoff equation and XL-Fit software. EXPERIMENTAL PROCEDURES Electrophysiology Animals Coronal slices were prepared from the frontal pole of the brain of Hartley guinea pigs (Charles River Laboratories, Wilmington, MA, guinea-pig neonates of either sex at 2–15 days of age, weighing USA) were used in these experiments. Animals were housed in a 40–200 g. Animals were decapitated after induction of deep an- temperature-controlled vivarium with free access to food and wa- esthesia with isoflurane. The brain was rapidly removed and im- ter. Animal experiments were approved by the AstraZeneca mersed in ice cold artificial cerebrospinal fluid (ACSF) gassed with IACUC and carried out in accordance with the National Institutes 95% O2–5% CO2. The brain was cut in half with a single coronal of Health Guide for the Care and Use of Laboratory Animals (NIH cut. The front half of the brain was mounted anterior side up in an Publications No. 80–23). The number of animals used and their ice cold gassed bath for slicing. Slices (200 ␮m) were cut using a suffering were minimized. Vibratome (Leica Microsystems). The first four slices were dis- carded. Subsequent slices were stored in a warm (30 °C) holding

chamber gassed with 95% O2–5% CO2. Slices were allowed to Autoradiography equilibrate for at least 1 h before recording. Individual slices were transferred to a recording chamber which was perfused with Naïve adult male Hartley guinea pigs were killed by decapitation gassed ACSF at 2.0 ml/min. Whole cell recordings of single neu- without anesthesia. Brains were removed and frozen in cooled rons were conducted at RT, ϳ23 °C, using an Axopatch 200C isopentane (Ϫ35 °C). Sections (20 ␮m) were cut in a cryostat at (Hamden, CT, USA) patch clamp amplifier. Electrodes with resis- Ϫ15 °C, mounted onto gelatin-coated slides, rapidly dried and tances of 4–8 M⍀ were filled with pipette solution. Drugs were stored at Ϫ80 °C until use. On the day of the experiment, slides applied by bath perfusion. were warmed to room temperature (RT, ϳ22–25 °C) and prein- The composition of solutions used for these experiments is cubated in 50 mM Tris–HCl, 0.005% polyethylamine, pH 7.4. To determine total binding the sections were incubated in the same listed below, in mM unless stated otherwise. ACSF: 130 NaCl, 3.5 3 KCl, 1.5 CaCl2, 1.5 MgSO4, 24 NaHCO3, 1.25 NaH2PO4,10 buffer with 3 mM MnCl2, 0.02% BSA, and 1 nM [ H] SB-222,200 (in house, specific activity 34.7 Ci/mmol) for 60 min. Non-specific glucose. K-containing pipette solution: 140 K gluconate, 2 MgSO4, binding was determined by adding 10 ␮M of talnetant (60 min, 0.1 CaCl2, 0.1 GTP, 2.0 ATP, 10 Hepes, 1 EGTA, pH 7.25. RT). Slides were rinsed, dried and then exposed to a phosphoim- Cs-containing pipette solution: 120 Cs methanesulfonate, 2 ager screen for 5 days. The screens were run on a Cyclone MgCl2, 0.3 GTP, 2.0 ATP, 10 Hepes, pH 7.35. Phosphoimager (PerkinElmer) and images were imported into Excitatory postsynaptic currents (EPSCs) were analyzed by Photoshop and are unretouched except for the lettering. the Minanalysis program. EC50 values were calculated using the ϭ ϩ logistic equation: y Emax·x/(EC50 x), where x is drug concentration. Of the neurons tested, 90% responded to senktide. Data Receptor binding were obtained from 52 cells in brain slices from 15 different animals. Data are expressed as the meanϮS.E.M. Naïve adult male Hartley guinea pigs were killed by decapitation without anesthesia, the brains were removed and frontal cortical Chemicals tissue was carefully dissected for membrane preparation. The tissue was homogenized in 20 mM Tris pH 7.4 plus protease [125I]-MePhe7-NKB, specific activity 2200 Ci/mmol, was pur- inhibitors and centrifuged at 1000ϫg for 5 min. The supernatant chased from PerkinElmer; NKA and SP were purchased from was transferred to a new tube and centrifuged at 100,000ϫg for Sigma (St. Louis, MO, USA); senktide, D-(Ϫ)-2-amino-5-phosphono- 30 min to pellet cellular membranes. The pellet was resuspended pentanoic acid (AP5) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydroben- in 20 mM Tris pH 7.4 buffer and protein concentration determined zo[f]quinoxaline-7-sulfonamide (NBQX) from Tocris (Ellisville, using a standard protein determination kit. MO, USA), and N-MePhe7-NKB from Bachem. SB-222,200 and Membranes were resuspended in assay buffer (20 mM osanetant were synthesized in house. All other chemicals were

