NeuropharmacologyVol. 30, No. 2, pp. 17%182, 1991 Printed in Great Britain
0028-3908/91 $3.00+ 0.00 Pergamon Press pie
THE EFFECTS OF APOMORPHINE ON THE HIPPOCAMPAL FIELD POTENTIAL IN FREELY MOVING RATS: PHARMACOLOGICAL EVIDENCE OF THE INVOLVEMENT OF D 2 RECEPTORS R. YANAGIHASHI,K. YAMANOUCHI and T. ISHIKAWA Department of Physiology and Biochemistry, School of Nursing, Chiba University, 1-8-1 Inohana, Chiba 280, Japan
(Accepted 31 July 1990) Summary--The effects of apomorphine on the hippocampal field potential of dentate granule cells were investigated in freely-moving male Sprague-Dawley rats. Five sequential field potentials were recorded from the dentate gyrus of the dorsal hippocampus, by stimulating the perforant path in the entorhinal cortex at 30 sec intervals. The slope of the population excitatory postsynaptic potential (EPSP) slope and the amplitude of the population spike of these field potentials were analyzed and averaged with a computer. The effects of apomorphine were observed at intervals of 15 min over 2 hr. Although the slope of the population EPSP showed no significant change after the administration of apomorphine (1.0 mg/kg, i.p.), the amplitude of the population spike was enhanced by about 30%. This enhancement continued for about 90 rain. These results suggest that the apomorphine does not change the synaptic input from the perforant path to the granule cells but enhances the excitability of the hippocampal dentate granule cells. This effect of apomorphine on the amplitude of the population spike was decreased by sulpiride (20 mg/kg, i.p.) but was not affected by SCH-23390 (0.1 mg/kg, i.p.). These results lead to the conclusion that the enhancement of the excitability of the dentate granule cells by apomorphine is caused by the activation of the postsynaptic D 2 receptors.
Key words--hippocampus, granule cell, field potential, apomorphine, D 2 receptor, freely-moving rat.
Many studies have suggested that the hippocampus contains only small amounts of dopamine and that it does not receive any dopaminergic innervation. Recent studies, however, have shown, using recently developed histochemical and biochemical techniques, that the hippocampus receives a dopaminergic innervation (Bischoff, Scatton and Korf, 1979; Fuxe, Agnati, Kalia, Goidstein, Andersson and Hartstrand, 1985; Ishikawa, Ott and McGaugh, 1982; Reymann, Pohle, Muller-Welde and Ott, 1983; Scatton, Simon, Le Moal and Bischoff, 1980). Electrophysiological studies, using the hippocampal slice technique, recently reported that spontaneous activity in hippocampal CA I cells was modulated by dopamine or other dopaminergic agonists (Smialowski and Bijak, 1987). Other studies have reported that the amplitude of the field potential in the CA1 area, evoked by the stimulation of mossy fibres, was changed by the infusion of dopamine (Gribkoff and Ashe, 1984; Marciani, Calabresi, Stanzione and Bernardi, 1984). These electrophysiological reports in vitro, using a hippocampal slice, suggest the possibility of a dopaminergic innervation of the hippocampus. However, it is not known whether dopamine receptors in the hippocampus have any physiological role in the in vivo non-anesthetized condition. Additionally, the recording site for these experiments was restricted to
the CA I area; there have been few studies in the granule cell layer. In the present experiment, freely moving rats were used and the hippocampal field potential in the dentate gyrus, evoked by the stimulation of the perforant path in the entorhinal cortex was examined. Furthermore, the effects of apomorphine, a dopamine agonist, on the entorhinal-granule cell synapse were investigated. METHODS
Preparation of subjects Male Sprague-Dawley rats, weighing 300-450 g (Charles River, Japan Inc.), were anesthetized with an intraperitoneal (i.p.) injection of amobarbital sodium (100 mg/kg). The skull was exposed and holes for the placement of electrodes were drilled with a dental burr. A monopolar recording electrode, consisting of a teflon-coated 50/~m diameter stainlesssteel wire, was placed in the left dentate gyrus of the dorsal hippocampus. Auditory monitoring of multiple-unit activity guided the placement (Lomo, 1971). A monopolar stimulating electrode, consisting of a teflon-coated lO0/~m diameter stainless-steel wire, was then lowered into the ipsilateral angular bundle of the perforant path (McNaughton and 177
skull, over the left side of the motor cortex, to serve as recording electrodes for the electroencephalogram (EEG). Three additional screws were placed on the left frontal, the left occipital portions and the right side of the skull, to serve as reference electrodes for recording and stimulation, and as the ground electrode• All electrodes were connected to 9 pin-connector strip. This assembly was attached to the skull with dental acrylic cement.
