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Introduction
l-Stepholidine (SPD), isolated from the Chinese herb
Stephania, is a tetrahydroprotoberberine alkaloid. Previous studies
in neuropharmacology, neurochemistry, electro-physiology, and behavioral experiments have demonstrated that SPD not
only acts as a partial D1
agonist[1_5], but also as a full
D2 antagonist[2,5,6]. SPD is the first known drug to
possess the dual properties toward the dopamine (DA) receptor. Clinical trials have implicated that SPD is effective in the treatment of both
positive and negative syndromes in
schizophrenia[7]. Furthermore, SPD was also found to enhance the antipsychiatry
effectiveness of other drugs and reduce tardive
dyskinesia[8]. However, the mechanism of SPD's anti-schizophrenia has not
been explored.
The prelimbic cortex (PL) is well known as a critical structure in cognitive function. It is also believed that functional
abnormalities in PL are associated with many neuropsychiatric disorders and symptoms including negative symptoms and
cognitive impairment in
schizophrenia[9_12]. The functional activity of the PL is mainly mediated by the intrinsic neurons and
their interactions with other brain regions. The dopaminergic project from the ventral tegmental area (VTA) to the PL
pyramidal cells located in layers V_VI is of
particular importance for modulating PL
function[13_19]. Particularly layers V
contains a high density of D1
receptors[20]. Furthermore, many studies documented the importance of
PL DA transmission via D1 receptors for optimal PL
function[21,22]. D1 receptor
stimulation is generally believed to promote
N-methyl-D-aspartate (NMDA) receptor function on the excitable neurons via the
medium spiny neuronal transmission, further supporting the
hypothesis that DA transmission at D1 receptors in the PL is
involved in the cognitive impairment and the negative
symptoms of schizophrenia[12,19,23,24].
Anatomical studies have shown that the dendritic and
somatic regions of PL pyramidal neurons receive synaptic
inputs from both glutamatergic terminals arising from
cortical and subcortical sources and dopaminergic afferents from
VTA[25_29]. Furthermore, ultrastructural studies demonstrate
that dopaminergic and glutamatergic axon terminals are in
direct apposition to each other on the same postsynaptic
pyramidal neuron in the PL which forms so-called "synaptic
triads", suggesting that both presynaptic and
postsynaptic inputs participate in the modulation of synaptic
transmission[30_32]. SPD is found to potently enhance the amplitude
of NMDA-mediated currents in PL pyramidal neurons in brain
slices via a postsynaptic mechanism[12]. However, the
potential role of presynaptic regulation in mediating the
effect of SPD is unknown and of interest since it has reported
that DA can act on presynaptic D1 receptors and increase
the frequency of sEPSC in PL pyramidal neurons in brain
slices[33].
The present work was designed to study the effect and
signaling mechanism for the SPD-modulated frequency of
sEPSC in PL pyramidal neurons. The results indicate that
presynaptic mechanism plays a critical role in the
SPD-elicited effect on the frequency of sEPSC in PL pyramidal cells
via D1 receptors.
Materials and methods
Preparation of prelimbic cortical slices Sprague-Dawley
rats (Shanghai Experimental Animal Center, Chinese
Academy of Sciences, Shanghai, China), weighing 30_50 g, were
housed under standard laboratory conditions with constant
temperature (22_23 °C) and humidity (50%_60%). All animal
experiments were conducted in compliance with the Guide
for the Care and Use of Laboratory Animals (National
Research Council, 1996). Prelimbic cortical slices were prepared
according to procedures described
previously[33]. The rats were anesthetized with chloral hydrate (400 mg/kg, ip).
Following decapitation, the brain was quickly removed and
submerged in ice-cold perfusive medium containing 130 mmol/L
NaCl, 5 mmol/L KCl, 2 mmol/L CaCl2, 2 mmol/L
MgSO4, 1.25 mmol/L
NaH2PO4, 26 mmol/L
NaHCO3, 10 mmol/L glucose, 10 mmol/L sucrose, and saturated with 95%
O2 and 5% CO2. The PL was removed and placed on a layer of moistened
filter paper glued to the cutting stage of automatic
oscillating tissue slicer (OTS-4000, Electron Microscopy Sciences,
Fort Washington, PA, USA). Serial coronal slices (380 µm)
were cut and transferred to an incubating chamber (28_30 °C)
for at least 1 h before recording.
