Adrenergic modulation of NMDA receptors
in prefrontal cortex is differentially regulated
by RGS proteins and spinophilin
Wenhua Liu*, Eunice Y. Yuen*, Patrick B. Allen†, Jian Feng*, Paul Greengard‡, and Zhen Yan*§
*Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214;†Department of
Psychiatry, Yale University School of Medicine, New Haven, CT 06508; and‡Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University,
New York, NY 10021
Edited by Richard L. Huganir, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 6, 2006 (received for review June 2, 2006)
The noradrenergic system in the prefrontal cortex (PFC) is involved
ing memory and mood control. To understand the functions of the
noradrenergic system, we examined the regulation of NMDA
receptors (NMDARs), key players in cognition and emotion, by ?1-
and ?2-adrenergic receptors (?1-ARs, ?2-ARs) in PFC pyramidal
inhibitor reduced the amplitude but not paired-pulse ratio of
NMDAR-mediated excitatory postsynaptic currents (EPSC) in PFC
slices. Specific ?1-AR or ?2-AR agonists also decreased NMDAR-
EPSC amplitude and whole-cell NMDAR current amplitude in dis-
sociated PFC neurons. The ?1-AR effect depended on the phos-
pholipase C–inositol 1,4,5-trisphosphate–Ca2?pathway, whereas
the ?2-AR effect depended on protein kinase A and the microtu-
bule-based transport of NMDARs that is regulated by ERK signal-
ing. Furthermore, two members of the RGS family, RGS2 and RGS4,
were found to down-regulate the effect of ?1-AR on NMDAR
currents, whereas only RGS4 was involved in inhibiting ?2-AR
regulation of NMDAR currents. The regulating effects of RGS2/4 on
?1-AR signaling were lost in mutant mice lacking spinophilin,
which binds several RGS members and G protein-coupled recep-
tors, whereas the effect of RGS4 on ?2-AR signaling was not
altered in spinophilin-knockout mice. Our work suggests that
activation of ?1-ARs or ?2-ARs suppresses NMDAR currents in PFC
neurons by distinct mechanisms. The effect of ?1-ARs is modified
by RGS2/4 that are recruited to the receptor complex by spinophi-
lin, whereas the effect of ?2-ARs is modified by RGS4 independent
adrenergic receptor ? RGS4 ? neuropsychiatric diseases ? G protein-coupled
receptor ? microtubule
sequence information, receptor pharmacology, and signaling
mechanisms. Although ?-ARs are located mainly in the cardio-
vascular system, most ?1- and ?2-AR subtypes (except ?1Cand
?2B) are highly expressed in CNS regions, e.g., prefrontal cortex
(PFC), hippocampus, and brainstem. Norepinephrine, through
the action of ?1-ARs and ?2-ARs, has been implicated in many
key functions of PFC, including working memory and emotional
control (1–3). An aberrant noradrenergic system, complement-
ing altered serotonergic or dopaminergic signaling, contributes
significantly to the pathophysiology of a variety of neuropsychi-
atric diseases associated with PFC dysfunction, such as depres-
sion, anxiety, schizophrenia, and attention-deficit hyperactivity
disorder (4–7). Therefore, modifying noradrenergic signaling
has been considered one of the key therapeutic actions of many
antidepressants, anxiolytic drugs, and antipsychotics (8, 9). To
understand the functional role of ?1- and ?2-ARs, we need to
know their cellular targets that are important for cognition and
emotion. The NMDAR channel has been implicated in normal
cognitive processes and mental disorders (10–12), which makes
drenergic receptors (ARs) can be divided into three main
types: ?1 (?1A–D), ?2 (?2A–C), and ? (1–3), based on
it a potentially important target by which ?1- and ?2-ARs may
regulate PFC functioning.
Activation of ?1-ARs or ?2-ARs Reduces NMDAR-Mediated Currents in
PFC Pyramidal Neurons. We first examined the effect of the
noradrenergic system on NMDAR-mediated excitatory postsyn-
aptic currents (NMDAR-EPSC) evoked by stimulation of syn-
aptic NMDARs in PFC slices. As shown in Fig. 1A, application
of 100 ?M natural neurotransmitter norepinephrine induced a
strong and persistent reduction in the amplitude of NMDAR-
EPSC, whereas in parallel control measurements where no
norepinephrine was administered, NMDAR-EPSC remained
stable throughout the recording. Application of 20 ?M desipra-
mine, a norepinephrine transporter inhibitor, to activate endog-
enous noradrenergic receptors also caused a potent and long-
lasting reduction of the NMDAR-EPSC amplitude (Fig. 1B).
The specific ?1-AR agonist cirazoline (40 ?M) produced a
similar reducing effect, which was blocked by 40 ?M prazosin, a
specific ?1-AR antagonist (Fig. 1C). In a sample of neurons we
tested, norepinephrine, norepinephrine transporter inhibitors,
and ?1-AR agonist all significantly suppressed synaptic
NMDAR-mediated responses (norepinephrine: 31.5 ? 8.5%,
n ? 4; desipramine: 34.2 ? 2.7%, n ? 6; nisoxetine (an
norepinephrine transporter inhibitor, 50 ?M): 36.3 ? 5.3%, n ?
