- Email: [email protected]
Effects of dynorphin on rat entopeduncular nucleus neurons in vitro
PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 3 5 5 - X
Neuroscience Vol. 114, No. 4, pp. 973^982, 2002 B 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
EFFECTS OF DYNORPHIN ON RAT ENTOPEDUNCULAR NUCLEUS NEURONS IN VITRO M. OGURA and H. KITA Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Memphis, 855 Monroe Avenue, Memphis, TN 38163, USA
Abstract/The entopeduncular nucleus (EP) receives dense neostriatal a¡erent axons that contain dynorphin (DYN, an endogenous U-receptor agonist), in addition to GABA and substance P. To examine the role of DYN in the EP, wholecell recordings were performed in rat brain slice preparations. Based on the physiological and morphological characteristics, all the neurons recorded were similar to the Type-I EP neuron described in a previous study. The U-receptor agonist dynorphin A (1-13) (DYN13) hyperpolarized and decreased the input resistance of approximately one-quarter of the EP neurons examined. The hyperpolarization was due to an increase in potassium conductance since current^voltage relationship curves obtained before and after DYN13 application crossed at the potassium equilibrium potential. In the presence of the glutamate blocker 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide and 3-(2-carboxypiperzin-4-yl)-propyl-1-phosphonic acid in arti¢cial cerebrospinal £uid, stimulation of the globus pallidus evoked bicuculline-sensitive multi-component GABAergic responses in EP neurons. Application of DYN13 equally reduced the amplitudes of the short-latency response, conceivably evoked by pallido-EP axons, and the medium-latency response, conceivably evoked by striato-EP axons. These e¡ects were reversed by bath application of a non-selective opioid antagonist naloxone or by a U-opioid receptor-selective antagonist nor-binaltorphimine dihydrochloride (nor-BNI), but not by the D-antagonist naltrindole or the W-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 . DYN13 also reduced the frequency of tetrodotoxin-insensitive miniature-inhibitory postsynaptic potential (mIPSPs) without changing their amplitude distributions. The decrease of the frequency of mIPSPs was reversible upon washing and was also completely blocked by nor-BNI. The results of the present study on the EP indicated that DYN released from striatal axons might exert at least three di¡erent e¡ects on these target nuclei. Firstly, DYN might provide negative feedback regulation of striatal GABAergic outputs at their termination sites. Secondly, DYN released from the striatal terminals might di¡use to the pallidal terminals, regulating their GABA release. Thirdly, DYN might exert a direct inhibition of EP neurons. Thus, DYN released from striatal axons might control the activity of EP neurons by reducing the GABAergic transmission and also by hyperpolarizing postsynaptic membrane. B 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dynorphin, entopeduncular nucleus, GABAergic responses, presynaptic modulation, postsynaptic e¡ect.
the GP (Kawaguchi et al., 1990). Thus, the GP also receives striatal ¢bers containing GABA and DYN. Although injection of U-receptor agonists into the sites where DYN containing ¢bers terminate shows various behavioral e¡ects (Slater and Longman, 1980; Slater, 1982; Herrera-Marschitz et al., 1984; Friederich et al., 1987; Maneuf et al., 1995), the physiological e¡ects of DYN have not been studied extensively. We have recently found that DYN exerts both pre- and postsynaptic e¡ects on the pallidal neurons in rat brain slice preparations (Ogura and Kita, 2000). Postsynaptically, DYN hyperpolarized about one-third of pallidal neurons and, presynaptically, DYN attenuated striato-GP and intrapallidal GABAergic synaptic transmissions. It is conceivable that the activity of EP neurons is also controlled by DYN. The EP is more densely innervated by DYN containing ¢bers than the GP (Vincent et al., 1982; Zamir et al., 1983; Weber and Barchas, 1983). The EP contains U-receptors (Sharif and Hughes, 1989; SimSelley et al., 1999). Injection of the U-receptor agonist enadoline into the rat EP reduced reserpine-induced akinesia (Maneuf et al., 1995). Similarly, injection of enadoline into the internal segment of the pallidum alleviated
The entopeduncular nucleus (EP) receives major inputs from the neostriatum (Str), the globus pallidus (GP) and the subthalamic nucleus and sends the major output to the dorsal thalamus. Thus, the EP is an output nucleus of the basal ganglia. The striatal neurons projecting to the EP contain GABA, dynorphin (DYN) and substance P (Reiner et al., 1999; Penny et al., 1986; Gerfen and Young, 1988; Lee et al., 1997). DYN is an endogenous ligand of the U-opioid receptor (Chavkin et al., 1982; Brookes and Bradley, 1984; Raynor et al., 1994). Striato-EP neurons also send their collateral axons to
*Corresponding author. Tel. : +1-901-448-5234; fax: +1-901-4487193. E-mail address: [email protected] (H. Kita). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; CPP, 3-(2-carboxypiperzin-4-yl)-propyl-1-phosphonic acid ; DYN, dynorphin ; EP, entopeduncular nucleus; GABA, Q-amino-butyric acid; GP, globus pallidus; (m)IPSC, (miniature-)inhibitory postsynaptic current; (m)IPSP, (miniature-)inhibitory postsynaptic potential ; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline7-sulfonamide ; nor-BNI, nor-binaltorphimine dihydrochloride; Str, neostriatum ; TTX, tetrodotoxin. 973
NSC 5779 25-9-02
974
M. Ogura and H. Kita
parkinsonian symptoms of the N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-treated marmoset (Maneuf et al., 1995). To verify the physiological e¡ects of DYN on EP neurons, we performed whole-cell recording experiments using rat brain slice preparations. Speci¢cally, we examined the e¡ects of the U-agonist dynorphin A (1-13) (DYN13) on the neuronal membrane and on GABAergic synaptic transmissions in the EP.
EXPERIMENTAL PROCEDURES
Opioid receptor ligand Opioid receptor ligands used in this study were: the U-agonist DYN13 (1 WM); the non-selective antagonist naloxone hydrochloride (5 WM); the U-receptor-selective antagonist nor-binaltorphimine dihydrochloride (nor-BNI, 1^5 WM); the D-receptorselective antagonist naltrindole hydrochloride (1^2 WM); and the W-receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-ThrPen-Thr-NH2 (CTOP, 1^4 WM). These drugs were dissolved in deoxygenated water as concentrated stocks. The stocks were aliquoted and stored at 320‡C. They were thawed and diluted immediately before use. These opioid receptor ligands were obtained from RBI.
Slice preparations
Histology
Sprague^Dawley juvenile rats (16^21 days old) of both sexes were used. Animals were anesthetized (i.p.) with a mixture of ketamine (85 mg/kg) and xylazine (15 mg/kg) and were perfused through the heart with cold oxygenated arti¢cial cerebrospinal £uid (ACSF). After decapitation, the brains were rapidly removed and blocks containing the EP were obtained. Parasagittal slices (300 Wm thick) were cut from the blocks on a Vibroslice (Campden, UK) in ice-cold ACSF. The slices were incubated in ACSF at 37‡C for 1 h before recording. The composition of ACSF (in mM) was NaCl 124, KCl 5.0, KH2 PO4 1.24, NaHCO3 26, CaCl2 2.4, MgSO4 1.3, and glucose 10.
After recording, the slices were ¢xed overnight with a mixture of 4% paraformaldehyde and 0.2% picric acid. The ¢xed slices were rinsed several times with bu¡ered saline, incubated overnight with avidin^biotin^horseradish peroxidase complex (1% in bu¡ered saline with 0.4% Triton X-100), rinsed, and then reacted with diaminobenzidine. The slices were post-¢xed with 0.5% osmium tetroxide, in¢ltrated with a plastic resin, and mounted onto glass slides. The stained neurons were drawn under the microscope BH2 (Olympus, Tokyo, Japan) equipped with a drawing tube and a U60 dry objective. Statistics
Recording and electrical stimulation The slices were transferred to a recording chamber with oxygenated ACSF continuously perfused at a £ow rate of 2 ml/min. All recordings were performed at room temperature. Whole-cell patch recording pipettes with a tip diameter of about 1.5 Wm were pulled from 1.5 mm, thin-wall, borosilicate glass capillaries on a horizontal electrode puller (P-87, Sutter Instruments, Navato, CA, USA). The recording pipettes were ¢lled with high-Cl electrolyte containing (in mM): K-gluconate 90, KCl 50, HEPES 10, Mg-ATP 2, Na-GTP 0.2, and Neurobiotin 0.2%, with pH adjusted to 7.2 with KOH. The chloride equilibrium potential of the cells was calculated to be 325 mV by the Nernst equation when the cytoplasm of the recorded cells was fully equilibrated with 50 mM chloride. The resistance of these recording pipettes ranged from 4.0 to 8.0 M6. Neurons and recording pipettes were visualized using an infrared^di¡erential interference contrast microscope Reichert Diastar (Leica, Deer¢eld, IL, USA) with a U40 water immersion objective (Carl Zeiss, Thornwood, NY, USA) and a CCD camera Model6412 (COHU). Current and voltage clamp recordings were obtained using an ampli¢er IR183 (Neurodata Instruments, New York, NY, USA) and an electrometer AXOPATCH 200B (Axon Instruments, Foster City, CA, USA), respectively. The output of the ampli¢ers was monitored with an oscilloscope D51 (Tektronix, Beaverton, OR, USA) and a chart recorder WindoGraf (Gould Instrument Systems, Valley View, OH, USA). All data were digitized with an NEURO-CORDER Model DR-484 (Neurodata Instruments) and stored on videotape. The data were analyzed with a Macintosh computer with the data analysis program Oscilloscope, written and generously provided by Dr. C.J. Wilson. To evoke inhibitory postsynaptic potentials (IPSPs) or inhibitory postsynaptic currents (IPSCs), electrical stimulation (0.2 ms in duration) was applied through a bipolar electrode to the GP. To isolate GABAergic responses from glutamatergic ones, the N-methyl-D-aspartate (NMDA) receptor antagonist, 3-(2carboxypiperzin-4-yl)-propyl-1-phosphonic acid (CPP, 10 WM), and the non-NMDA receptor antagonist, 1,2,3,4-tetrahydro6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX, 5 WM), were applied to the bath. To record action potentialindependent miniature-IPSCs (mIPSCs), tetrodotoxin (TTX, 1 WM) was applied in addition to the glutamate blockers. The GABAA antagonist gabazine (10 WM) was used to con¢rm GABAergic responses.