Visualization of pyramidal cells To visualize the cell,
the slice was placed in a recording chamber and viewed with
a fixed stage, upright microscope (BX51WI, Olympus, Tokyo,
Japan). To increase the clarity of the cell, infrared light was
used to illuminate the slice. The resultant infrared
differential interference contrast (DIC) images were visualized on a
black_white TV monitor through the use of a light sensitive
charge coupled device (CCD) camera. Recordings were made
from pyramidal cells located in layers V_VI. They were
identified by their pyramidal shape, large soma, and presence of
apical dendrites (Figure 1).
Whole-cell recording The slice was continuously
perfused with the above perfusive medium and saturated with
95% O2 and 5% CO2. Electrodes (4_6
MW), pulled from glass capillaries with a Sutter micropipette puller (P-97, Novato,
CA, USA), were filled with a solution (pH 7.25) containing
140 mmol/L K-gluconate, 0.1 mmol/L
CaCl2, 2 mmol/L MgCl2, 1 mmol/L ethylene glycol tetraacetic acid (EGTA), 2 mmol/L
ATP·K2, 0.1 mmol/L
GTP·Na3, and 10 mmol/L N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)
(HEPES). Voltage and current signals were recorded with an
Axon patch 700A amplifier (Union City, CA, USA) connected
to a Digidata 1322A interface (Union City, CA, USA). The
data were digitized and stored on disks using Clampfit
(version 8.2.0.228, Axon, USA). Resting membrane potential
and action potential were recorded under the current clamp
mode. The recording pyramidal cell had a resting membrane
potential less than -50 mV, an action potential amplitude
greater than 80 mV, and no spontaneous action potentials.
sEPSC, which had variable amplitude with a fast rising phase
and a slower decay, were recorded at a holding potential of
-70 mV (Figure 2A). These currents were blocked by an
α-amino-3-hydroxy-5-methylisoxazole-4-propionic
acid (AMPA)/kainite glutamate receptor antagonist
2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, 10 µmol/L).
The series resistance was monitored by measuring the
instantaneous current in response to a 5 mV voltage
step command. Series resistance compensation was not used,
and cells whose series resistance changed more than 15%
were discarded.
Offline data analysis Offline data analysis was performed
using the Mini Analysis Program 5.0 (Synaptosoft, Fort Lee,
NJ, USA) and Origin 7.0 (OriginLab Corporation, Northampton,
MA, USA). The record of the sEPSC was shown with high
resolution, and events that did not show a typical sEPSC
waveform were rejected. The frequency and the inter-event
intervals of sEPSC were measured. The effects of drugs
were compared before and after drug injection with ANCOVA.
The covariate was the baseline value prior to the drug
injection. Statistical significance of distribution was made
with the Kolmogorov-Smirnov (K-S) test. All numerical data
were expressed as mean±SEM. In all cases,
n refers to the number of cells studied.
Drugs SPD (Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, China) was dissolved in 0.1
mmol/L H2SO4, then diluted and neutralized with 0.1 mmol/L NaOH
(pH 5.0). NBQX, K-gluconate,
ATP·K2, GTP·Na3, SCH23390,
N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfona
mide hydrochloride (H-89), SKF38393, sulpiride, and
chelerythrine were purchased from Sigma (St Louis, MO, USA).
All drugs were applied through bath perfusion.
Results
SPD increases frequency of sEPSC In PL pyramidal
cells, bath application of SPD (50 µmol/L) resulted in an
apparent increase in sEPSC frequency. A typical current trace
is shown at Figure 2A. A plotting cumulative distribution of
sEPSC intervals with the K-S statistical analysis indicated
that SPD induced a clear shift towards shorter intervals in
the cumulative curve (Figure 2B). On average, sEPSC
frequency significantly increased after SPD
(P<0.05, n=13 cells, Figure 2C). A SPD-elicited increase in sEPSC frequency
was concentration dependent (n=5 cells, Figure 2D). A
significant increase in the sEPSC (125.2%±6.8%,
P<0.05) at 10 µmol/L was already observed; it reached its maximum at 100
µmol/L (P<0.01).