The recording of the hippocampal field potential Fig. 1. Schematic design of implanted electrodes for recording of field potentials. CAI and CA2/3 indicate fields CA1 and CA2/3 of Ammon's horn, respectively; CC and DG indicate the corpus callosum and the dentate gyrus, respectively. Barnes, 1977). Figure 1 shows a schematic diagram of the sites for the implantation of these electrodes. The implantations in the dentate and the perforant path were performed, relative to the bregrna, 3.8 mm posterior, 2.3 mm lateral and 7.8 mm posterior, 4.5 mm lateral, according to the stereotaxic coordinates of Paxinos and Watson (1986). The exact depth of both electrodes was determined by the laminar analysis of field potentials, evoked by stimulation of the perforant path (Fig. 2). A pair of small stainless-steel screws (l.25mm in diameter) were affixed to the
All experiments were carried out following a recovery period of at least 1 month. The hippocampal field potentials were amplified with filter settings of 1.5 Hz and 3 kHz and monitored by an oscilloscope (VC-10, NIHON KOHDEN). A dual-channel field effect transistor (FET; 2SKI8A, Toshiba Electric, Tokyo, Japan) was mounted directly on the head of the recording cable in order to eliminate movement artifacts. Field potentials were digitized at sampling intervals of 40 # sec by a mini-computer (ATAC-450, NIHON KOHDEN), then loaded onto the floppy disks of a personal computer (PC-9801, NEC) through a GPIB interface• The cortical EEG was monitored and recorded on paper. Stimulation of the perforant path was delivered by an electronic stimulator (SEN-7103, NIHON
2.60 " 2.90 -
~CA1 3.06 "
3.28 " 3.29 "
CA 1-3: fields CA 1-3 of Ammon's horn corpus callosum dentate gyrus HiF: hippocampal fissure
Fig. 2. The depth profile of hippocampal field potentials by stimulation of the perforant path. During the implanting operation, a laminar analysis of the hippocampal field potential was carried out in each rat, in order to determine the ventral position of both recording and stimulating electrodes. The reversal of the field potential was used as a marker and the recording electrode was fixed in a position where the amplitude of the population spike of the field potential was largest. St indicates stimulation of the perforant path.
Effect of apomorphine on hippocampus KOHDEN), with an isolator (SS-201J, N I H O N KOHDEN). The stimulation parameters consisted of constant current single biphasic square wave pulses, each with a 50--400/z sec half-duration and a 100-500 # A peak-to-peak amplitude. In the preliminary experiment, the relationship between the strength of the stimulus to the perforant path and the field potential was examined by increasing the pulse duration, in order to determine the intensity of stimulation to be used in the experiment (Fig. 3B). A stimulation intensity (pulse duration) which caused about 50% of the maximum response was determined for individual rats and was used in the subsequent experiments. In order to examine the relationship between the slope of the population EPSP and the amplitude of the population spike, various intensities of stimulation were used in a few rats.
Experimental design All sessions of the recording of field potentials were carried out at intervals of 15 min. Five sequential field potentials in each session were recorded at 30 sec intervals and were averaged by a computer. After the control response was recorded for 1 hr (4 sessions), apomorphine (0.1, 0.5 and l mg/kg) or saline (I ml/kg) was administered intraperitoneally and changes in the field potentials were observed for A
2hr (8 sessions). For the antagonist studies, the D~ antagonist, R(+)-8-chloro-2,3,4,5-tetrahydro-3methyl-5-phenyl-lH-3-benzazepin-7-ol HCI (SCH23390, 0.1 mg/kg) or the D2 antagonist, sulpiride (20 mg/kg), was administered 15 min prior to the injection of apomorphine. A muscarinic antagonist, atropine (3 mg/kg) was administered 1 hr prior to the injection of apomorphine.