All group data were expressed as a mean T standard error of the mean (S.E.M.) and analyzed statistically using a Student’s t-test or an analysis of variance (ANOVA). The Kolmogorov^ Smirnov (K^S) test (Press et al., 1986) was used to determine the statistical signi¢cance of the amplitude distributions of mIPSCs.
RESULTS
E¡ects of DYN13 on the membrane potential and the input resistance Twenty-three EP neurons with the resting membrane potential more negative than 355 mV or with the amplitude of spontaneous action potentials exceeding 60 mV were tested with bath application of DYN13. When the neurons ¢red low-frequency spikes spontaneously or upon injection of a low-intensity intracellular current, each spike was followed by a large after-hyperpolarization (Fig. 1B, D). All the recorded neurons were capable of generating repetitive ¢ring without accommodation upon strong intracellular current stimulation (data not shown), and thus were considered to be Type-I EP neurons (Nakanishi et al., 1990). Neurons exhibiting spontaneous spikes were current clamped at 365 mV before application of DYN13. DYN13 (1 WM) hyperpolarized (5.4 T 1.0 mV) the somatic membrane of ¢ve of the 23 neurons tested. The DYN13-induced hyperpolarization was accompanied by a decrease (25.2 T 1.8%, n = 5) in the input resistance (Fig. 1), and was reversed by washing (n = 2) or application of nor-BNI (1 WM, n = 3). The neurons that were hyperpolarized less than 3 mV or that had an ambiguous response with no recovery were not included in the above analysis. The DYN13 did not induce depolarization in any of the neurons tested. The current^voltage relationship curves obtained from the
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
975
Fig. 1. E¡ects of DYN13 on the resting membrane potential and the input resistance of an EP neuron. (A) A slow sweep trace of a current clamp recording of an EP neuron using a high-Cl electrolyte containing pipette. The GP was stimulated to induce depolarizing IPSPs. Current pulses with an intensity of 330 pA and a duration of 250 ms were injected to monitor the input resistance (also see F). To block glutamatergic responses, NBQX (5 WM) and CPP (10 WM) were added to the ACSF. The noisy appearance of the trace is due to spontaneous depolarizing IPSPs. Bath application of DYN13 (1 WM) hyperpolarized this EP neuron by approximately 5 mV. Decreases in the input resistance and the amplitude of IPSPs accompanied the hyperpolarization. (B^D) Responses to depolarizing and hyperpolarizing current pulses were recorded before DYN13 application (B), immediately after (C), and after a 30 min wash (D). Supra-threshold current pulses triggered repetitive spikes (B, D). (E) Current^voltage relationship curves obtained from this neuron. The membrane potentials at the terminations of current pulses were measured on four to ¢ve traces with no observable IPSPs at that time and the averages were plotted. The DYN13-induced hyperpolarization was accompanied by a decrease in the input resistance. The I^V curves for control and during DYN13 appliction cross at approximately 381 mV. (F) Examples of fast sweep tracing show responses to 330 pA current pulses and pallidal stimulation induced IPSPs before and immediately after DYN13 application.
DYN13-sensitive neurons before and after DYN13 application crossed at 383.6 T 2.3 mV (n = 5). DYN13 diminishes inhibitory postsynaptic response EP neurons receive GABAergic a¡erent inputs from both the Str and the GP. The conduction velocity of
the striatal axons is slower than that of the pallidal axons. Thus, it was expected that stimulation of the GP would evoke long- and short-latency postsynaptic responses in EP neurons. All the experiments described below were performed in the presence of NBQX (5 WM) and CPP (10 WM) in ACSF to block glutamatergic responses. Stimulation of the GP evoked bicuculline-sen-
NSC 5779 25-9-02
976
M. Ogura and H. Kita
Fig. 2. An example of an EP neurons that was not hyperpolarized by DYN13. (A) A slow sweep trace of a current clamp recording of an EP neuron using a high-Cl electrolyte containing pipette. Current pulses with the intensity of 330 pA and the duration of 250 ms were injected to monitor the input resistance. The GP was stimulated to induce depolarizing IPSPs. To block glutamatergic responses, NBQX (5 WM) and CPP (10 WM) were added to the ACSF. The noisy appearance of the trace is due to a large number of spontaneous depolarizing IPSPs. Bath application of DYN13 (1 WM) failed to hyperpolarize this EP neuron but did decrease the amplitude of the IPSPs. This DYN13 e¡ect was blocked by nor-BNI (1 WM), but not by CTOP (1 WM). (B, C) Examples of fast sweep tracing show responses to 330 pA current pulses and pallidal stimulation induced IPSPs before and immediately after DYN13 application.
sitive GABAergic responses in 17 EP neurons. Since the recording pipettes contained a high-Cl electrolyte, IPSPs and IPSCs were observed as depolarizations and inward current events, respectively. Stimulation of the GP evoked clearly separable multi-component responses in some EP neurons. For example, the neuron shown in Fig. 3 exhibited short- and very long-latency IPSCs after stimulation with a low intensity. The long-latency, exceeding 10 ms, small IPSCs were observed in only two other neurons. In all three of these neurons, the longlatency responses became inseparable when the stimulus intensity was increased. When the stimulus intensity was increased above 400 WA, IPSCs with a latency of 7.5 ms were evoked, as shown in the neuron in Fig. 3. Clearly separable short- and medium-latency IPSCs were observed in seven other neurons. In another nine neurons, only short-latency responses without apparent de£ections on the decay phase were observed. The latencies of the GP-induced short- and medium-latency IPSPs/IPSCs were 4.6 T 0.6 (n = 17) and 7.8 T 0.3 ms (n = 8), respectively. The e¡ects of DYN13 on the GABAergic responses were assessed from neurons that did not show signi¢cant changes in their membrane potentials or in their input resistance during DYN13 application (Fig. 2). Supramaximal stimulation was applied to evoke steady IPSPs or IPSCs and was tested by bath application of DYN13 (1 WM). When two separable latency responses were evoked, DYN13 almost equally reduced the amplitudes of both the short- and medium-latency responses (for an example, see Fig. 3). The data on compound IPSPs and IPSCs were grouped in here because DYN13 reduced their peak amplitudes by 33.9 T 3.3% (n = 17) in a similar degree to of the control (Fig. 4). This e¡ect was reversed
by bath application of a non-selective opioid antagonist naloxone (5 WM), and by the U-opioid receptor-selective antagonist nor-BNI (1 WM) (Fig. 2). In contrast, the D-antagonist naltrindole (1 WM) and the W-antagonist CTOP (1 WM) did not block the DYN13 e¡ect. Naloxone, nor-BNI, naltrindole and CTOP had no e¡ect on the evoked IPSPs (data not shown). These results suggest that DYN13 reduces the amplitude of evoked IPSPs through U-opioid receptor-mediated mechanisms. E¡ects of DYN13 on TTX-insensitive mIPSPs GABAergic mIPSPs were recorded in ACSF containing CPP (10 WM), NBQX (5 WM) and TTX (1 WM). Data were obtained from four EP neurons whose resting membrane potentials were not altered by DYN13. We measured spontaneous mIPSPs with the amplitude of more than twice the noise level, typically 0.3 mV. Most of the mIPSPs were less than 2 mV but occasional mIPSPs over 10 mV were also observed (Fig. 5D). The mIPSPs had approximately 6 ms rise times and 30^150 ms decay times. These four neurons exhibited mIPSPs with the frequency ranging from 0.47 to 1.84 Hz. DYN13 (1 WM) reduced the frequency of the mIPSPs to a range of 0.24^0.88 Hz. This change was statistically signi¢cant (n = 4, P 6 0.05, paired t-test). The decrease of the frequency of mIPSPs was reversible upon washing (n = 2) and was also completely blocked by nor-BNI (2 WM; n = 2; data not shown). DYN13 did not change the mean amplitude (Fig. 5B) or the amplitude distribution (P s 0.01, K^S test; Fig. 5C). These results suggest that DYN13 does not a¡ect postsynaptic GABA sensitivity, but instead modulates GABA release.
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
977
Fig. 3. E¡ects of DYN13 on pallidal stimulation induced short- and medium-latency IPSCs. Recordings were made with high-Cl electrolyte containing pipettes. Electrical stimulation was applied to the GP through a bipolar electrode. To block glutamatergic responses, NBQX (5 WM) and CPP (10 WM) were added to the ACSF. (A^C) IPSCs were recorded from an EP neuron voltage clamped at 370 mV. (A) Sample traces show GP stimulation-induced IPSCs with a threshold slightly above that of the short-latency IPSCs. The traces also show small- and long-, approximately 15 ms, latency IPSCs in this neuron. The long-latency responses could not be detected when the stimulus intensities were increased (see B, C). (B) Responses evoked by stimulus with the intensity threshold for the medium-latency IPSCs. (C) Responses evoked by stimulus with the intensity supra-maximal for the short- and medium-latency IPSCs. (D) Plots of the amplitude and the latency of IPSCs against the stimulus intensity. The amplitudes of the IPSCs were measured as shown in the inset. (E) DYN13 (1 WM) diminished the amplitudes of both the short- and medium-latency IPSCs. Washing the tissue reversed the DYN13 e¡ect. Each point in the graph represents a normalized (percentage of control) average amplitude T S.E.M. of three IPSCs.