SPD-induced increase in sEPSC frequency is mediated
by the D1 receptor The above results showed that SPD
produced an increase of sEPSC frequency. We then
determined the potential mechanism involved in the action of SPD.
The application of the selective D1 receptor antagonist
SCH23390 (10 µmol/L) significantly blunted the SPD-induced
increase in sEPSC frequency (Figure 3A, 3B). The
stimulation on sEPSC frequency was recovered while SCH23390
was washed out. This was further supported by data that
the D1 partial agonist SKF38393 (10 µmol/L) mimicked the
increase of SPD on the frequency of sEPSC
(P<0.05, Figure 3E,F), while the
D2/3 receptor antagonist sulpiride (10
µmol/L) elicited no effects on sEPSC frequency
(n=6 cells, Figure 3C,D). Thus, it is clear that the
D1 receptor, not the D2/3 receptor, mediated the effect of SPD on sEPSC frequency.
Both PKA and PKC are involved in SPD-induced increase
in the frequency of sEPSC It is known that
D1 receptor stimulation induces the activation of protein kinase A (PKA)
and protein kinase C (PKC). We determined which
intracellular pathway was involved in the SPD-altered frequency of
sEPSC. As shown in Figure 4, when the specific PKA
antagonist H-89 (10 µmol/L) was applied prior to or during SPD
incubation, the SPD-elicited increase in the frequency of
sEPSC was abolished. This was also true when the PKC
inhibitor chelerythrine (2.5 µmol/L) was employed (Figure
4C, D). The results therefore implicated that both PKA and
PKC were involved in the regulatory effect of SPD on sEPSC
frequency.
Discussion
The present study demonstrated that SPD significantly
increased the frequency of sEPSC in a
concentration-dependent manner. Selective
D1 receptor antagonist SCH23390 blocked SPD-mediated effects, whereas
D1 agonist SKF38393, but not the
D2/3 antagonist sulpiride mimicked a
SPD-mediated increase in the frequency of sEPSC. Moreover, both
PKA inhibitor H-89 and PKC inhibitor chelerythrine
attenuated the effect of SPD on sEPSC.
Previous studies from our laboratory and others have
shown that SPD displays high affinity to both
D1- and D2-like receptors. It is clear now that SPD possesses a dual
property towards DA receptors. It acts as an agonist on the
D1 receptor and functions as an antagonist on the
D2/3 receptor[5,34-40]. Our data demonstrated that enhancement of sEPSC
frequency in the PL pyramidal cells by SPD was mediated by
the D1 receptor, whereas
D2/3 receptors appeared not to
associate with the action.
It is known that sEPSC are mainly resulted from the
action potential-dependent glutamate release and thereby the
increase in sEPSC frequency could be due to the activation
of some neurotransmitter receptors, such as the
D1 receptor, that result in synaptic transmission from a presynaptic
action[33,41-43]. In agreement with previous finding that
D1 receptors could modulate presynaptic glutamate
release[44,45], the present study showed that SPD increased sEPSC
frequency via D1 receptors. It therefore appeared that the
underlying mechanism for the SPD-mediated increase in sEPSC
frequency is associated with the drug-induced presynaptic
release of glutamate, and subsequently, the modulation of
the synaptic inputs to PL pyramidal cells.
Both PKA and PKC are known to be the important
signaling molecules that transduce D1-like receptor
signals[46,47]. A previous study demonstrated that DA stimulated the
frequency of sEPSC through PKA and PKC in rat PL pyramidal
cells via the presynaptic D1
receptor[33]. Present data also indicate that PKA and PKC play an important role in
SPD-mediated modulation on sEPSC frequency. Therefore, it
appears that both DA and SPD share common signaling
pathways via the activation of the D1 receptor in the modulation
of sEPSC frequency.