Hepes, pH 8.4, 5 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.5 mM purchased from Sigma or Fisher Scientific (Pittsburgh, PA, USA). ␮ MnCl2, 0.1% BSA) containing thiorphan (10 M), homogenized For the electrophysiological experiments, senktide stock solution briefly and resuspended at 0.278 mg/ml for a final concentration of at a concentration of 5 mM and SB-222,200 at a concentration of 50 ␮g/well in 180 ␮l. For saturation binding experiments, 20 ␮l 10 mM were prepared in DMSO. At the highest concentration of [125I]-MePhe7-NKB was added to triplicate wells at final concen- senktide tested (2 ␮M), the concentration of DMSO would be trations ranging from 0.003 nM to 0.75 nM. Non-specific binding 0.04%. To test for effects of this vehicle, 0.05% DMSO was was defined by including cold N-MePhe7-NKB (Bachem, Tor- applied to brain slices. No change in current or sEPSCs was rance, CA, USA), osanetant, or senktide (1–10 ␮M) with the observed (nϭ4 neurons from two different slices). M. A. Simmons et al. / Neuroscience 156 (2008) 987–994 989

RESULTS NK3 receptor binding Both saturation and competition studies have been con- NK3 receptor localization ducted to characterize the NK3 receptor binding sites in 3 NK3 receptors were labeled by autoradiography with [ H]- guinea-pig cerebral cortex. For saturation binding studies,

labeled SB-222,200 (1 nM), an NK3 receptor antagonist membranes were isolated from guinea-pig frontal cortex. 125 7 (Sarau et al., 2000). The speciﬁcity of the binding was [ I]-MePhe -NKB, a selective NK3 receptor agonist, determined by incubation with an excess of unlabeled showed saturable binding consistent with a single high ␮ talnetant (10 M), a structurally related NK3 antagonist. afﬁnity site (Fig. 2). Non-linear regression analysis of

Except for an intense band of staining that outlined the ﬁve independent experiments yielded a mean Kd of Ϯ Ϯ cerebral ventricles, labeling was conﬁned to the cerebral 0.073 nM 0.02 and a Bmax of 11 2.71 fmol/mg protein.

cortices. Dense speciﬁc bands of labeling were observed To further characterize this binding site, Ki values in layers II/III and in layer V of the cerebral cortex, including were obtained from competition binding studies with

medial prefrontal areas (Fig. 1). NK1,NK2 and NK3 receptor-selective ligands versus

3 Fig. 1. Localization of NK3 receptors in guinea-pig cerebral cortex by autoradiography with [ H]-SB-222,200. (A) Horizontal section at mid level showing pattern of [3H]-SB-222,200 labeling. (B) Sagittal section at the midline showing pattern of [3H]-SB-222,200 labeling. Prominent labeling was observed in the area comprising layers II/III of the cortex as well as in layer V. Binding was also observed lining the third and fourth ventricles. (C, D) Block of [3H]-SB-222,200 labeling in presence of 10 ␮M talnetant. The scale barϭ5 mm in A and refers to all panels. 990 M. A. Simmons et al. / Neuroscience 156 (2008) 987–994