Morphology of the recorded neurons Nineteen of the 23 neurons recorded in this study were intracellularly stained with Neurobiotin. All the stained neurons had morphological properties similar to that described for Type-I neurons in the previous study (Nakanishi et al., 1990; Kita, 2001). The neurons were of medium size, fusiform or round in shape, with the shortest diameter being 11.3 T 1.5 Wm and the longest being 24.0 T 4.9 Wm (Fig. 6). The neurons had two to ¢ve thick, slowly tapering, smooth primary dendrites that branched into secondary and tertiary dendrites with spines. These dendrites had tortuous trajectories. Many of the dendrites tapered slowly and extended for a long distance while some dendrites formed complex arborizations with abundant spines and appendages
(Fig. 6). The dendritic ¢eld of these neurons extended for 267 T 52 Wm from the soma in the sagittal plane. Their axons originated from a primary dendrite or soma and were thin, smooth and of constant diameter. Local collateral axons were not found in these neurons.
DISCUSSION
Although the EP receives a massive DYN containing ¢ber projection from the Str, the physiological e¡ects of DYN on the EP have not been previously studied. We have shown in a previous study that DYN13 exerts both post- and presynaptic e¡ects on the pallidal neurons in slice preparations (Ogura and Kita, 2000). From that study, we anticipated that DYN13 might have similar
NSC 5779 25-9-02
978
M. Ogura and H. Kita
Fig. 4. U-Receptor antagonists block the DYN13 e¡ect on evoked IPSPs/IPSCs. DYN13 (1 WM) reduced the amplitude of compound IPSPs/IPSCs to 33.9 T 3.3% (n = 17) of the control. This DYN13 e¡ect was blocked by the non-selective opioid receptor antagonist naloxone (5 WM, n = 3, 89.4 T 8.5% of the controls, P 6 0.0001, ANOVA) and by the U-opioid receptor-selective antagonist norBNI (1 WM, n = 7, 100.3 T 3.6% of the controls, P 6 0.0001, ANOVA). The W-antagonist CTOP (1 WM, 29.6 T 6.1 of the controls, n = 3) and the D-antagonist naltrindole (1 WM, 41.8 T 6.5 of the controls, n = 3) had no e¡ect (P s 0.05, ANOVA). Data are expressed as mean T S.E.M.
e¡ects on the EP neurons. The present study was undertaken to con¢rm this assumption using a whole-cell recording method in slice preparations. However, recording from EP neurons was more di⁄cult than from pallidal neurons under similar conditions. Therefore, the present study was limited to the examination of DYN13 e¡ects on the resting membrane potential and on GABAergic synaptic transmission. We also used a lower recording temperature than with the previous study in the GP to prolong the survival time of the EP neurons. The data presented here con¢rmed that DYN13 exerts both post- and presynaptic e¡ects in the EP which are very similar to those observed in pallidal neurons in slice preparations (Ogura and Kita, 2000). Types of the EP neurons recorded All the neurons recorded in the present study had ¢ring properties and somato-dendritic morphology similar to the Type-I EP neuron reported in an earlier study (Nakanishi et al., 1990). These observations suggest that the morphological and physiological properties of Type-I neurons in juvenile rats are indistinguishable from those in adult rats. In the present study, we did not obtained any Type-II neurons that are characterized by having a strong spike adaptation and strong A-current (Nakanishi et al., 1990). The failure to obtain TypeII neurons may be because their population is small and because the recordings obtained from them did not meet our recording criteria. Postsynaptic e¡ects of DYN13 DYN13 caused a hyperpolarization and a decrease in
the input resistance in ¢ve of the 23 EP neurons recorded. It is unclear whether only a subpopulation of the Type-I EP neurons was sensitive to DYN13 or whether all were sensitive but the detection method used in this study was not sensitive enough to reveal small responses. The hyperpolarization was most likely due to an activation of U-receptors because only the U-receptor-selective antagonist, not D- or W-antagonists, blocked the response. The hyperpolarization with a decrease in the input resistance was considered to be due to an increase in potassium conductance, as with the pallidal neurons. The current^voltage relationship curves before and after DYN13 application crossed at approximately 384 mV, which is very close to the potassium equilibrium potential of these neurons, as estimated by the Nernst equation. The hyperpolarization should be independent of chloride channel activation because the chloride equilibrium potential of the neurons was elevated to about 325 mV to evoke large depolarizing IPSPs. Activation of the potassium conductance by U-agonists has been reported in other brain areas (Hayar and Guyenet, 1998; Grudt and Williams, 1993). It has been shown that U-receptors and inwardly rectifying potassium channels coexpressed by Xenopus oocytes formed functional couplings (Ikeda et al., 1995; Henry et al., 1995). DYN13 e¡ects on GABAergic synaptic transmission The striato-EP ¢bers containing GABA, DYN and substance P terminate on the distal portion of the dendrites of EP neurons (Hazrati and Parent, 1992). In contrast, the pallido-EP GABA containing ¢bers terminate on the soma and proximal dendrites of the EP neurons (Hazrati et al., 1990; Kincaid et al., 1991; Smith et al., 1998). The conduction velocity of the striatal axons is signi¢cantly slower than that of the pallidal axons (Walker et al., 1989; Nakanishi et al., 1991; Kita and Kitai, 1991; Kita, 2001). Previous intracellular recording studies have shown that activation of striato-EP axons induces small- and long-latency IPSPs and activation of pallido-EP axons induces large, short-latency IPSPs in EP neurons (Park et al., 1982; Jaeger et al., 1994; Nambu and Llinas, 1994; Nakanishi et al., 1991; Kita, 2001). The latency of the striato-EP axon- and pallidoEP axon-induced IPSPs were 2.8 and 5.9 ms, respectively (Kita, 2001). In the present study using juvenile rats brain slices, pallidal stimulation also induced multicomponent IPSPs/IPSCs. Pallidal stimulation induced short-, medium- and occasional long-latency IPSPs/ IPSCs and their latencies were 4.6, 7.8 and approximately 15 ms, respectively. We consider that pallido-EP and striato-EP axons induced the short- and mediumlatency responses, respectively, although the latencies were longer than those obtained in the previous study. The longer latencies are probably due to younger rat age and to a cooler recording temperature, room temperature versus 35‡C, used in the present study. The long-, approximately 15 ms, latency responses were unable to be characterized in detail and the origin was unknown.
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
979
Fig. 5. DYN13 changed the frequency of mIPSPs without a¡ecting the amplitude distribution. mIPSPs were recorded from four EP neurons with pipettes containing the high-Cl electrolyte. The ACSF contained TTX (1 WM), NBQX (5 WM) and CPP (10 WM) to block action potentials and glutamatergic responses. (A, B) The frequency and the amplitude of mIPSCs was determined from 10 min of continuous recording under each condition. The frequency of the mIPSPs of the four neurons ranged from 0.47 to 1.84 Hz with a mean of 0.94 Hz. DYN13 (1 WM) reduced the frequency of mIPSPs to a range of 0.24^ 0.88 Hz with a mean of 0.51 Hz and this change was statistically signi¢cant (n = 4, P 6 0.05, paired t-test). The decrease of the frequency of mIPSPs was reversible upon washing. (B, C) DYN13 did not change the mean amplitude or the amplitude distribution (P s 0.01, K^S test). (D) Examples of mIPSPs. Most of the mIPSPs were less than 2 mV but mIPSPs over 10 mV were also observed. The rise time of the mIPSPs was approximately 6 ms.
DYN13 (1 WM) greatly reduced the amplitude of postsynaptic GABAergic responses in all of the EP neurons tested. This e¡ect was most likely mediated by U-receptors because the U-receptor antagonist blocked the e¡ect but D- or W-antagonists did not. Our previous study revealed that 1 WM DYN13 did not act as a partial W-agonist in the GP (Ogura and Kita, 2000). Large mIPSPs with amplitudes exceeding 10 mV were recorded from EP neurons. Based on their large amplitudes and short rise times, it was likely that pallidal axon terminals that form synapses at somata or proximal dendrites induced the large mIPSPs observed in EP neurons. DYN13 decreased the frequency of mIPSCs without changing the amplitude. This result suggested that DYN13 presynaptically reduced GABA release from the pallidal axon terminals. The reduction of neurotransmitter release by U-agonists has been reported in several brain areas including GABAergic inputs to the GP
(Ogura and Kita, 2000), glutamatergic mossy ¢bers in the hippocampus (Gannon and Terrian, 1991; Wagner et al., 1993; Weisskopf et al., 1993; Simmons and Chavkin, 1996), glutamatergic inputs to bulbospinal neurons (Hayar and Guyenet, 1998), dopaminergic ¢bers in the Str (Mulder et al., 1984), glutamate release in striatal synaptosomes (Hill and Brotchie, 1995), and the dorsal root ganglion in culture (MacDonald and Nelson, 1978).
CONCLUSION
The results of the present study on the EP and the previous study on the GP both indicated that DYN released from striatal axons might exert at least three e¡ects at the target nuclei. Firstly, DYN might provide negative feedback regulation of striatal GABAergic out-
NSC 5779 25-9-02
980
M. Ogura and H. Kita
Fig. 6. An example of a DYN-sensitive EP neuron intracellularly stained with Neurobiotin. (A) Camera-lucida drawing of the neuron shows that the neuron has a medium-sized, fusiform soma and thick, smooth primary dendrites without spines. Secondary and tertiary dendrites had spines and had tortuous trajectories. Some dendrites formed complex arborizations with abundant spines and appendages. The axon originating from the soma was cut at the surface of the section. (B, C) Photomontages of a dendrite forming complex arborizations and of the soma-proximal dendrites.
puts at their termination sites. Secondly, DYN released from the striatal terminals might di¡use to the pallidal terminals and regulate their GABA release. Thirdly, DYN might exert a direct inhibition of both GP and EP neurons. Thus, DYN released from striatal axons might control the activity of EP neurons by reducing
the GABAergic transmission and also by hyperpolarizing postsynaptic membrane. Acknowledgements.We thank Ms. Dawn Merrick for editing the manuscript. This study was supported by NIH grants NS26473 and NS-42762.