More importantly, the present data provides novel
evidence for the functional interaction between the
D1 and NMDA receptors in neurons. It is known that the
interaction between the D1 and NMDA receptors plays a critical
functional role in the frontal cortex. It is interesting to note
that the suggested alterations of the
D1_NMDA receptor
interaction in the PL are important pathological mechanisms
underlying the neurochemical imbalance in schizo-
phrenia[12,19]. Both the D1 and NMDA receptors in the frontal
cortex play a critical role in synaptic plasticity, memory, and
cognition. Dysfunction of NMDA and D1 receptor is
believed to associate with the negative symptoms and
cognitive impairment in
schizophrenia[48-50]. Our results show
that SPD increases the frequency of sEPSC via the
activation of D1 receptors located in the PL V_VI layers,
presumably through enhancing the presynaptic glutamate release.
Considering the advantageous benefits of SPD on negative
symptoms of schizophrenic patients[7], the present study
provides a potential molecular mechanism for the treatment of
SPD on negative symptoms of schizophrenia. Furthermore,
unique pharmacological properties of SPD (the
D1 agonist in the PL and the
D2/D3 antagonist in the subcortex) lead us to
believe that SPD is a promising candidate as a novel
antipsychotic agent.
References
1 Dong ZJ, Chen LJ, Jin GZ, Creese I. Gtp regulation of
(-)-stepholidine binding to R(h) of D1 dopamine receptors in calf
striatum. Biochem Pharmacol 1997; 54: 227_32.
2 Jin GZ, Zhu ZT, Fu Y. (-)-stepholidine: A potential novel
antipsychotic drug with dual D1 receptor agonist and
D2 receptor antagonist actions. Trends Pharmacol Sci 2002; 23: 4_7.
3 Shi WX, Chen Y, Jin GZ. Effect of
l-stepholidine on rotational behavior in rats. Acta Pharmacol Sin 1984; 5: 222_5.
4 Xu SX, Yu LP, Han YR, Chen Y, Jin GZ. Effects of
tetrahydro-protoberberines on dopamine receptor subtypes in brain. Acta
Pharmacol Sin 1989; 10: 104_10.
5 Zou LL, Liu J, Jin GZ. Involvement of receptor reserve in
D1 agonistic action of (-)-stepholidine in lesioned rats. Biochem
Pharmacol 1997; 54: 233_40.
6 Dong ZJ, Guo X, Chen LJ, Han YF, Jin GZ. Dual actions of
(-)-stepholidine on the dopamine receptor-mediated adenylate
cyclase activity in rat corpus striatum. Life Sci 1997; 61: 465_72.
7 Wu DC, Xing XZ, Wang WA, Xie H, Wang YH, Wang XY,
et al. A double-blind comparison trial of
l-stepholidine and perphenazine in treatmeat of schizophrenia. Chin J New Drugs Clin Remedies
2003; 22: 155_9.
8 Cai N. A controlled study on the treatment of tardive dyskinesia
using l-stepholidine. Zhonghua Shen Jing Jing Shen Ke Za Zhi
1988; 21: 281_3.
9 Goldman-Rakic PS. The cortical dopamine system: Role in
memory and cognition. Adv Pharmacol 1998; 42: 707_11.
10 Goldman-Rakic PS, Castner SA, Svensson TH, Siever LJ,
Williams GV. Targeting the dopamine D1 receptor in schizophrenia:
Insights for cognitive dysfunction. Psychopharmacology (Berl)
2004; 174: 3_16.
11 Seamans JK, Yang CR. The principal features and mechanisms
of dopamine modulation in the prefrontal cortex. Prog Neurobiol
2004; 74: 1_58.
12 Yang CR, Chen L. Targeting prefrontal cortical dopamine
D1 and n-methyl-d-aspartate receptor interactions in schizophrenia
treatment. Neuroscientist 2005; 11: 452_70.
13 Brozoski TJ, Brown RM, Rosvold HE, Goldman PS. Cognitive
deficit caused by regional depletion of dopamine in prefrontal
cortex of rhesus monkey. Science 1979; 205: 929_32.