Fig. 2. Saturation binding of [125I]-MePhe7NKB to membranes from guinea-pig frontal cortex. The graph shows the results from triplicate Fig. 3. Localization and morphology of cells used for electrophysiol- determinations of a single experiment. The inset shows a Scatchard ogy. The left panel shows a high magnification of a biocytin-filled layer plot of the data fit by non-linear regression. This was one of five V pyramidal neuron. The position of this cell in the brain slice is independent experiments which yielded a mean Kd of 0.073 nM and illustrated in the right panel. The box is 875 ␮mϫ490 ␮m. The location Bmax of 11 fmol/mg protein. of this cell is at the most dorsal end of the region from which cells were chosen for electrophysiology. All cells chosen were visually identified 125 7 I-MePhe NKB (Table 1). The NK3 receptor-selective as layer V pyramidal neurons from this medial portion of the prefrontal ligands MePhe7-NKB, senktide, and SB-222,200 all com- cortex. 125 7 peted for I-MePhe NKB binding with Kis in the low nM Senktide effects on membrane current. In voltage range. The NK agonist, SP, and the NK agonist, NKA, ϭϪ ϩ 1 2 clamp mode (VH 70 mV) with a Cs -containing pipette were less potent. These binding properties are character- solution, senktide produced an inward current and a dra- istic of the NK3 receptor. matic increase in EPSCs, as shown in Fig. 5A and B. The amplitude of the inward current produced was dependent Electrophysiology on the senktide concentration (Fig. 5C). The concentration Whole cell recordings were made from single neurons in response curve for inward current was well fit by the logis- slices of prefrontal cortex from guinea-pig brains. Record- tic equation with a Hill coefficient of 1. The EC50 from this ings were made from pyramidal neurons located in layer V curve was 836 nM. of the medial portion of the slice. An example of a biocytin- Effects of senktide on EPSCs. The effect of senktide filled cell and its location in the slice are shown in Fig. 3.To was to increase both the amplitude and the frequency of avoid confounding the data with desensitization to agonist EPSCs. Histograms of the number of events observed for or prolonged effects of antagonist, a recording was made 5 min periods under control conditions and in the presence from only a single cell in each brain slice exposed to drug. of senktide (500 nM) show the increase in EPSC frequency Effects of senktide on membrane potential. In current (Fig. 5D). Plots of cumulative EPSC amplitude under con- clamp mode with a Kϩ-containing pipette solution, senk- trol conditions and in the presence of senktide reveal that tide depolarized the pyramidal neurons, as illustrated in there were a greater proportion of larger amplitude events Fig. 4. The response of the neuron shown in the figure was in the presence of senktide (Fig. 5E). typical in that the depolarization in response to senktide was sufficient to lead to repetitive firing of action potentials Pharmacological analysis of the senktide response and that the action potentials persisted for several minutes Concentration-response curve for senktide effect on following washout of senktide. A similar response was EPSCs. Since senktide increased both the amplitude observed in four other neurons. and frequency of EPSCs the response was quantified by integrating the amplitude of the EPSCs as a function of Table 1. Competition binding to guinea-pig frontal cortex membranes time to obtain the charge transferred and to fully account for the effect of the drug. This value was measured for the Compound Mean K ϮS.E.M. (nM) n i EPSCs that occurred during stable 1 min periods during MePhe-NKB 0.35Ϯ0.13 3 exposure to various concentrations of drug. These data Senktide 6.37Ϯ1.3 3 are plotted as the total area of EPSCs in fC versus senk- SB-222200 1.44Ϯ0.89 3 tide concentration (Fig. 6C). Unlike the concentration re- SP 435Ϯ86 3 sponse curve for inward current, the concentration re- NKA 1117Ϯ215 3 sponse curve for EPSC activation did not have an appar- ent sigmoidal shape and could not be fit by the logistic Ki values were obtained from competition binding studies versus 125I-MePhe7NKB. Unlabeled MePhe7-NKB, senktide, and SB-222,200 equation. Based on a peak effect of senktide of 11,000 fC/min, the 50% effect was obtained at a senktide concen- all competed for binding with Kis in the low nM range, while the NK1 ϳ agonist, SP, and the NK2 agonist, NKA, were less potent. tration of 200 nM. M. A. Simmons et al. / Neuroscience 156 (2008) 987–994 991

Fig. 4. Effect of senktide on membrane potential. The electrode was filled with Kϩ-containing solution. After obtaining the whole cell recording configuration, the amplifier was switched to current clamp mode. Application of senktide (200 nM) as indicated by the bar led to depolarization and the generation of repeated action potentials. The firing gradually slowed following washout of the drug.