REFERENCES
Brookes, A., Bradley, P.B., 1984. Electrophysiological evidence for kappa-agonist activity of dynorphin in rat brain. Neuropharmacology 23, 207^ 210. Chavkin, C., James, I.F., Goldstein, A., 1982. Dynorphin is a speci¢c endogenous ligand of the kappa opioid receptor. Science 215, 413^ 415. Friederich, M.W., Friederich, D.P., Walker, J.M., 1987. E¡ects of dynorphin (1-8) on movement : non-opiate e¡ects and structure-activity relationship. Peptides 8, 837^840.
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
981
Gannon, R.L., Terrian, D.M., 1991. U-50, 488H inhibits dynorphin and glutamate release from guinea pig hippocampal mossy ¢ber terminals. Brain Res. 548, 242^247. Gerfen, C.R., Young, W.S., 1988. Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments : an in situ hybridization histochemistry and £uorescent retrograde tracing study. Brain Res. 460, 161^167. Grudt, T.J., Williams, J.T., 1993. Kappa-opioid receptors also increase potassium conductance. Proc. Natl. Acad. Sci. USA 90, 11429^ 11432. Hayar, A., Guyenet, P.G., 1998. Pre- and postsynaptic inhibitory actions of methionine-enkephaline on identi¢ed bulbospinal neurons of the rat RVL. J. Neurophysiol. 80, 2003^2014. Hazrati, L.N., Parent, A., 1992. Di¡erential patterns of arborization of striatal and subthalamic ¢bers in the two pallidal segments in primates. Brain Res. 598, 311^315. Hazrati, L.N., Parent, A., Mitchell, S., Haber, S.N., 1990. Evidence for interconnections between the two segments of the globus pallidus in primates : a PHA-L anterograde tracing study. Brain Res. 533, 171^175. Henry, D.J., Grandy, D.K., Lester, H.A., Davidson, N., Chavkin, C., 1995. Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes. Mol. Pharmacol. 47, 551^557. Herrera-Marschitz, M., Hokfelt, T., Ungerstedt, U., Terenius, L., Goldstein, M., 1984. E¡ect of intranigral injections of dynorphin, dynorphin fragments and alpha-neoendorphin on rotational behavior in the rat. Eur. J. Pharmacol. 102, 213^227. Hill, M.P., Brotchie, J.M., 1995. Modulation of glutamate release by a kappa-opioid receptor agonist in rodent and primate striatum. Eur. J. Pharmacol. 281, R1^R2. Ikeda, K., Kobayashi, T., Ichikawa, T., Usui, H., Kumanishi, T., 1995. Functional couplings of the delta- and the kappa-opioid receptors with the G-protein-activated Kþ channel. Biochem. Biophys. Res. Commun. 208, 302^308. Jaeger, D., Kita, H., Wilson, C.J., 1994. Surround inhibition among projection neurons is weak or nonexistent in the rat neostriatum. J. Neurophysiol. 72, 2555^2558. Kawaguchi, Y., Wilson, C.J., Emson, P.C., 1990. Projection subtypes of rat neostriatal matrix cells revealed by intracellular injection of biocytin. J. Neurosci. 10, 3421^3438. Kincaid, A.E., Penney, J.B.J., Young, A.B., Newman, S.W., 1991. Evidence for a projection from the globus pallidus to the entopeduncular nucleus in the rat. Neurosci. Lett. 128, 121^125. Kita, H., 2001. Neostriatal and globus pallidus stimulation induced inhibitory postsynaptic potentials in entopeduncular neurons in rat brain slice preparations. Neuroscience 105, 871^879. Kita, H., Kitai, S.T., 1991. Intracellular study of rat globus pallidus neurons: membrane properties and responses to neostriatal, subthalamic and nigral stimulation. Brain Res. 564, 296^305. Lee, T., Kaneko, T., Taki, K., Mizuno, N., 1997. Preprodynorphin-, preproenkephalin-, and preprotachykinin-expressing neurons in the rat neostriatum : an analysis by immunocytochemistry and retrograde tracing. J. Comp. Neurol. 22, 229^244. MacDonald, R.L., Nelson, P.G., 1978. Speci¢c-opiate-induced depression of transmitter release from dorsal root ganglion cells in culture. Science 199, 1449^1451. Maneuf, Y.P., Mitchell, I.J., Crossman, A.R., Woodru¡, G.N., Brotchie, J.M., 1995. Functional implications of kappa opioid receptor-mediated modulation of glutamate transmission in the output regions of the basal ganglia in rodent and primate models of Parkinson’s disease. Brain Res. 683, 102^108. Mulder, A.H., Wardeh, G., Hogenboom, F., Frankhuyzen, A.L., 1984. Kappa- and delta-opioid receptor agonists di¡erentially inhibit striatal dopamine and acetylcholine release. Nature 308, 278^280. Nakanishi, H., Kita, H., Kitai, S.T., 1990. Intracellular study of rat entopeduncular nucleus neurons in an in vitro slice preparation: electrical membrane properties. Brain Res. 527, 81^88. Nakanishi, H., Kita, H., Kitai, S.T., 1991. Intracellular study of rat entopeduncular nucleus neurons in an in vitro slice preparation: response to subthalamic stimulation. Brain Res. 549, 285^291. Nambu, A., Llinas, R., 1994. Electrophysiology of globus pallidus neurons in vitro. J. Neurophysiol. 72, 1127^1139. Ogura, M., Kita, H., 2000. Dynorphin exerts both postsynaptic and presynaptic e¡ects in the globus pallidus of the rat. J. Neurophysiol. 83, 3366^ 3376. Park, M.R., Falls, W.M., Kitai, S.T., 1982. An intracellular HRP study of the rat globus pallidus. I. Responses and light microscopic analysis. J. Comp. Neurol. 211, 284^294. Penny, G.R., Afsharpour, S., Kitai, S.T., 1986. The glutamate decarboxylase-, leucine enkephalin-, methionine enkephalin- and substance P-immunoreactive neurons in the neostriatum of the rat and cat: evidence for partial population overlap. Neuroscience 17, 1011^1045. Press, W.H., Flannery, B.P., Teukolsky, S.A., Vetterling, W.T., 1986. Are two distributions di¡erent? Kolmogorov^Smirnov test. In: Numerical Recipes: The Art of Scienti¢c Computing. Cambridge University Press, New York, pp. 472^475. Raynor, K., Kong, H., Chen, Y., Yasuda, K., Yu, L., Bell, G.I., Reisine, T., 1994. Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors. Mol. Pharmacol. 45, 330^334. Reiner, A., Medina, L., Haber, S.N., 1999. The distribution of dynorphinergic terminals in striatal target regions in comparison to the distribution of substance P-containing and enkephalinergic terminals in monkeys and humans. Neuroscience 88, 775^793. Sharif, N.A., Hughes, J., 1989. Discrete mapping of brain mu and delta opioid receptors using selective peptides: quantitative autoradiography, species di¡erences and comparison with kappa receptors. Peptides 10, 499^522. Simmons, M.L., Chavkin, C., 1996. U-Opioid receptor activation of a dendrotoxin-sensitive potassium channel mediates presynaptic inhibition of mossy ¢ber neurotransmitter release. Mol. Pharmacol. 50, 80^85. Sim-Selley, L.J., Daunais, J.B., Porrino, L.J., Childers, S.R., 1999. Mu and kappa1 opioid-stimulated [35S]guanylyl-5P-O-(gamma-thio)-triphosphate binding in cynomolgus monkey brain. Neuroscience 94, 651^662. Slater, P., 1982. Role of globus pallidus GABA and opiate receptors in apomorphine circling in nigro-striatal lesioned rat. Naunyn-Schmiedeberg’s Arch. Pharmacol. 319, 43^47. Slater, P., Longman, D.A., 1980. E¡ects of intrapallidal opiate receptor agonists on striatally evoked head turning. Neuropharmacology 19, 1153^ 1156. Smith, Y., Shink, E., Sidibe, M., 1998. Neuronal circuitry and synaptic connectivity of the basal ganglia. Neurosurg. Clin. N. Am. 9, 203^222. Vincent, S., Hokfelt, T., Christensson, I., Terenius, L., 1982. Immunohistochemical evidence for a dynorphin immunoreactive striato-nigral pathway. Eur. J. Pharmacol. 85, 251^252. Wagner, J.J., Terman, G.W., Chavkin, C., 1993. Endogenous dynorphins inhibit excitatory neurotransmission and block LTP induction in the hippocampus. Nature 363, 451^454. Walker, R.H., Arbuthnott, G.W., Wright, A.K., 1989. Electrophysiological and anatomical observations concerning the pallidostriatal pathway in the rat. Exp. Brain Res. 74, 303^310.