14 Jentsch JD, Roth RH, Taylor JR. Role for dopamine in the
behavioral functions of the prefrontal corticostriatal system:
Implications for mental disorders and psychotropic drug action.
Prog Brain Res 2000; 126: 433_53.
15 Lindstrom LH, Gefvert O, Hagberg G, Lundberg T, Bergstrom M,
Langstrom B, et al. Increased dopamine synthesis rate in medial
prefrontal cortex and striatum in schizophrenia indicated by
l-(beta-11c) dopa and pet. Biol Psychiatry 1999; 46: 681_8.
16 Murphy BL, Arnsten AF, Goldman-Rakic PS, Roth RH. Increased
dopamine turnover in the prefrontal cortex impairs spatial working
memory performance in rats and monkeys. Proc Natl Acad Sci
USA 1996; 93: 1325_9.
17 Tzschentke TM. Pharmacology and behavioral pharmacology
of the mesocortical dopamine system. Prog Neurobiol 2001; 63:
241_320.
18 Winn P. Schizophrenia research moves to the prefrontal cortex.
Trends Neurosci 1994; 17: 265_8.
19 Abi-Dargham A, Laruelle M. Mechanisms of action of second
generation antipsychotic drugs in schizophrenia: Insights from
brain imaging studies. Eur Psychiatry 2005; 20: 15_27.
20 Goldman-Rakic PS, Lidow MS, Gallager DW. Overlap of
dopaminergic, adrenergic, and serotoninergic receptors and
complementarity of their subtypes in primate prefrontal cortex.
J Neurosci 1990; 10: 2125_38.
21 Goldman-Rakic PS, Muly EC 3rd, Williams GV.
D1 receptors in prefrontal cells and circuits. Brain Res Rev 2000; 31: 295_301.
22 Sawaguchi T, Goldman-Rakic PS.
D1 dopamine receptors in prefrontal cortex: Involvement in working memory. Science 1991;
251: 947_50.
23 Davis KL, Kahn RS, Ko G, Davidson M. Dopamine in
schizo-phrenia: A review and reconceptualization. Am J Psychiatry
1991; 148: 1474_86.
24 Weinberger DR. Implications of normal brain development for
the pathogenesis of schizophrenia. Arch Gen Psychiatry 1987;
44: 660_9.
25 Berger B, Thierry AM, Tassin JP, Moyne MA. Dopaminergic
innervation of the rat prefrontal cortex: A fluorescence
histochemical study. Brain Res 1976; 106: 133_45.
26 Conde F, Audinat E, Maire-Lepoivre E, Crepel F. Afferent
connections of the medial frontal cortex of the rat. A study using
retrograde transport of fluorescent dyes. I. Thalamic afferents.
Brain Res Bull 1990; 24: 341_54.
27 Herkenham M. Laminar organization of thalamic projections
to the rat neocortex. Science 1980; 207: 532_5.
28 Penit-Soria J, Audinat E, Crepel F. Excitation of rat prefrontal
cortical neurons by dopamine: An in vitro electrophysiological
study. Brain Res 1987; 425: 263_74.
29 Ray JP, Price JL. The organization of the thalamocortical
connections of the mediodorsal thalamic nucleus in the rat, related
to the ventral forebrain-prefrontal cortex topography. J Comp
Neurol 1992; 323: 167_97.
30 Carr DB, Sesack SR. Hippocampal afferents to the rat prefrontal
cortex: Synaptic targets and relation to dopamine terminals. J
Comp Neurol 1996; 369: 1_15.
31 Goldman-Rakic PS, Leranth C, Williams SM, Mons N, Geffard
M. Dopamine synaptic complex with pyramidal neurons in
primate cerebral cortex. Proc Natl Acad Sci USA 1989; 86:
9015_19.
32 Smiley JF, Goldman-Rakic PS. Heterogeneous targets of
dopamine synapses in monkey prefrontal cortex demonstrated by serial
section electron microscopy: A laminar analysis using the
silver-enhanced diaminobenzidine sulfide (SEDS) immunolabeling
technique. Cereb Cortex 1993; 3: 223_38.