3; cirazoline: 36.2 ? 3.3%, n ? 4). We further examined the
noradrenergic effect on NMDAR-EPSC evoked by paired
pulses, a measure that is sensitive to changes in the probability
of transmitter release (13). As shown in Fig. 1D, application of
desipramine reduced the amplitudes of NMDAR-EPSC trig-
gered by both pulses, but it did not cause a significant change in
the ratio of the paired-pulse facilitation (control: 1.72 ? 0.1;
desipramine: 1.73 ? 0.1, n ? 5). This observation suggests that
activation of noradrenergic receptors in PFC pyramidal neurons
is likely to induce a change in postsynaptic NMDARs rather than
To verify the potential influence of ?1-ARs on NMDARs, we
examined the effect of ?1-ARs on NMDAR-mediated whole-
cell currents in dissociated PFC pyramidal neurons. As shown in
Fig. 1E, application of 40 ?M cirazoline caused a reversible
Author contributions: J.F. and Z.Y. designed research; W.L. and E.Y.Y. performed research;
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: 2APB, 2-aminoethoxydiphenyl borane; AR, adrenergic receptor; EPSC, ex-
citatory postsynaptic currents; GPCR, G protein-coupled receptor; IP3, inositol 1,4,5-
trisphosphate; NMDAR, NMDA receptor; NR2B, NMDAR 2B; PFC, prefrontal cortex; PLC,
phospholipase C; RGS, regulators of G protein signaling.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
November 28, 2006 ?
vol. 103 ?
uation of the ?1-AR effect on NMDARs was lost in spinophilin-
knockout mice, leading to a bigger reduction of NMDAR
currents by ?1-AR agonists in spinophilin-deficient neurons. In
contrast, the RGS4-mediated suppression of the ?2-AR effect
on NMDARs was intact in spinophilin-knockout mice, despite
lin (28, 41). This observation suggests that spinophilin may
selectively regulate Gq-coupled receptor signaling by improving
access of RGS2/4 to the receptors and facilitating the interaction
of RGS2/4 with G?q.
Materials and Methods
Electrophysiological Recordings in Slices.Toevaluatetheregulation
of NMDAR-EPSC in pyramidal neurons located in deep layers
(V–VI) of PFC, slices from young adult (3–5 weeks postnatal)
Sprague–Dawley rats were used for recordings with the whole-
cell voltage-clamp technique (16, 32). The brain slice (300 ?m)
was submerged in oxygenated artificial cerebrospinal fluid
(ACSF) containing 20 ?M CNQX and 10 ?M bicuculline. Cells
were visualized with a water-immersion lens (magnification,
?40) and illuminated with near-IR light. A bipolar stimulating
electrode was positioned ?100 ?m from the neuron under
recording. Before stimulation, cells (clamped at ?70 mV) were
depolarized to 60 mV for 3 s to relieve fully the voltage-
dependent Mg2?block of NMDAR channels. The Clampfit
program (Axon Instruments, Union City, CA) was used to
analyze evoked synaptic activity.
Whole-Cell Recordings in Dissociated Neurons. Acutely dissociated
PFC pyramidal neurons were prepared by using procedures
described in refs. 16 and 32. Spinophilin-knockout mice were
produced as detailed before (30). Recordings of whole-cell
ligand-gated ion channel currents used standard voltage-clamp
techniques (16). The cell membrane potential was held at ?60
mV. NMDA (100 ?M) was applied for 2 s every 30 s. Drugs
were applied with a gravity-fed sewer-pipe system. The array
of application capillaries was positioned near the cell under
study. Solution changes were effected by the SF-77B fast-step
solution stimulus delivery device (Warner Instrument Co.,
To test the impact of RGS proteins on GPCR signaling, the
antibody raised against a peptide mapping at the highly specific
N terminus of RGS2 or RGS4 (Santa Cruz Biotechnology,
Santa Cruz, CA) was dialyzed into neurons through the patch
electrode for 10 min before electrophysiological recordings
started. Data analyses were performed with AxoGraph (Axon
Instruments) and Kaleidagraph (Albeck Software, Reading,
PA). ANOVA tests were performed to compare the differen-
tial degrees of current modulation between groups subjected
to different treatments.
We thank Xiaoqing Chen for technical support. This work was supported
by National Institutes of Health Grants MH63128 and NS48911 and a
National Alliance for Research on Schizophrenia and Depression In-
dependent Investigator Award (to Z.Y.).
1. Birnbaum S, Gobeske KT, Auerbach J, Taylor JR, Arnsten AF (1999) Biol
2. Schramm NL, McDonald MP, Limbird LE (2001) J Neurosci 21:4875–4882.
3. Franowicz JS, Kessler LE, Borja CM, Kobilka BK, Limbird LE, Arnsten AF
(2002) J Neurosci 22:8771–8777.