NSC 5779 25-9-02
982
M. Ogura and H. Kita
Weber, E., Barchas, J.D., 1983. Immunohistochemical distribution of dynorphin B in rat brain: relation to dynorphin A and a-neo-endorphin systems. Proc. Natl. Acad. Sci. USA 80, 1125^1129. Weisskopf, M.G., Zalutsky, R.A., Nicoll, R.A., 1993. The opioid peptide dynorphin mediates heterosynaptic depression of hippocampal mossy ¢ber synapses and modulates long-term potentiation. Nature 362, 423^427. Zamir, N., Palkovits, M., Brownstein, M.J., 1983. Distribution of immunoreactive dynorphin in the central nervous system of the rat. Brain Res. 280, 81^93. (Accepted 23 April 2002)
NSC 5779 25-9-02
Neuroscience Vol. 114, No. 4, pp. 973^982, 2002 B 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
EFFECTS OF DYNORPHIN ON RAT ENTOPEDUNCULAR NUCLEUS NEURONS IN VITRO M. OGURA and H. KITA Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee Memphis, 855 Monroe Avenue, Memphis, TN 38163, USA
Abstract/The entopeduncular nucleus (EP) receives dense neostriatal a¡erent axons that contain dynorphin (DYN, an endogenous U-receptor agonist), in addition to GABA and substance P. To examine the role of DYN in the EP, wholecell recordings were performed in rat brain slice preparations. Based on the physiological and morphological characteristics, all the neurons recorded were similar to the Type-I EP neuron described in a previous study. The U-receptor agonist dynorphin A (1-13) (DYN13) hyperpolarized and decreased the input resistance of approximately one-quarter of the EP neurons examined. The hyperpolarization was due to an increase in potassium conductance since current^voltage relationship curves obtained before and after DYN13 application crossed at the potassium equilibrium potential. In the presence of the glutamate blocker 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide and 3-(2-carboxypiperzin-4-yl)-propyl-1-phosphonic acid in arti¢cial cerebrospinal £uid, stimulation of the globus pallidus evoked bicuculline-sensitive multi-component GABAergic responses in EP neurons. Application of DYN13 equally reduced the amplitudes of the short-latency response, conceivably evoked by pallido-EP axons, and the medium-latency response, conceivably evoked by striato-EP axons. These e¡ects were reversed by bath application of a non-selective opioid antagonist naloxone or by a U-opioid receptor-selective antagonist nor-binaltorphimine dihydrochloride (nor-BNI), but not by the D-antagonist naltrindole or the W-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 . DYN13 also reduced the frequency of tetrodotoxin-insensitive miniature-inhibitory postsynaptic potential (mIPSPs) without changing their amplitude distributions. The decrease of the frequency of mIPSPs was reversible upon washing and was also completely blocked by nor-BNI. The results of the present study on the EP indicated that DYN released from striatal axons might exert at least three di¡erent e¡ects on these target nuclei. Firstly, DYN might provide negative feedback regulation of striatal GABAergic outputs at their termination sites. Secondly, DYN released from the striatal terminals might di¡use to the pallidal terminals, regulating their GABA release. Thirdly, DYN might exert a direct inhibition of EP neurons. Thus, DYN released from striatal axons might control the activity of EP neurons by reducing the GABAergic transmission and also by hyperpolarizing postsynaptic membrane. B 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: dynorphin, entopeduncular nucleus, GABAergic responses, presynaptic modulation, postsynaptic e¡ect.
the GP (Kawaguchi et al., 1990). Thus, the GP also receives striatal ¢bers containing GABA and DYN. Although injection of U-receptor agonists into the sites where DYN containing ¢bers terminate shows various behavioral e¡ects (Slater and Longman, 1980; Slater, 1982; Herrera-Marschitz et al., 1984; Friederich et al., 1987; Maneuf et al., 1995), the physiological e¡ects of DYN have not been studied extensively. We have recently found that DYN exerts both pre- and postsynaptic e¡ects on the pallidal neurons in rat brain slice preparations (Ogura and Kita, 2000). Postsynaptically, DYN hyperpolarized about one-third of pallidal neurons and, presynaptically, DYN attenuated striato-GP and intrapallidal GABAergic synaptic transmissions. It is conceivable that the activity of EP neurons is also controlled by DYN. The EP is more densely innervated by DYN containing ¢bers than the GP (Vincent et al., 1982; Zamir et al., 1983; Weber and Barchas, 1983). The EP contains U-receptors (Sharif and Hughes, 1989; SimSelley et al., 1999). Injection of the U-receptor agonist enadoline into the rat EP reduced reserpine-induced akinesia (Maneuf et al., 1995). Similarly, injection of enadoline into the internal segment of the pallidum alleviated
The entopeduncular nucleus (EP) receives major inputs from the neostriatum (Str), the globus pallidus (GP) and the subthalamic nucleus and sends the major output to the dorsal thalamus. Thus, the EP is an output nucleus of the basal ganglia. The striatal neurons projecting to the EP contain GABA, dynorphin (DYN) and substance P (Reiner et al., 1999; Penny et al., 1986; Gerfen and Young, 1988; Lee et al., 1997). DYN is an endogenous ligand of the U-opioid receptor (Chavkin et al., 1982; Brookes and Bradley, 1984; Raynor et al., 1994). Striato-EP neurons also send their collateral axons to
*Corresponding author. Tel. : +1-901-448-5234; fax: +1-901-4487193. E-mail address: [email protected] (H. Kita). Abbreviations : ACSF, arti¢cial cerebrospinal £uid; CPP, 3-(2-carboxypiperzin-4-yl)-propyl-1-phosphonic acid ; DYN, dynorphin ; EP, entopeduncular nucleus; GABA, Q-amino-butyric acid; GP, globus pallidus; (m)IPSC, (miniature-)inhibitory postsynaptic current; (m)IPSP, (miniature-)inhibitory postsynaptic potential ; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline7-sulfonamide ; nor-BNI, nor-binaltorphimine dihydrochloride; Str, neostriatum ; TTX, tetrodotoxin. 973
NSC 5779 25-9-02
974
M. Ogura and H. Kita
parkinsonian symptoms of the N-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-treated marmoset (Maneuf et al., 1995). To verify the physiological e¡ects of DYN on EP neurons, we performed whole-cell recording experiments using rat brain slice preparations. Speci¢cally, we examined the e¡ects of the U-agonist dynorphin A (1-13) (DYN13) on the neuronal membrane and on GABAergic synaptic transmissions in the EP.
EXPERIMENTAL PROCEDURES
Opioid receptor ligand Opioid receptor ligands used in this study were: the U-agonist DYN13 (1 WM); the non-selective antagonist naloxone hydrochloride (5 WM); the U-receptor-selective antagonist nor-binaltorphimine dihydrochloride (nor-BNI, 1^5 WM); the D-receptorselective antagonist naltrindole hydrochloride (1^2 WM); and the W-receptor-selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-ThrPen-Thr-NH2 (CTOP, 1^4 WM). These drugs were dissolved in deoxygenated water as concentrated stocks. The stocks were aliquoted and stored at 320‡C. They were thawed and diluted immediately before use. These opioid receptor ligands were obtained from RBI.
Slice preparations
Histology
Sprague^Dawley juvenile rats (16^21 days old) of both sexes were used. Animals were anesthetized (i.p.) with a mixture of ketamine (85 mg/kg) and xylazine (15 mg/kg) and were perfused through the heart with cold oxygenated arti¢cial cerebrospinal £uid (ACSF). After decapitation, the brains were rapidly removed and blocks containing the EP were obtained. Parasagittal slices (300 Wm thick) were cut from the blocks on a Vibroslice (Campden, UK) in ice-cold ACSF. The slices were incubated in ACSF at 37‡C for 1 h before recording. The composition of ACSF (in mM) was NaCl 124, KCl 5.0, KH2 PO4 1.24, NaHCO3 26, CaCl2 2.4, MgSO4 1.3, and glucose 10.
After recording, the slices were ¢xed overnight with a mixture of 4% paraformaldehyde and 0.2% picric acid. The ¢xed slices were rinsed several times with bu¡ered saline, incubated overnight with avidin^biotin^horseradish peroxidase complex (1% in bu¡ered saline with 0.4% Triton X-100), rinsed, and then reacted with diaminobenzidine. The slices were post-¢xed with 0.5% osmium tetroxide, in¢ltrated with a plastic resin, and mounted onto glass slides. The stained neurons were drawn under the microscope BH2 (Olympus, Tokyo, Japan) equipped with a drawing tube and a U60 dry objective. Statistics
Recording and electrical stimulation The slices were transferred to a recording chamber with oxygenated ACSF continuously perfused at a £ow rate of 2 ml/min. All recordings were performed at room temperature. Whole-cell patch recording pipettes with a tip diameter of about 1.5 Wm were pulled from 1.5 mm, thin-wall, borosilicate glass capillaries on a horizontal electrode puller (P-87, Sutter Instruments, Navato, CA, USA). The recording pipettes were ¢lled with high-Cl electrolyte containing (in mM): K-gluconate 90, KCl 50, HEPES 10, Mg-ATP 2, Na-GTP 0.2, and Neurobiotin 0.2%, with pH adjusted to 7.2 with KOH. The chloride equilibrium potential of the cells was calculated to be 325 mV by the Nernst equation when the cytoplasm of the recorded cells was fully equilibrated with 50 mM chloride. The resistance of these recording pipettes ranged from 4.0 to 8.0 M6. Neurons and recording pipettes were visualized using an infrared^di¡erential interference contrast microscope Reichert Diastar (Leica, Deer¢eld, IL, USA) with a U40 water immersion objective (Carl Zeiss, Thornwood, NY, USA) and a CCD camera Model6412 (COHU). Current and voltage clamp recordings were obtained using an ampli¢er IR183 (Neurodata Instruments, New York, NY, USA) and an electrometer AXOPATCH 200B (Axon Instruments, Foster City, CA, USA), respectively. The output of the ampli¢ers was monitored with an oscilloscope D51 (Tektronix, Beaverton, OR, USA) and a chart recorder WindoGraf (Gould Instrument Systems, Valley View, OH, USA). All data were digitized with an NEURO-CORDER Model DR-484 (Neurodata Instruments) and stored on videotape. The data were analyzed with a Macintosh computer with the data analysis program Oscilloscope, written and generously provided by Dr. C.J. Wilson. To evoke inhibitory postsynaptic potentials (IPSPs) or inhibitory postsynaptic currents (IPSCs), electrical stimulation (0.2 ms in duration) was applied through a bipolar electrode to the GP. To isolate GABAergic responses from glutamatergic ones, the N-methyl-D-aspartate (NMDA) receptor antagonist, 3-(2carboxypiperzin-4-yl)-propyl-1-phosphonic acid (CPP, 10 WM), and the non-NMDA receptor antagonist, 1,2,3,4-tetrahydro6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX, 5 WM), were applied to the bath. To record action potentialindependent miniature-IPSCs (mIPSCs), tetrodotoxin (TTX, 1 WM) was applied in addition to the glutamate blockers. The GABAA antagonist gabazine (10 WM) was used to con¢rm GABAergic responses.