Analysis of data A monosynaptic field potential, evoked by a single pulse stimulation of the perforant path, was divided into two components; a positive-going extracellular excitatory synaptic potential, called "the population EPSP" and a superimposed negative-going "population spike", reflecting the synchronous discharge of dentate granule cells (Fig. 3A). The maximum slope of the population EPSP and the amplitude of the population spike were calculated using a computer (NEC, PC-9801). The relationship between the slope of the population EPSP and the amplitude of the population spike, was examined in order to determine whether the effects of apomorphine primarily reflected the presynaptic or the postsynaptic effect. A paired-pulse stimulation of the perforant path was delivered in order to examine the strength of the feed-back inhibition in several rats. The ratio of the 2nd stimulation of the population spike to the 1st stimulation of the population spike (P2/P1) was calculated. The duration of the EEG desynchronization induced, by apomorphine was measured from the EEG record. This duration was termed as the interval from the time of injection of apomorphine, to the first appearance time of slow or spindle wave on the cortical EEG.
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Fig. 3. (A) An example of the hippocampal field potentials evoked by stimulation of the perforant path. The pEPSP indicates the slope of the population EPSP and PS indicates the amplitude of the population spike. (B) An example of the relationship between the stimulus intensity (pulse duration) and the response (the pEPSP or the PS). The right and the left ordinates show the amplitude of the population spike and the slope of the population EPSP, respectively. The abscissa shows the strength of the stimulation of the perforant path (pulse duration). Each dot represents a mean value of 5 responses. NP
Apomorphine hydrochloride and atropine sulfate were purchased from Sigma Chemical, while the SCH-23390 was purchased from Research Biochemicals Inc. and the sulpiride (Dogrnatyl injection) was purchased from Fujisawa Pharmaceutical Co., Ltd. The apomorphine, SCH-23390 and atropine were dissolved in saline (1, 0.1 and 3 mg/ml, respectively). The sulpiride was diluted with saline (20 mg/ml).
The slope of the population EPSP, the amplitude of the population spike and the duration of EEG desynchronization are shown as means + SEM, The significance of difference between the control and drug-treated groups was calculated using analysis of variance. RESULTS
After intraperitoneal administration of apomorphine (1 mg/kg), the slope of the population EPSP
R. YANAGIHASHIet al.
showed no significant change, but the amplitude of the population spike was enhanced by about 30% over the control. This enhancement in the amplitude of the population spike continued for about 90min (Fig. 4). The injection of 0.1 or 0.5mg/kg of apomorphine showed no significant change in the amplitude of the population spike, but tended to enhance it. Figure 5 shows the relationship between the slope of the population EPSP and the amplitude of the population spike. The regression line shifted to the left but its slope was not changed by apomorphine (1 mg/kg, i.p.). The X-intercept of the control was 3.15 mV/msec and the X-intercept after administration of apomorphine was 1.47 mV/msec, in this sample. The effect of dopaminergic antagonism on the effect of apomorphine was examined. The enhancing effect of apomorphine on the amplitude of the population spike was diminished by pre-treatment with sulpiride (20 mg/kg i.p.), the D2 antagonist but not by the SCH-23390 (0.1 mg/kg i.p.), the D~ antagonist (Fig. 6). The pre-administration of atropine (3 mg/kg, i.p.) did not affect the enhancing effect of apomorphine. That is, the amplitude of the population spike was significantly increased by about 30% for at least ~50
60 time ( min )
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Fig. 4. Effects of the apomorphine (1 mg/kg, i.p.) on the hippocampal field potential. (A) Shows the effects on the slope of the population EPSP (pEPSP) and (B) the effects on the amplitude of the population spike (PS). The ordinates show the ratio (%) of the response after the administration of apomorphine or saline to the response prior to administration and the abscissa shows the time after the administration of the apomorphine. Each dot represents a mean value of 5 rats and the vertical bars show the SEM. Asterisks (*) mark significant deviations from the control group (P < 0.05).
