Two different 19-mer antisense oligodeoxynucleotides complementary to the initial coding regions of dopamine D2 or D3 receptor mRNA were infused unilaterally into the substantia nigra of rats for 3–6 d to suppress synthesis of D2 and/or D3 receptors on substantia nigra dopaminergic neurons, thereby producing specific reductions of D2 and/or D3 receptors. Autoradiographic receptor binding revealed that D2 and D3antisense oligodeoxynucleotides specifically and significantly reduced D2 or D3 binding in the ipsilateral substantia nigra, respectively, without affecting dopamine receptor binding in the neostriatum. Either D2 or D3 antisense oligodeoxynucleotides greatly attenuated the ability of apomorphine to inhibit dopaminergic neurons in vivo, an effect that was potentiated by simultaneous administration of D2 and D3 antisenses. Despite these effects, neither the rate nor the pattern of spontaneous activity of antisense-treated nigrostriatal neurons differed from those in the control groups. The proportion of antidromic responses consisting of full spikes from antisense-treated rats was significantly greater, and the mean antidromic threshold was significantly lower than in controls, indicating that autoreceptor knockdown increased both somatodendritic and terminal excitability. These data demonstrate that selective reduction of specific dopamine receptor subtypes by antisense infusion can be effected in vivo, and that nigrostriatal neurons express both D2 and D3 autoreceptors at their somatodendritic and axon terminal regions. Although the somatodendritic and terminal autoreceptors modulate dendritic and terminal excitability, respectively, the interaction of endogenously released dopamine with somatodendritic autoreceptors does not appear to exert a significant effect on spontaneous activity in anesthetized rats.
- antisense oligodeoxynucleotide
- dopamine receptor subtype
- D2 receptor
- D3 receptor
- substantia nigra
Mesencephalic dopaminergic neurons express receptors for their own neurotransmitter, dopamine (DA), in their somatodendritic and axon terminal regions. Stimulation of somatodendritic autoreceptors inhibits the spontaneous activity of dopaminergic neurons (Bunney et al., 1973; Groves et al., 1975), whereas stimulation of the axon terminal autoreceptors reduces the excitability of DAergic axon terminals and inhibits DA synthesis and release (Tepper et al., 1987; Starke et al., 1989; Wolf and Roth, 1990).
DA receptors were originally classified as D1 or D2 receptors based on their differing affinities for various ligands and linkage to intracellular signaling pathways (Kebabian and Calne, 1979). Within this classification, the somatodendritic autoreceptor was identified as a D2receptor on pharmacological (Morelli et al., 1988), electrophysiological (Lacey et al., 1987), and molecular biological (Meador-Woodruff et al., 1991) grounds. The axon terminal autoreceptor was similarly identified as a D2 receptor pharmacologically (Boyar and Altar, 1987; Starke et al., 1989) and electrophysiologically (Tepper et al., 1984a,b).
However, advances in molecular biology have rendered this “identification” uncertain, because it is now clear that twofamilies of DA receptors exist (Sibley and Monsma, 1992). The D1-class family consists of D1 and D5receptors and the D2-class family of D2L or Sisoforms and D3 and D4 receptors. (Nonsubscripted receptor subtypes, e.g., D1-class, refer to the receptor family, whereas subscripted receptor subtypes, e.g., D2, refer to the molecular biologically defined specific receptor subtypes.)
Relatively large amounts of D2 mRNA exist in the midbrain, localized to dopaminergic neurons; however, the existence of D3 receptors in midbrain is more controversial. Some studies report D3 mRNA in the substantia nigra–ventral tegmental area (Sokoloff et al., 1990; Bouthenet et al., 1991), whereas others failed to find D3 mRNA or found it only in trace amounts (Valerio et al., 1994; Diaz et al., 1995; Le Moine and Bloch, 1996). On the other hand, it has recently been argued on pharmacological grounds that some DA autoreceptors may, in fact, be D3 receptors (Meller et al., 1993; Tang et al., 1994; Chesi et al., 1995; Kreiss et al., 1995; Lejeune and Millan, 1995; Nissbrandt et al., 1995). Because the K Ds for most agonists and antagonists are within one order of magnitude between D2 and D3 receptors (Gingrich and Caron, 1993;Seeman and Van Tol, 1994; Burris et al., 1995), they cannot be used to discriminate, at least in a physiologically useful manner, among members within the D2-class family. Thus, the precise subtype(s) of the somatodendritic and terminal autoreceptors remains unknown.