All group data were expressed as a mean T standard error of the mean (S.E.M.) and analyzed statistically using a Student’s t-test or an analysis of variance (ANOVA). The Kolmogorov^ Smirnov (K^S) test (Press et al., 1986) was used to determine the statistical signi¢cance of the amplitude distributions of mIPSCs.
RESULTS
E¡ects of DYN13 on the membrane potential and the input resistance Twenty-three EP neurons with the resting membrane potential more negative than 355 mV or with the amplitude of spontaneous action potentials exceeding 60 mV were tested with bath application of DYN13. When the neurons ¢red low-frequency spikes spontaneously or upon injection of a low-intensity intracellular current, each spike was followed by a large after-hyperpolarization (Fig. 1B, D). All the recorded neurons were capable of generating repetitive ¢ring without accommodation upon strong intracellular current stimulation (data not shown), and thus were considered to be Type-I EP neurons (Nakanishi et al., 1990). Neurons exhibiting spontaneous spikes were current clamped at 365 mV before application of DYN13. DYN13 (1 WM) hyperpolarized (5.4 T 1.0 mV) the somatic membrane of ¢ve of the 23 neurons tested. The DYN13-induced hyperpolarization was accompanied by a decrease (25.2 T 1.8%, n = 5) in the input resistance (Fig. 1), and was reversed by washing (n = 2) or application of nor-BNI (1 WM, n = 3). The neurons that were hyperpolarized less than 3 mV or that had an ambiguous response with no recovery were not included in the above analysis. The DYN13 did not induce depolarization in any of the neurons tested. The current^voltage relationship curves obtained from the
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
975
Fig. 1. E¡ects of DYN13 on the resting membrane potential and the input resistance of an EP neuron. (A) A slow sweep trace of a current clamp recording of an EP neuron using a high-Cl electrolyte containing pipette. The GP was stimulated to induce depolarizing IPSPs. Current pulses with an intensity of 330 pA and a duration of 250 ms were injected to monitor the input resistance (also see F). To block glutamatergic responses, NBQX (5 WM) and CPP (10 WM) were added to the ACSF. The noisy appearance of the trace is due to spontaneous depolarizing IPSPs. Bath application of DYN13 (1 WM) hyperpolarized this EP neuron by approximately 5 mV. Decreases in the input resistance and the amplitude of IPSPs accompanied the hyperpolarization. (B^D) Responses to depolarizing and hyperpolarizing current pulses were recorded before DYN13 application (B), immediately after (C), and after a 30 min wash (D). Supra-threshold current pulses triggered repetitive spikes (B, D). (E) Current^voltage relationship curves obtained from this neuron. The membrane potentials at the terminations of current pulses were measured on four to ¢ve traces with no observable IPSPs at that time and the averages were plotted. The DYN13-induced hyperpolarization was accompanied by a decrease in the input resistance. The I^V curves for control and during DYN13 appliction cross at approximately 381 mV. (F) Examples of fast sweep tracing show responses to 330 pA current pulses and pallidal stimulation induced IPSPs before and immediately after DYN13 application.
DYN13-sensitive neurons before and after DYN13 application crossed at 383.6 T 2.3 mV (n = 5). DYN13 diminishes inhibitory postsynaptic response EP neurons receive GABAergic a¡erent inputs from both the Str and the GP. The conduction velocity of
the striatal axons is slower than that of the pallidal axons. Thus, it was expected that stimulation of the GP would evoke long- and short-latency postsynaptic responses in EP neurons. All the experiments described below were performed in the presence of NBQX (5 WM) and CPP (10 WM) in ACSF to block glutamatergic responses. Stimulation of the GP evoked bicuculline-sen-
NSC 5779 25-9-02
976
M. Ogura and H. Kita
Fig. 2. An example of an EP neurons that was not hyperpolarized by DYN13. (A) A slow sweep trace of a current clamp recording of an EP neuron using a high-Cl electrolyte containing pipette. Current pulses with the intensity of 330 pA and the duration of 250 ms were injected to monitor the input resistance. The GP was stimulated to induce depolarizing IPSPs. To block glutamatergic responses, NBQX (5 WM) and CPP (10 WM) were added to the ACSF. The noisy appearance of the trace is due to a large number of spontaneous depolarizing IPSPs. Bath application of DYN13 (1 WM) failed to hyperpolarize this EP neuron but did decrease the amplitude of the IPSPs. This DYN13 e¡ect was blocked by nor-BNI (1 WM), but not by CTOP (1 WM). (B, C) Examples of fast sweep tracing show responses to 330 pA current pulses and pallidal stimulation induced IPSPs before and immediately after DYN13 application.
sitive GABAergic responses in 17 EP neurons. Since the recording pipettes contained a high-Cl electrolyte, IPSPs and IPSCs were observed as depolarizations and inward current events, respectively. Stimulation of the GP evoked clearly separable multi-component responses in some EP neurons. For example, the neuron shown in Fig. 3 exhibited short- and very long-latency IPSCs after stimulation with a low intensity. The long-latency, exceeding 10 ms, small IPSCs were observed in only two other neurons. In all three of these neurons, the longlatency responses became inseparable when the stimulus intensity was increased. When the stimulus intensity was increased above 400 WA, IPSCs with a latency of 7.5 ms were evoked, as shown in the neuron in Fig. 3. Clearly separable short- and medium-latency IPSCs were observed in seven other neurons. In another nine neurons, only short-latency responses without apparent de£ections on the decay phase were observed. The latencies of the GP-induced short- and medium-latency IPSPs/IPSCs were 4.6 T 0.6 (n = 17) and 7.8 T 0.3 ms (n = 8), respectively. The e¡ects of DYN13 on the GABAergic responses were assessed from neurons that did not show signi¢cant changes in their membrane potentials or in their input resistance during DYN13 application (Fig. 2). Supramaximal stimulation was applied to evoke steady IPSPs or IPSCs and was tested by bath application of DYN13 (1 WM). When two separable latency responses were evoked, DYN13 almost equally reduced the amplitudes of both the short- and medium-latency responses (for an example, see Fig. 3). The data on compound IPSPs and IPSCs were grouped in here because DYN13 reduced their peak amplitudes by 33.9 T 3.3% (n = 17) in a similar degree to of the control (Fig. 4). This e¡ect was reversed
by bath application of a non-selective opioid antagonist naloxone (5 WM), and by the U-opioid receptor-selective antagonist nor-BNI (1 WM) (Fig. 2). In contrast, the D-antagonist naltrindole (1 WM) and the W-antagonist CTOP (1 WM) did not block the DYN13 e¡ect. Naloxone, nor-BNI, naltrindole and CTOP had no e¡ect on the evoked IPSPs (data not shown). These results suggest that DYN13 reduces the amplitude of evoked IPSPs through U-opioid receptor-mediated mechanisms. E¡ects of DYN13 on TTX-insensitive mIPSPs GABAergic mIPSPs were recorded in ACSF containing CPP (10 WM), NBQX (5 WM) and TTX (1 WM). Data were obtained from four EP neurons whose resting membrane potentials were not altered by DYN13. We measured spontaneous mIPSPs with the amplitude of more than twice the noise level, typically 0.3 mV. Most of the mIPSPs were less than 2 mV but occasional mIPSPs over 10 mV were also observed (Fig. 5D). The mIPSPs had approximately 6 ms rise times and 30^150 ms decay times. These four neurons exhibited mIPSPs with the frequency ranging from 0.47 to 1.84 Hz. DYN13 (1 WM) reduced the frequency of the mIPSPs to a range of 0.24^0.88 Hz. This change was statistically signi¢cant (n = 4, P 6 0.05, paired t-test). The decrease of the frequency of mIPSPs was reversible upon washing (n = 2) and was also completely blocked by nor-BNI (2 WM; n = 2; data not shown). DYN13 did not change the mean amplitude (Fig. 5B) or the amplitude distribution (P s 0.01, K^S test; Fig. 5C). These results suggest that DYN13 does not a¡ect postsynaptic GABA sensitivity, but instead modulates GABA release.
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
977
Fig. 3. E¡ects of DYN13 on pallidal stimulation induced short- and medium-latency IPSCs. Recordings were made with high-Cl electrolyte containing pipettes. Electrical stimulation was applied to the GP through a bipolar electrode. To block glutamatergic responses, NBQX (5 WM) and CPP (10 WM) were added to the ACSF. (A^C) IPSCs were recorded from an EP neuron voltage clamped at 370 mV. (A) Sample traces show GP stimulation-induced IPSCs with a threshold slightly above that of the short-latency IPSCs. The traces also show small- and long-, approximately 15 ms, latency IPSCs in this neuron. The long-latency responses could not be detected when the stimulus intensities were increased (see B, C). (B) Responses evoked by stimulus with the intensity threshold for the medium-latency IPSCs. (C) Responses evoked by stimulus with the intensity supra-maximal for the short- and medium-latency IPSCs. (D) Plots of the amplitude and the latency of IPSCs against the stimulus intensity. The amplitudes of the IPSCs were measured as shown in the inset. (E) DYN13 (1 WM) diminished the amplitudes of both the short- and medium-latency IPSCs. Washing the tissue reversed the DYN13 e¡ect. Each point in the graph represents a normalized (percentage of control) average amplitude T S.E.M. of three IPSCs.