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Fig. 5. The relationship between the slope of the population EPSP (pEPSP) and the amplitude of the population spike (PS) on the hippocampal field potential. The broken and solid lines show the regression lines of an apomorphinetreated rat (r = 0.95, n = 26) and a control rat (r = 0.91, n = 18), respectively. "k" marks the slope of these regression lines.
90min after the administration of apomorphine (1 mg/kg, i.p.), in atropine-treated rats. In order to examine the strength of the recurrent inhibition, the paired-pulse depression was investigated at 30 msec interpuise intervals. In apomorphine-treated rats, the ratio of P2/PI (%) was about 50% in the control. This ratio showed no significant change in any sessions after the administration of apomorphine. There was no significant difference between the saline-treated and the apomorphine-treated rats. The cortical EEG showed an arousal pattern after the administration of apomorphine. The desynchronization continued for about 50 min after the administration of apomorphine (1 mg/kg) (Table 1). The duration of the desynchronization of the cortical EEG was shortened to half by pretreatment
Fig. 6. Effects of pre-treatment with SCH-23390(0.1 mg/kg, i.p.) or sulpiride (20 mg/kg, i.p.) on the hippocampal field potential, after the administration of apomorphinc (1 mg/kg, i.p.). The ordinate shows the ratio (%) of the amplitude of the population spike (PS) after the administration of apomorphine to the amplitude of the population spike before administration. A, B, C and D indicate the control, the apomorphine-treated, the SCH-23390 pretreated and the sulpiride pre-treated group, respectively. Each column shows a mean of +SEM (n = 5) at 60min after the administration of apomorphine, and the asterisks (*) indicate significant deviations from the control groups (P < 0.05).
Effect of apomorphine on hippocampus Table 1. Effects of pre-treatment with SCH-23390 (0.1 mg/kg, i.p.) or sulpiride (20 mg/kg, i.p.) on the duration of the EEG desynchronization after administration of apomorphine (I mg/kg, i.p.)
suppressed by D~ receptors and activated by D2 receptors. The present experiment suggests that the dentate Mean + SEM Number granule cells, as well as the CA1 pyramidal cells, Drugs (rain) of rats receive a dopaminergic innervation. Although it was Saline 19.5 + 3.5 4 Apomorphine 54.0 + 9.5* 5 suggested that the dentate granule cells were excited SCH-23390 + apomorphine 24.6 + 13.3 5 by the activation of D2 receptors, the present experSulpiride + apomorphine 54.4 + 9.9* 5 iment, which was carried out under physiological *P < 0.01 compared to saline. conditions using freely moving rats, did not demonstrate the involvement of D~ receptors in the dentate with SCH-23390 (0.1 mg/kg) but not by sulpiride granule cells. It is believed that the dose (0.1 mg/kg) of SCH-23390, which was used in the present exper(20 mg/kg). iment, was sufficient to antagonize the D~ receptor, because the EEG arousal induced by apomorphine, DISCUSSION was antagonized by the injection of 0.1 mg/kg of In the field potential evoked by stimulation of the SCH-23390. This agrees with the paper by Ongini, perforant path, the amplitude of the population spike Caporali and Massotti (1986), reporting that the significantly increased with apomorphine, but the EEG desynchronization induced by apomorphine slope of the population EPSP did not show any (1 mg/kg, i.v.), was prevented by SCH-23390 (0.003, significant change. The I/O curve (the relationship 0.01 and 0.1 mg/kg; i.v.) in rabbits. It is possible between the slope of the population EPSP and the that the D~ receptors have another physiological role amplitude of the population spike) shifted to the left in vivo in the dentate granule cells. There is the possibility that the effects of apomorafter the administration of apomorphine. That is, the X-intercept after the administration of apomorphine phine are indirect and are mediated by other systems. was significantly less than before the administration, Gribkoff and Ashe (1984b) reported that the enhancalthough the slope of this curve did not change. Maru ing effect of dopamine on the population spike in and Goddard (1987) reported that cellular excitability the CA I area, was caused by the reduction of the could be defined as the ratio of the cellular output to inhibitory influences of the paired-pulse paradigm. the synaptic input. Furthermore, they