Antisense knockdown refers to the ability of specifically designed short sequences of oligodeoxynucleotides to bind to their complementary mRNA and stop translation, thereby preventing the expression of the protein that the mRNA codes for. It is highly specific, and can be used to probe the functions of receptor subtypes for which specific and selective agonists or antagonists do not exist (Zhang and Creese, 1993). Here we describe the electrophysiological consequences of antisense knockdown of DA D2, and/or D3receptors in DAergic nigrostriatal neurons.
MATERIALS AND METHODS
Subjects. Eighty-three male Harlan Sprague Dawley rats (Zivic-Miller) weighing between 150 and 250 gm at the time of initial surgery were used in these experiments. All animals were treated in strict accordance with guidelines set forth in the Public Health Service manual “Guide for the Care and Use of Laboratory Animals.”
Antisense treatment. Rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (15 mg/kg) intraperitoneally and placed in a stereotaxic apparatus. Under aseptic conditions, the scalp was reflected and a small burr hole was drilled overlying the left substantia nigra [anterior: 2.0 (from lambda); lateral: 4.7 (at a 20° angle; see below)]. A 28 ga stainless steel infusion guide cannula (Plastics One, Roanoke, VA) was lowered 5.9 mm from the cortical surface and affixed with cyanoacrylate glue and dental cement. After a 24 hr recovery period, the inner 33 ga injection cannula, which was 1 mm longer than the guide, was filled, inserted into the guide, and connected to a length of Teflon tubing connected through an Instech Lab fluid swivel to a Harvard Apparatus Model digital 22 syringe pump. To minimize mechanical damage to the pars compacta, the nigral cannula was inserted at an angle of 20° to the midline such that the tip of the inner cannula was positioned ∼500 μm dorsal and lateral to the center of the pars compacta. Oligodeoxynucleotides (10–20 μg/μl) or saline vehicle were infused continuously at 0.1 μl/hr for 3–6 d while the animals were housed in individual circular Plexiglas cages with access to food and water ad libitum. During the course of the antisense treatment, some animals were observed for two 30 min periods each day (at approximately 10:00 A.M. and 7:00 P.M.), and rotational behavior was recorded manually. A rotation was scored when a rat traversed 360° in either direction, and the total number of rotations was calculated by subtracting the number of ipsilateral rotations from the number of contralateral rotations that occurred during both 30 min periods.
Antisense oligodeoxynucleotides. The D2antisense oligodeoxynucleotide (AODN) was a 19-mer complementary to codons 2–8 (nucleotides 4–22) of the D2 receptor mRNA with sequence 5′-AGGACAGGTTCAGTGGATC-3′. The D3 AODN, also directed against codons 2–8 (nucleotides 4–22), had the sequence 5′-TTATCTGGCTCAGAGGTGC-3′. There were two AODN controls: a D2 random AODN consisted of the same bases as in the D2 AODN in pseudorandom order with 11 of the 19 bases mismatched from the sense mRNA (5′-AGAACGGCACTTAGTGGGT-3′) and a D3 random oligodeoxynucleotide that consisted of the same bases as in the D3 antisense AODN but with 14 bases mismatched (5′-ATTCTGAGTTCGGTCAGCG-3′). Neither of the two random oligodeoxynucleotides is complementary to any other known sequence. All oligodeoxynucleotides consisted ofS-oligodeoxynucleotides, in which the phosphodiester backbone of the nucleotide was modified by the inclusion of a phosphorothioate to increase the resistance of the nucleotide to degradation by endogenous nucleases (Agrawal et al., 1991; Sklarczyk and Kaczmarek, 1995). All AODNs were purchased from Oligos, Etc. (Wilsonville, OR).
Stimulating and recording. For acute recording experiments, rats were anesthetized with urethane (1.3 gm/kg, i.p.), the left femoral vein or a lateral tail vein was cannulated, and the rat was installed into a stereotaxic frame. Body temperature was maintained at 37 ± 1°C with a thermostatically controlled solid-state heating device. All wound edges and contact points between the animal and the stereotaxic frame were infiltrated with lidocaine ointment (5%) or solution (2%).