Morphology of the recorded neurons Nineteen of the 23 neurons recorded in this study were intracellularly stained with Neurobiotin. All the stained neurons had morphological properties similar to that described for Type-I neurons in the previous study (Nakanishi et al., 1990; Kita, 2001). The neurons were of medium size, fusiform or round in shape, with the shortest diameter being 11.3 T 1.5 Wm and the longest being 24.0 T 4.9 Wm (Fig. 6). The neurons had two to ¢ve thick, slowly tapering, smooth primary dendrites that branched into secondary and tertiary dendrites with spines. These dendrites had tortuous trajectories. Many of the dendrites tapered slowly and extended for a long distance while some dendrites formed complex arborizations with abundant spines and appendages
(Fig. 6). The dendritic ¢eld of these neurons extended for 267 T 52 Wm from the soma in the sagittal plane. Their axons originated from a primary dendrite or soma and were thin, smooth and of constant diameter. Local collateral axons were not found in these neurons.
DISCUSSION
Although the EP receives a massive DYN containing ¢ber projection from the Str, the physiological e¡ects of DYN on the EP have not been previously studied. We have shown in a previous study that DYN13 exerts both post- and presynaptic e¡ects on the pallidal neurons in slice preparations (Ogura and Kita, 2000). From that study, we anticipated that DYN13 might have similar
NSC 5779 25-9-02
978
M. Ogura and H. Kita
Fig. 4. U-Receptor antagonists block the DYN13 e¡ect on evoked IPSPs/IPSCs. DYN13 (1 WM) reduced the amplitude of compound IPSPs/IPSCs to 33.9 T 3.3% (n = 17) of the control. This DYN13 e¡ect was blocked by the non-selective opioid receptor antagonist naloxone (5 WM, n = 3, 89.4 T 8.5% of the controls, P 6 0.0001, ANOVA) and by the U-opioid receptor-selective antagonist norBNI (1 WM, n = 7, 100.3 T 3.6% of the controls, P 6 0.0001, ANOVA). The W-antagonist CTOP (1 WM, 29.6 T 6.1 of the controls, n = 3) and the D-antagonist naltrindole (1 WM, 41.8 T 6.5 of the controls, n = 3) had no e¡ect (P s 0.05, ANOVA). Data are expressed as mean T S.E.M.
e¡ects on the EP neurons. The present study was undertaken to con¢rm this assumption using a whole-cell recording method in slice preparations. However, recording from EP neurons was more di⁄cult than from pallidal neurons under similar conditions. Therefore, the present study was limited to the examination of DYN13 e¡ects on the resting membrane potential and on GABAergic synaptic transmission. We also used a lower recording temperature than with the previous study in the GP to prolong the survival time of the EP neurons. The data presented here con¢rmed that DYN13 exerts both post- and presynaptic e¡ects in the EP which are very similar to those observed in pallidal neurons in slice preparations (Ogura and Kita, 2000). Types of the EP neurons recorded All the neurons recorded in the present study had ¢ring properties and somato-dendritic morphology similar to the Type-I EP neuron reported in an earlier study (Nakanishi et al., 1990). These observations suggest that the morphological and physiological properties of Type-I neurons in juvenile rats are indistinguishable from those in adult rats. In the present study, we did not obtained any Type-II neurons that are characterized by having a strong spike adaptation and strong A-current (Nakanishi et al., 1990). The failure to obtain TypeII neurons may be because their population is small and because the recordings obtained from them did not meet our recording criteria. Postsynaptic e¡ects of DYN13 DYN13 caused a hyperpolarization and a decrease in
the input resistance in ¢ve of the 23 EP neurons recorded. It is unclear whether only a subpopulation of the Type-I EP neurons was sensitive to DYN13 or whether all were sensitive but the detection method used in this study was not sensitive enough to reveal small responses. The hyperpolarization was most likely due to an activation of U-receptors because only the U-receptor-selective antagonist, not D- or W-antagonists, blocked the response. The hyperpolarization with a decrease in the input resistance was considered to be due to an increase in potassium conductance, as with the pallidal neurons. The current^voltage relationship curves before and after DYN13 application crossed at approximately 384 mV, which is very close to the potassium equilibrium potential of these neurons, as estimated by the Nernst equation. The hyperpolarization should be independent of chloride channel activation because the chloride equilibrium potential of the neurons was elevated to about 325 mV to evoke large depolarizing IPSPs. Activation of the potassium conductance by U-agonists has been reported in other brain areas (Hayar and Guyenet, 1998; Grudt and Williams, 1993). It has been shown that U-receptors and inwardly rectifying potassium channels coexpressed by Xenopus oocytes formed functional couplings (Ikeda et al., 1995; Henry et al., 1995). DYN13 e¡ects on GABAergic synaptic transmission The striato-EP ¢bers containing GABA, DYN and substance P terminate on the distal portion of the dendrites of EP neurons (Hazrati and Parent, 1992). In contrast, the pallido-EP GABA containing ¢bers terminate on the soma and proximal dendrites of the EP neurons (Hazrati et al., 1990; Kincaid et al., 1991; Smith et al., 1998). The conduction velocity of the striatal axons is signi¢cantly slower than that of the pallidal axons (Walker et al., 1989; Nakanishi et al., 1991; Kita and Kitai, 1991; Kita, 2001). Previous intracellular recording studies have shown that activation of striato-EP axons induces small- and long-latency IPSPs and activation of pallido-EP axons induces large, short-latency IPSPs in EP neurons (Park et al., 1982; Jaeger et al., 1994; Nambu and Llinas, 1994; Nakanishi et al., 1991; Kita, 2001). The latency of the striato-EP axon- and pallidoEP axon-induced IPSPs were 2.8 and 5.9 ms, respectively (Kita, 2001). In the present study using juvenile rats brain slices, pallidal stimulation also induced multicomponent IPSPs/IPSCs. Pallidal stimulation induced short-, medium- and occasional long-latency IPSPs/ IPSCs and their latencies were 4.6, 7.8 and approximately 15 ms, respectively. We consider that pallido-EP and striato-EP axons induced the short- and mediumlatency responses, respectively, although the latencies were longer than those obtained in the previous study. The longer latencies are probably due to younger rat age and to a cooler recording temperature, room temperature versus 35‡C, used in the present study. The long-, approximately 15 ms, latency responses were unable to be characterized in detail and the origin was unknown.
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
979
Fig. 5. DYN13 changed the frequency of mIPSPs without a¡ecting the amplitude distribution. mIPSPs were recorded from four EP neurons with pipettes containing the high-Cl electrolyte. The ACSF contained TTX (1 WM), NBQX (5 WM) and CPP (10 WM) to block action potentials and glutamatergic responses. (A, B) The frequency and the amplitude of mIPSCs was determined from 10 min of continuous recording under each condition. The frequency of the mIPSPs of the four neurons ranged from 0.47 to 1.84 Hz with a mean of 0.94 Hz. DYN13 (1 WM) reduced the frequency of mIPSPs to a range of 0.24^ 0.88 Hz with a mean of 0.51 Hz and this change was statistically signi¢cant (n = 4, P 6 0.05, paired t-test). The decrease of the frequency of mIPSPs was reversible upon washing. (B, C) DYN13 did not change the mean amplitude or the amplitude distribution (P s 0.01, K^S test). (D) Examples of mIPSPs. Most of the mIPSPs were less than 2 mV but mIPSPs over 10 mV were also observed. The rise time of the mIPSPs was approximately 6 ms.
DYN13 (1 WM) greatly reduced the amplitude of postsynaptic GABAergic responses in all of the EP neurons tested. This e¡ect was most likely mediated by U-receptors because the U-receptor antagonist blocked the e¡ect but D- or W-antagonists did not. Our previous study revealed that 1 WM DYN13 did not act as a partial W-agonist in the GP (Ogura and Kita, 2000). Large mIPSPs with amplitudes exceeding 10 mV were recorded from EP neurons. Based on their large amplitudes and short rise times, it was likely that pallidal axon terminals that form synapses at somata or proximal dendrites induced the large mIPSPs observed in EP neurons. DYN13 decreased the frequency of mIPSCs without changing the amplitude. This result suggested that DYN13 presynaptically reduced GABA release from the pallidal axon terminals. The reduction of neurotransmitter release by U-agonists has been reported in several brain areas including GABAergic inputs to the GP
(Ogura and Kita, 2000), glutamatergic mossy ¢bers in the hippocampus (Gannon and Terrian, 1991; Wagner et al., 1993; Weisskopf et al., 1993; Simmons and Chavkin, 1996), glutamatergic inputs to bulbospinal neurons (Hayar and Guyenet, 1998), dopaminergic ¢bers in the Str (Mulder et al., 1984), glutamate release in striatal synaptosomes (Hill and Brotchie, 1995), and the dorsal root ganglion in culture (MacDonald and Nelson, 1978).
CONCLUSION
The results of the present study on the EP and the previous study on the GP both indicated that DYN released from striatal axons might exert at least three e¡ects at the target nuclei. Firstly, DYN might provide negative feedback regulation of striatal GABAergic out-
NSC 5779 25-9-02
980
M. Ogura and H. Kita
Fig. 6. An example of a DYN-sensitive EP neuron intracellularly stained with Neurobiotin. (A) Camera-lucida drawing of the neuron shows that the neuron has a medium-sized, fusiform soma and thick, smooth primary dendrites without spines. Secondary and tertiary dendrites had spines and had tortuous trajectories. Some dendrites formed complex arborizations with abundant spines and appendages. The axon originating from the soma was cut at the surface of the section. (B, C) Photomontages of a dendrite forming complex arborizations and of the soma-proximal dendrites.
puts at their termination sites. Secondly, DYN released from the striatal terminals might di¡use to the pallidal terminals and regulate their GABA release. Thirdly, DYN might exert a direct inhibition of both GP and EP neurons. Thus, DYN released from striatal axons might control the activity of EP neurons by reducing
the GABAergic transmission and also by hyperpolarizing postsynaptic membrane. Acknowledgements.We thank Ms. Dawn Merrick for editing the manuscript. This study was supported by NIH grants NS26473 and NS-42762.