Block by NK3 antagonist and glutamate antagonists. (SR-142801) (Langlois et al., 2001). Both agonist and

The effects of senktide were blocked by the NK3 antago- antagonist showed the same pattern of distribution with nist SB-222,200. When added during a senktide response, particularly high densities of binding described in the mid- SB-222,200 attenuated the inward current and EPSC ac- cortical layer and deep cortical layer, corresponding to our tivation produced by senktide (Fig. 6A). When the slice layer II/III and layer V binding. A similar pattern of staining

was pre-incubated with SB-222,200 for 15 min, the effect was seen with an NK3 receptor antibody (Yip and Chahl, of senktide was almost completely blocked (Fig. 6B). The 2001). There appears to be a similar cortical distribution of average effect of SB-222,200 is plotted on the senktide NK3 receptors in mouse (Duarte et al., 2006), rat (Dam et concentration response curve (square on Fig. 6C). al., 1990; Langlois et al., 2001; Saffroy et al., 2003; Shugh- The glutamatergic nature of the EPSCs induced by rue et al., 1996; Mileusnic et al., 1999), gerbil (Langlois et senktide is demonstrated by block by a 5 min preincuba- al., 2001), monkey (Nagano et al., 2006) and human (Mile- tion with the combination of the NMDA receptor antagonist usnic et al., 1999; Tooney et al., 2000), although there are ␮ AP5 (50 M) and the AMPA receptor antagonist NBQX species differences in NK receptor localization in other ␮ 3 (10 M) (Fig. 7). These concentrations have been shown brain areas. to block glutamatergic EPSCs in previous studies (Wuarin The NK3 receptor is distinguished by its preference for and Dudek, 1991; Sheardown, 1993). This result was re- NKB-like ligands over SP and NKA, which preferentially peated in ﬁve other cells. The inward current in response to bind to NK1 and NK2 receptors, respectively. In addition, senktide was still observed in the presence of AP5 and SB-222,200 is a selective NK antagonist. The receptor NBQX. The increase in EPSCs was blocked. 3 binding data indicate the presence of an NK3 receptor in guinea-pig cerebral cortical membranes and are consistent DISCUSSION with previous studies of NK3 receptor binding properties. Suman-Chauhan et al., 1994, observed a K of 0.362 nM In this study we have shown that the NK3 receptor system d 125 7 has a strong excitatory effect on layer V pyramidal neurons for I-MePhe NKB binding to guinea-pig cortical mem- of guinea-pig medial prefrontal cortex. Despite the fact that branes. This is higher than the value of 0.073 nM determined here, but within the expected range of error. Both the role of NK3 receptors in modulating cortical function is of considerable interest, the effects of NK3 receptor ago- their saturation data and competition data are about two to nists and antagonists in guinea-pig cortex have not been three times higher than our values for all of the ligands previously shown. tested. Furthermore, the relative potency proﬁles for differ- The pattern of receptor autoradiographic binding of ent ligands reported by Suman-Chauhan et al. are the SB-222,200 shown here is consistent with previous studies same as reported here.

of NK3 receptor autoradiography with other ligands includ- NK3 receptors have been shown to modulate release ing the agonist senktide and the antagonist osanetant of amine neurotransmitters at several sites in guinea-pig 992 M. A. Simmons et al. / Neuroscience 156 (2008) 987–994

Fig. 5. Effect of senktide on membrane current of a layer V pyramidal neuron. (A) An inward current and a large increase in EPSCs were produced by senktide (500 nM) application, as indicated by the bar. Time calibration ϭ 1 min. (B) At a faster time base, a few small EPSCs are observed in the control condition, prior to senktide application. The response to senktide is shown in the bottom tracing. (C) The amplitude of inward current in ϭ response to various concentrations of senktide is shown. n 3–5 for each point. Fitting this data to the logistic equation yielded an EC50 of 836 nM ϭ Ϯ (pEC50 6.08 0.31). (D) EPSC amplitude histograms before and after senktide (500 nM) application to a single neuron are shown. EPSC amplitudes were measured during 5 min periods prior to (left panel labeled “Control”) and during application of 500 nM senktide (right panel labeled “Senktide”). Comparison of these plots clearly illustrates a dramatic increase in the frequency of EPSCs in the presence of senktide. (E) Cumulative frequency plots of EPSC amplitude recorded from a single neuron before and after senktide application. In the presence of senktide, larger-amplitude events make up a larger fraction of the EPSCs. brain. Injection of senktide into the substantia nigra leads recording from layer V pyramidal neurons in medial ento- to increased dopamine release in the striatum, injection rhinal cortex of the rat revealed that senktide (50–500 nM) into the ventral tegmental area increases dopamine re- increased glutamatergic EPSCs. This effect was blocked lease in the nucleus accumbens and injection into the by osanetant. septal area increase hippocampal release of acetylcholine The data presented here are consistent with the