After removal of the scalp, a small burr hole was drilled overlying the neostriatum (1.0 mm anterior to bregma, 3.7 mm lateral to the midline) for the insertion of stimulating electrodes. Bipolar stimulating electrodes with a tip separation of ∼150 μm and in vitroimpedances of ∼10 kΩ were formed from 100-μm-diameter enamel-coated stainless steel wires (California Fine Wire). After releasing some CSF by puncturing the atlanto-occipital membrane, stimulating electrodes were lowered 4.0 mm below the cortical surface and cemented in place with cyanoacrylate glue and dental cement. For recording, holes ∼3.0 mm in diameter were drilled above the substantia nigra at coordinates 2.1 mm anterior to lambda and 2.0 mm lateral to the midline, and the infusion cannulae were carefully removed.
Recording electrodes were fabricated from 2.0 mm outer diameter capillary tubing (World Precision Instruments, Kwik-filr) on a Narishige PE-2 vertical pipette puller and possessed in vitro impedances of ∼20 MΩ when filled with 2 mNaCl. The electrode impedance was lowered to between 4 and 10 MΩ by passing 500 msec 150 V direct current pulses (Grass stimulator, model S-48) through the electrode in vitro.
Constant current electrical stimuli were generated with a Winston A-65 timer/stimulator and SC-100 constant current stimulus isolators. Neostriatal stimuli consisted of monophasic square-wave pulses of 0.2–3.0 mA intensity and 100–500 μsec duration and were delivered at a rate of 0.67 Hz. The antidromic threshold current was defined as the minimum current sufficient to elicit antidromic responses on 100% of the trials on which a collision with a spontaneous spike did not occur (Tepper et al., 1984a). Single unit extracellular recordings were amplified with a Neurodata IR-183 preamplifier and displayed on a Tektronix 5113A storage oscilloscope. All data were recorded on magnetic tape for off-line analysis on a Macintosh IIfx computer with a National Instruments MIO16L multifunction board and custom-designed software.
DAergic neurons were identified by their extracellular waveforms, often characterized by a prominent notch in the initial positive phase and having a duration of 2–5 msec, slow spontaneous activity, and long latency antidromic responses evoked from neostriatum that consisted mostly of initial segment (IS) only spikes (Deniau et al., 1978;Guyenet and Aghajanian, 1978; Tepper et al., 1984a,b). The pattern of firing of each neuron was classified as pacemaker, random, or bursty based on the neuron’s autocorrelation histogram. Neurons showing three or more peaks in the autocorrelogram were classified as pacemaker, those showing an initial peak followed by a decay to a steady state were classified as bursty, and the remainder as random (Tepper et al., 1995). The proportion of antidromic spikes evoked at the threshold current for each neuron that consisted of the full spike (initial segment plus somatodendritic components) was taken as an index of somatodendritic excitability (Trent and Tepper, 1991).
Inhibitory responses after striatal stimulation were analyzed by constructing peristimulus time histograms and cumulative sum histograms (CUSUMs) (Ellaway, 1977) generated from spike trains. Changes in the slope of CUSUMs were used to delineate the onset, offset, and duration of stimulus-evoked effects as described in detail by Tepper et al. (1995). The average firing rate during the interval so defined was calculated by dividing the average number of spikes per bin during this interval by the prestimulus baseline activity.
Apomorphine dose–response measurements. After the establishment of a stable baseline firing rate for at least 5 min, a dose of apomorphine HCl that was double the previous dose was injected intravenously every 2 min, starting with either 1 or 2 μg/kg. This was continued until complete inhibition of spontaneous activity was obtained, a cumulative dose of 2048 μg/kg was reached, or until the cell was lost. In some cases in which complete inhibition was obtained, haloperidol lactate (50–200 μg/kg, i.v.) was subsequently administered in an attempt to reverse the inhibition. In most cases, the effects of apomorphine were studied on only one cell per animal. However, in a few cases, dose–response experiments were conducted on two cells, one ipsilateral to the infused side and one on the contralateral side. In these cases, haloperidol was not administered to the first cell studied, and a minimum of 3 hr elapsed between the last injection of apomorphine for the first cell and the beginning of the collection of baseline data for the second neuron.