33 Wang Z, Feng XQ, Zheng P. Activation of presynaptic
D1 dopamine receptors by dopamine increases the frequency of
spontaneous excitatory postsynaptic currents through protein kinase A
and protein kinase C in pyramidal cells of rat prelimbic cortex.
Neuroscience 2002; 112: 499_508.
34 Liu J, Guo X, Wang BC, Jin GZ. Increased phosphorylation of
DARPP-32 by D1 agonistic action of
l-stepholidine in the 6-OHDA-lesioned rat striatum. Acta Physiol Sin 1999; 51: 65_
72.
35 Sun BC, Zhang XX, Jin GZ. (-)-Stepholidine acts as a
D1 partial agonist on firing activity of substantia nigra pars reticulata
neurons in 6-hydroxydopamine-lesioned rats. Life Sci 1996; 59:
299_306.
36 Zhang XX, Liu J, Fu Y, Hu GY, Jin GZ. Action sites of rotation
and unit firing induced by l-stepholidine and DA agonists in basal
ganglia of 6-OHDA-lesioned rats. Acta Pharmacol Sin 1999; 20:
979_86.
37 Zhu ZT, Fu Y, Hu GY, Jin GZ. Electrophysiological study on
biphasic firing activity elicited by D1
agonistic-D2 antagonistic action of (-)-stepholidine in nucleus accumbens. Acta Physiol
Sin 2000; 52: 123_30.
38 Zhu ZT, Fu Y, Hu GY, Jin GZ. Modulation of medical prefrontal
cortical D1 receptors on the excitatory firing activity of nucleus
accumbens neurons elicited by (-)-stepholidine. Life Sci 2000;
67: 1265_74.
39 Zhu ZT, Wu WR, Fu Y, Jin GZ. l-stepholidine facilitates
inhibition of mPFC DA receptors on subcortical NAc DA release. Acta
Pharmacol Sin 2000; 21: 663_7.
40 He Y, Yu LP, Jin GZ. Altered rotation behavior of sensitized rats
to effect of l-stepholidine: Implication of dopamine
D3R antagonist. Pharmacol Biochem Behav 2007: in press.
41 Feng XQ, Dong Y, Fu YM, Zhu YH, Sun JL, Zheng P,
et al. Progesterone inhibition of dopamine-induced increase in
frequency of spontaneous excitatory postsynaptic currents in rat
prelimbic cortical neurons. Neuropharmacology 2004; 46:
211_22.
42 Parfitt KD, Madison DV. Phorbol esters enhance synaptic
transmission by a presynaptic, calcium-dependent mechanism in rat
hippocampus. J Physiol 1993; 471: 245_68.
43 Wang Z, Zheng P. Characterization of spontaneous excitatory
synaptic currents in pyramidal cells of rat prelimbic cortex. Brain
Res 2001; 901: 303_13.
44 Bouron A, Reuter H. The D1 dopamine receptor agonist
SKF-38393 stimulates the release of glutamate in the hippocampus.
Neuroscience 1999; 94: 1063_70.
45 Hu XT, White FJ. Dopamine enhances glutamate-induced
excitation of rat striatal neurons by cooperative activation of
D1 and D2 class receptors. Neurosci Lett 1997; 224: 61_5.
46 Kebabian JW, Petzold GL, Greengard P. Dopamine-sensitive
adenylate cyclase in caudate nucleus of rat brain, and its
similarity to the "Dopamine receptor". Proc Natl Acad Sci USA 1972;
69: 2145_9.
47 Hussain T, Lokhandwala MF. Renal dopamine receptor function
in hypertension. Hypertension 1998; 32: 187_97.
48 Kretschmer BD. Modulation of the mesolimbic dopamine
system by glutamate: Role of nmda receptors. J Neurochem 1999;
73: 839_48.
49 Olney JW, Newcomer JW, Farber NB. NMDA receptor
hypofunction model of schizophrenia. J Psychiatr Res 1999; 33:
523_33.
50 Svensson TH. Dysfunctional brain dopamine systems induced by
psychotomimetic NMDA-receptor antagonists and the effects
of antipsychotic drugs. Brain Res Rev 2000; 31: 320_9.
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