(Marco et al., 1998). The role of NK3 receptors in regulat- model presented by Stacey et al. (2002) (see Fig. 8 of ing midbrain dopaminergic neurons is further supported by their paper). In this model, the effect of activating NK3 studies showing a block of activation by osanetant receptors is to excite layer V pyramidal neurons. At the

(Gueudet et al., 1999). Our data show that NK3 receptors cell from which the recording is being made, this is also modulate synaptic transmission by direct actions in observed as a depolarization and increased spiking in prefrontal cortex. current clamp mode and an inward current under voltage Previous electrophysiological studies have examined clamp. This leads to increased glutamate release onto

NK3 receptor-mediated effects in rat entorhinal cortex (Sta- neighboring pyramidal neurons via recurrent axon col- cey et al., 2002) and gerbil cingulate cortex (Rekling, laterals. Thus, the increase in EPSCs observed after

2004). In the latter study, current clamp recordings from NK3 receptor activation is from neighboring pyramidal layer V pyramidal neurons revealed a depolarization and neurons that have been depolarized and excited by increase in excitatory postsynaptic potentials in response senktide and are now releasing glutamate onto the cell to senktide (500 nM). In the former study, voltage clamp being monitored. M. A. Simmons et al. / Neuroscience 156 (2008) 987–994 993

Fig. 6. Pharmacological analysis of the effects of senktide. (A) The NK3 antagonist SB-222,200, applied during a response to senktide (200 nM) resulted in a decreased inward current and a partial block of the increase in EPSCs produced by senktide. This effect was observed in four neurons tested. (B) When SB-222,200 was applied to the slice for 15 min prior to and during the senktide application, senktide was without effect. This effect was repeated in a total of four cells. The average senktide effect in these cells in plotted in C. (C) Concentration-response curve for senktide. nϭ3–6 For each point.

The NK3 receptor system has been shown to have a 2000). Animal studies have shown NK3 involvement in variety of psychopharmacological actions (Massi et al., anxiety, depression and reward systems. Human clinical

Fig. 7. Glutamatergic and synaptic nature of the EPSCs. The glutamate receptor antagonists AP5 (50 ␮M) and NBQX (10 ␮M) were applied to the slice for 5 min. Senktide (200 nM) was then added (top panel). Senktide produced an inward current but the increase in EPSCs was blocked. After a 10 min washout of AP5 and NBQX, reapplication of senktide leads to increased EPSCs. 994 M. A. Simmons et al. / Neuroscience 156 (2008) 987–994

studies have been conducted with two NK3 receptor an- Marco N, Thirion A, Mons G, Bougault I, Le Fur G, Soubrié P, Stein- tagonists, osanetant and talnetant. The results have berg R (1998) Activation of dopaminergic and cholinergic neuro- transmission by tachykinin NK3 receptor stimulation: an in vivo shown that treatment with an NK3 antagonist leads to improvements in the positive symptoms of schizophrenia microdialysis approach in guinea pig. Neuropeptides 32:481–488. Massi M, Panocka I, de Caro G (2000) The psychopharmacology of (Spooren et al., 2005). The site and mechanism of this tachykinin NK-3 receptors in laboratory animals. Peptides 21: effect are not fully understood. Given the proposed role of 1597–1609. prefrontal cortical areas in psychosis, our data suggest that Mileusnic D, Lee JM, Magnuson DJ, Hejna MJ, Krause JE, Lorens JB, at least some of the positive actions of NK3 antagonists in Lorens SA (1999) Neurokinin-3 receptor distribution in rat and human brain: an immunohistochemical study. Neuroscience schizophrenia could be related to a block of NK3 receptors in the cortex. 89:1269–1290. Nagano M, Saitow F, Haneda E, Konishi S, Hayashi M, Suzuki H (2006) Distribution and pharmacological characterization of pri- Acknowledgments—We thank our colleagues at AstraZeneca for mate NK-1 and NK-3 tachykinin receptors in the central nervous supporting this work. Of particular note are Ed Johnson for ar- system of the rhesus monkey. Br J Pharmacol 147:316–323. ranging this collaboration; and Craig Moore and Jeff Smith for Regoli D, Boudon A, Fauchére JL (1994) Receptors and antagonists providing superb guidance for this project. 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(Accepted 19 August 2008) (Available online 23 August 2008)