stated that this Robinson, Malthe-Sjorenssen, Wood and Commiscould be estimated from the regression of the ampli- sioning (1979) suggested that the septo-hippocampal tudes of the population spikes to the magnitudes of cholinergic pathway was controlled by a dopaminthe population EPSP, at various intensities of stimu- ergic system. In the present experiment, however, the lation. The results of the present experiment suggest enhancing effect of apomorphine on the granule cells, that apomorphine did not change the synaptic input was not the result of a reduction in inhibitory influfrom the perforant path to the granule cells but ences, since the P2/P1 values, which were calculated enhanced the excitability of the hippocampal dentate in this experiment using paired-pulse stimulation, did granule cells. not change with the administration of apomorphine. The enhancing effect of apomorphine on the The pre-administration of atropine did not affect amplitude of the population spike was antagonized the enhancing effect of the apomorphine, suggesting by sulpiride (20 mg/kg, i.p.) but not by SCH-23390 that the effect of apomorphine was not mediated (0.1 mg/kg, i.p.). It is well known that sulpiride is a by a cholinergic action. The results of the present selective D2 antagonist and that SCH-23390 is a experiments suggest that the effect of apomorphine selective D~ antagonist. Therefore, it is believed that on the hippocampal field potential is likely to be a the enhancing effect of apomorphine on the excit- direct dopaminergic effect on the dentate granule ability of the dentate granule cells was the result of cells rather than a secondary effect caused by other the activity of the postsynaptic D 2 receptors. systems (e.g.),-aminobutyric acid (GABAergic) or Gribkoff and Ashe (1984a) reported that the cholinergic systems). population response in CA1 pyramidal cells, evoked Recent biochemical and histochemical studies reby stimulation of the Schaffer-collateral, showed an port the existence of a dopaminergic innervation of initial decrease, followed by a profound increase in the hippocampus. Bischoff et al. (1979) reported that the hippocampal slice and that this increased effect the intraperitoneal injection of haloperidol in aneswas blocked by the D 2 antagonist, spiroperidol. thetized rats, produced an increase in the levels of 8"miatowski and Bijak (1987) reported that a D 1 3,4-dihydroxyphenylacetic acid (DOPAC) in the hipagonist suppressed the spontaneous firing in CA! pocampus and that treatment with apomorphine pyramidal cells and that a D 2 agonist, pergolide produced a decrease in the level of the DOPAC in the enhanced it in the hippocampal slice. These studies hippocampus. They also reported that a pharmacoand an intracellular study by Bernardo and Prince logical sympathectomy, followed by an injection of (1982), suggested that some CA1 pyramidal cells 6-hydroxydopamine (6-OHDA), laterally into the in the hippocampus were innervated by dopamin- pedunculus cerebellaris superior, produced a decrease ergic neurons and that these CA I cells were in the level of dopamine in the hippocampus. They
R. YANAGIHASHIet al.
concluded that these findings reliably reflected the presence of dopaminergic neurons in the hippocampus. In another biochemical study, Ishikawa et al. (1982) reported that the concentration of dopamine in the dorsal part of the hippocampus was found to be 4--10 times greater than in other parts and that the ratio of homovanillic acid (HVA)/DOPAC increased after treatment with haloperidol. These findings suggest that a dopaminergic innervation exists in at least the dorsal part of the hippocampus. Scatton et al. (1980), using lesion studies of the ventral tegmental area (A10) or substantia nigra (A9), reported that the dopaminergic afferents to the hippocampal formation originated from A10 and A9 dopaminergic cell groups. However, in a histochemical study, using the retrograde labelling method of Granule Blue, Reymann et al. (1983) reported that the mesencephalic dopaminergic fibers which innervate the hippocampus arose from the raphe nuclei, rather than from the ventral tegmental area. Although the present study does not reveal an origin, it can be speculated that dopaminergic neurons, from AI0 and A9 cell groups in the dorsal tegmental area or from the raphe nuclei, can modulate the activity of dentate granule cells.
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