REFERENCES
Brookes, A., Bradley, P.B., 1984. Electrophysiological evidence for kappa-agonist activity of dynorphin in rat brain. Neuropharmacology 23, 207^ 210. Chavkin, C., James, I.F., Goldstein, A., 1982. Dynorphin is a speci¢c endogenous ligand of the kappa opioid receptor. Science 215, 413^ 415. Friederich, M.W., Friederich, D.P., Walker, J.M., 1987. E¡ects of dynorphin (1-8) on movement : non-opiate e¡ects and structure-activity relationship. Peptides 8, 837^840.
NSC 5779 25-9-02
Dynorphin e¡ects on the entopeduncular nucleus
981
Gannon, R.L., Terrian, D.M., 1991. U-50, 488H inhibits dynorphin and glutamate release from guinea pig hippocampal mossy ¢ber terminals. Brain Res. 548, 242^247. Gerfen, C.R., Young, W.S., 1988. Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments : an in situ hybridization histochemistry and £uorescent retrograde tracing study. Brain Res. 460, 161^167. Grudt, T.J., Williams, J.T., 1993. Kappa-opioid receptors also increase potassium conductance. Proc. Natl. Acad. Sci. USA 90, 11429^ 11432. Hayar, A., Guyenet, P.G., 1998. Pre- and postsynaptic inhibitory actions of methionine-enkephaline on identi¢ed bulbospinal neurons of the rat RVL. J. Neurophysiol. 80, 2003^2014. Hazrati, L.N., Parent, A., 1992. Di¡erential patterns of arborization of striatal and subthalamic ¢bers in the two pallidal segments in primates. Brain Res. 598, 311^315. Hazrati, L.N., Parent, A., Mitchell, S., Haber, S.N., 1990. Evidence for interconnections between the two segments of the globus pallidus in primates : a PHA-L anterograde tracing study. Brain Res. 533, 171^175. Henry, D.J., Grandy, D.K., Lester, H.A., Davidson, N., Chavkin, C., 1995. Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes. Mol. Pharmacol. 47, 551^557. Herrera-Marschitz, M., Hokfelt, T., Ungerstedt, U., Terenius, L., Goldstein, M., 1984. E¡ect of intranigral injections of dynorphin, dynorphin fragments and alpha-neoendorphin on rotational behavior in the rat. Eur. J. Pharmacol. 102, 213^227. Hill, M.P., Brotchie, J.M., 1995. Modulation of glutamate release by a kappa-opioid receptor agonist in rodent and primate striatum. Eur. J. Pharmacol. 281, R1^R2. Ikeda, K., Kobayashi, T., Ichikawa, T., Usui, H., Kumanishi, T., 1995. Functional couplings of the delta- and the kappa-opioid receptors with the G-protein-activated Kþ channel. Biochem. Biophys. Res. Commun. 208, 302^308. Jaeger, D., Kita, H., Wilson, C.J., 1994. Surround inhibition among projection neurons is weak or nonexistent in the rat neostriatum. J. Neurophysiol. 72, 2555^2558. Kawaguchi, Y., Wilson, C.J., Emson, P.C., 1990. Projection subtypes of rat neostriatal matrix cells revealed by intracellular injection of biocytin. J. Neurosci. 10, 3421^3438. Kincaid, A.E., Penney, J.B.J., Young, A.B., Newman, S.W., 1991. Evidence for a projection from the globus pallidus to the entopeduncular nucleus in the rat. Neurosci. Lett. 128, 121^125. Kita, H., 2001. Neostriatal and globus pallidus stimulation induced inhibitory postsynaptic potentials in entopeduncular neurons in rat brain slice preparations. Neuroscience 105, 871^879. Kita, H., Kitai, S.T., 1991. Intracellular study of rat globus pallidus neurons: membrane properties and responses to neostriatal, subthalamic and nigral stimulation. Brain Res. 564, 296^305. Lee, T., Kaneko, T., Taki, K., Mizuno, N., 1997. Preprodynorphin-, preproenkephalin-, and preprotachykinin-expressing neurons in the rat neostriatum : an analysis by immunocytochemistry and retrograde tracing. J. Comp. Neurol. 22, 229^244. MacDonald, R.L., Nelson, P.G., 1978. Speci¢c-opiate-induced depression of transmitter release from dorsal root ganglion cells in culture. Science 199, 1449^1451. Maneuf, Y.P., Mitchell, I.J., Crossman, A.R., Woodru¡, G.N., Brotchie, J.M., 1995. Functional implications of kappa opioid receptor-mediated modulation of glutamate transmission in the output regions of the basal ganglia in rodent and primate models of Parkinson’s disease. Brain Res. 683, 102^108. Mulder, A.H., Wardeh, G., Hogenboom, F., Frankhuyzen, A.L., 1984. Kappa- and delta-opioid receptor agonists di¡erentially inhibit striatal dopamine and acetylcholine release. Nature 308, 278^280. Nakanishi, H., Kita, H., Kitai, S.T., 1990. Intracellular study of rat entopeduncular nucleus neurons in an in vitro slice preparation: electrical membrane properties. Brain Res. 527, 81^88. Nakanishi, H., Kita, H., Kitai, S.T., 1991. Intracellular study of rat entopeduncular nucleus neurons in an in vitro slice preparation: response to subthalamic stimulation. Brain Res. 549, 285^291. Nambu, A., Llinas, R., 1994. Electrophysiology of globus pallidus neurons in vitro. J. Neurophysiol. 72, 1127^1139. Ogura, M., Kita, H., 2000. Dynorphin exerts both postsynaptic and presynaptic e¡ects in the globus pallidus of the rat. J. Neurophysiol. 83, 3366^ 3376. Park, M.R., Falls, W.M., Kitai, S.T., 1982. An intracellular HRP study of the rat globus pallidus. I. Responses and light microscopic analysis. J. Comp. Neurol. 211, 284^294. Penny, G.R., Afsharpour, S., Kitai, S.T., 1986. The glutamate decarboxylase-, leucine enkephalin-, methionine enkephalin- and substance P-immunoreactive neurons in the neostriatum of the rat and cat: evidence for partial population overlap. Neuroscience 17, 1011^1045. Press, W.H., Flannery, B.P., Teukolsky, S.A., Vetterling, W.T., 1986. Are two distributions di¡erent? Kolmogorov^Smirnov test. In: Numerical Recipes: The Art of Scienti¢c Computing. Cambridge University Press, New York, pp. 472^475. Raynor, K., Kong, H., Chen, Y., Yasuda, K., Yu, L., Bell, G.I., Reisine, T., 1994. Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors. Mol. Pharmacol. 45, 330^334. Reiner, A., Medina, L., Haber, S.N., 1999. The distribution of dynorphinergic terminals in striatal target regions in comparison to the distribution of substance P-containing and enkephalinergic terminals in monkeys and humans. Neuroscience 88, 775^793. Sharif, N.A., Hughes, J., 1989. Discrete mapping of brain mu and delta opioid receptors using selective peptides: quantitative autoradiography, species di¡erences and comparison with kappa receptors. Peptides 10, 499^522. Simmons, M.L., Chavkin, C., 1996. U-Opioid receptor activation of a dendrotoxin-sensitive potassium channel mediates presynaptic inhibition of mossy ¢ber neurotransmitter release. Mol. Pharmacol. 50, 80^85. Sim-Selley, L.J., Daunais, J.B., Porrino, L.J., Childers, S.R., 1999. Mu and kappa1 opioid-stimulated [35S]guanylyl-5P-O-(gamma-thio)-triphosphate binding in cynomolgus monkey brain. Neuroscience 94, 651^662. Slater, P., 1982. Role of globus pallidus GABA and opiate receptors in apomorphine circling in nigro-striatal lesioned rat. Naunyn-Schmiedeberg’s Arch. Pharmacol. 319, 43^47. Slater, P., Longman, D.A., 1980. E¡ects of intrapallidal opiate receptor agonists on striatally evoked head turning. Neuropharmacology 19, 1153^ 1156. Smith, Y., Shink, E., Sidibe, M., 1998. Neuronal circuitry and synaptic connectivity of the basal ganglia. Neurosurg. Clin. N. Am. 9, 203^222. Vincent, S., Hokfelt, T., Christensson, I., Terenius, L., 1982. Immunohistochemical evidence for a dynorphin immunoreactive striato-nigral pathway. Eur. J. Pharmacol. 85, 251^252. Wagner, J.J., Terman, G.W., Chavkin, C., 1993. Endogenous dynorphins inhibit excitatory neurotransmission and block LTP induction in the hippocampus. Nature 363, 451^454. Walker, R.H., Arbuthnott, G.W., Wright, A.K., 1989. Electrophysiological and anatomical observations concerning the pallidostriatal pathway in the rat. Exp. Brain Res. 74, 303^310.
NSC 5779 25-9-02
982
M. Ogura and H. Kita
Weber, E., Barchas, J.D., 1983. Immunohistochemical distribution of dynorphin B in rat brain: relation to dynorphin A and a-neo-endorphin systems. Proc. Natl. Acad. Sci. USA 80, 1125^1129. Weisskopf, M.G., Zalutsky, R.A., Nicoll, R.A., 1993. The opioid peptide dynorphin mediates heterosynaptic depression of hippocampal mossy ¢ber synapses and modulates long-term potentiation. Nature 362, 423^427. Zamir, N., Palkovits, M., Brownstein, M.J., 1983. Distribution of immunoreactive dynorphin in the central nervous system of the rat. Brain Res. 280, 81^93. (Accepted 23 April 2002)
NSC 5779 25-9-02