4. Dubini A, Bosc M, Polin V (1997) J Psychopharmacol 11:S17–S23.
5. Charney DS, Redmond DE, Jr (1983) Neuropharmacology 22:1531–1536.
6. Arnsten AF (2004) Psychopharmacology 174:25–31.
7. Russell V, Allie S, Wiggins T (2000) Behav Brain Res 117:69–74.
8. Ninan PT (1999) J Clin Psychiatry 60(Suppl 22):12–17.
9. Hertel P, Fagerquist MV, Svensson TH (1999) Science 286:105–107.
10. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) Pharmacol Rev 51:7–61.
11. Jentsch JD, Roth RH (1999) Neuropsychopharmacology 20:201–225.
12. Tsai G, Coyle JT (2002) Annu Rev Pharmacol Toxicol 42:165–179.
13. Manabe T, Wyllie DJ, Perkel DJ, Nicoll RA (1993) J Neurophysiol 70:1451–1459.
14. Harrison JK, Pearson WR, Lynch KR (1991) Trends Pharmacol Sci 12:62–67.
15. Wenthold RJ, Prybylowski K, Standley S, Sans N, Petralia RS (2003) Annu Rev
Pharmacol Toxicol 43:335–358.
16. Yuen EY, Jiang Q, Chen P, Gu Z, Feng J, Yan Z (2005) J Neurosci 25:
17. Yuen EY, Jiang Q, Feng J, Yan Z (2005) J Biol Chem 280:29420–29427.
18. Vries LD, Zheng B, Fischer T, Elenko E, Farquhar MG (2000) Annu Rev
Pharmacol Toxicol 40:235–271.
19. Li Y, Hashim S, Anand-Srivastava MB (2005) Cardiovasc Res 66:503–511.
20. Diverse-Pierluissi MA, Fischer T, Jordan JD, Schiff M, Ortiz DF, Farquhar
MG, De Vries L (1999) J Biol Chem 274:14490–14494.
21. Ingi T, Krumins AM, Chidiac P, Brothers GM, Chung S, Snow BE, Barnes CA,
Lanahan AA, Siderovski DP, Ross EM, et al. (1998) J Neurosci 18:7178–7188.
22. Erdely HA, Lahti RA, Lopez MB, Myers CS, Roberts RC, Tamminga CA,
Vogel MW (2004) Eur J Neurosci 19:3125–3128.
23. Chowdari KV, Mirnics K, Semwal P, Wood J, Lawrence E, Bhatia T,
Deshpande SN, BK T, Ferrell RE, Middleton FA, et al. (2002) Hum Mol Genet
24. Williams NM, Preece A, Spurlock G, Norton N, Williams HJ, McCreadie RG,
Buckland P, Sharkey V, Chowdari KV, Zammit S, et al. (2004) Biol Psychiatry
25. Morris DW, Rodgers A, McGhee KA, Schwaiger S, Scully P, Quinn J, Meagher
D, Waddington JL, Gill M, Corvin AP (2004) Am J Med Genet Neuropsychiatr
26. Harrison PJ, Owen MJ (2003) Lancet 361:417–419.
27. Smith FD, Oxford GS, Milgram SL (1999) J Biol Chem 274:19894–19900.
28. Richman JG, Brady AE, Wang Q, Hensel JL, Colbran RJ, Limbird LE (2001)
J Biol Chem 276:15003–15008.
29. Wang X, Zeng W, Soyombo AA, Tang W, Ross EM, Barnes AP, Milgram SL,
Penninger JM, Allen PB, Greengard P, et al. (2005) Nat Cell Biol 7:405–411.
30. Feng J, Yan Z, Ferreira AB, Tomizawa K, Liauw JA, Zhuo M, Allen PB,
Ouimet CC, Greengard P (2000) Proc Natl Acad Sci USA 97:9287–9292.
31. Gerber U (2002) Neuropharmacology 42:587–592.
32. Wang X, Zhong P, Gu Z, Yan Z (2003) J Neurosci 23:9852–9861.
33. Hallett PJ, Spoelgen R, Hyman BT, Standaert DG, Dunah AW (2006)
J Neurosci 26:4690–4700.
34. Berman DM, Wilkie TM, Gilman AG (1996) Cell 86:445–452.
35. Zheng B, De Vries L, Gist Farquhar M (1999) Trends Biochem Sci 24:411–414.
36. Huang C, Hepler JR, Gilman AG, Mumby SM (1997) Proc Natl Acad Sci USA
37. Hepler JR, Berman DM, Gilman AG, Kozasa T (1997) Proc Natl Acad Sci USA
38. Yan Y, Chi PP, Bourne HR (1997) J Biol Chem 272:11924–11927.
39. Zeng W, Xu X, Popov S, Mukhopadhyay S, Chidiac P, Swistok J, Danho W,
Yagaloff KA, Fisher SL, Ross EM, et al. (1998) J Biol Chem 273:34687–
40. Xu X, Zeng W, Popov S, Berman DM, Davignon I, Yu K, Yowe D, Offermanns
S, Muallem S, Wilkie TM (1999) J Biol Chem 274:3549–3556.
41. Wang Q, Zhao J, Brady AE, Feng J, Allen PB, Lefkowitz RJ, Greengard P,
Limbird LE (2004) Science 304:1940–1944.
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