Autoradiography. For receptor autoradiography, animals were euthanized by overdose of urethane, and the brains were rapidly removed and either frozen in isopentane cooled with dry ice or placed in a −80°C freezer. Subsequently, 20 μm coronal sections were taken on a cryostat at −18°C, thaw-mounted on gelatin-subbed slides, and stored at −80°C. Immediately before beginning the labeling incubation, the sections were gradually brought to room temperature, preincubated for 30 min in 50 mm Tris-HCl buffer, pH 7.4, containing 120 mm NaCl, 2 mm CaCl2, 5 mm KCl, 1 mm MgCl2, and 0.001% ascorbic acid (for D2-class binding) or 20 mm3-(N-morpholino)propanesulfonic acid, pH 7.2, containing 50 mm NaCl, 1 mm EDTA, 10 μmpargyline, and 0.1% ascorbic acid (for D3 binding), and then incubated for 60 min at 22°C in the same buffer containing 1.2 nm 3H-spiperone for D2-class binding or 3 nm 3H-7-OH-N,N-di-n-propyl-2-aminotetralin (7-OHDPAT) for D3 binding. Nonspecific D2-class binding was determined by incubation in the presence of 1 μm (+) flupentixol, and nonspecific D3 binding was determined in the presence of 1 μm eticlopride. Incubation was terminated by rinsing the slides twice for 5 min with ice-cold buffer. After washing, the slides were dipped quickly in ice-cold water and dried under a stream of cold air. For D1-class binding, the buffer, incubation, and exposure times were the same as for D2-class binding, except that 1 nm 3H-SCH23390 was used in place of 3H-spiperone. Slides were then placed in x-ray cassettes, together with [3H] microscales (Amersham), and exposed to Hyperfilm-[3H] (Amersham) for 2 weeks (for3H-spiperone binding) or for 2 months (for3H-7-OHDPAT binding) at 4°C. Average binding densities were determined with a computerized image analysis system (Microcomputer Imaging Device, Imaging Research, St. Catharine’s, Ontario, Canada).
Histological analyses. At the end of the recording experiment, animals that were not used for autoradiographic determination of the extent of receptor knockdown were deeply anesthetized with urethane and perfused intracardially with 50 ml of saline followed by 250 ml of 4% paraformaldehyde. The brain was then removed and placed into fixative overnight. Frozen coronal sections (60–80 μm) through substantia nigra and neostriatum were cut and processed either for tyrosine hydroxylase (TH) immunocytochemistry using a primary antibody from Eugene Tech International (Allendale, NJ) and the ABC protocol from Vector Laboratories (Burlingame, CA) as described previously (Tepper et al., 1994) or stained with neutral red for verification of recording and stimulating sites.
Data analysis. There were five types of controls for the local AODN infusions: recordings from substantia nigra DAergic neurons ipsilateral to saline infusions, ipsilateral to D2 random AODN infusions, ipsilateral to D3 random AODN infusions, contralateral to infusion of AODNs, and from untreated control animals. Most parametric electrophysiological data were analyzed with ANOVA followed by Fisher’s protected least significant difference (PLSD)post hoc test at the p < 0.05 level of significance. Numbers in the text are expressed as mean ± SEM. Because these initial ANOVAs showed no significant differences in any of the parameters measured among the five different control groups, they were pooled into a single control group to increase the power of the statistical analyses. The distribution of firing pattern was analyzed with the χ2 test. Dose–response and rotation data were first analyzed with a repeated-measures ANOVA followed by a factorial ANOVA to determine specific differences among the different treatment groups by dose or day of treatment. ED50s were log-transformed before ANOVA because the raw data exhibited heterogeneity of variance. Binding data were analyzed with pairedt tests.
This report is based on data obtained from 175 neurons recorded from 83 rats. All neurons selected for study were presumed to be substantia nigra DAergic neurons on the basis of long duration bi- or triphasic extracellularly recorded waveform (>2.0 msec); spontaneous firing rate between 0.7 and 10.7 spikes/sec; pacemaker, random, or bursty firing pattern; and location in the midbrain. Of these neurons, 91 (52%) could be driven antidromically from neostriatum with characteristics (antidromic latency range, 7.70–23.5 msec; mean latency, 14.18 ± 0.38 msec; 66.4 ± 3.1% of all antidromic action potentials consisting of initial segment-only spike) that identify them conclusively as DAergic neurons (Deniau et al., 1978;Guyenet and Aghajanian, 1978). Representative examples of antidromically identified nigrostriatal DAergic neurons from an untreated control rat, a D2 random AODN-, a D2AODN-, and a D3 AODN-treated rat are shown in Figure1.
Administration of either the D2 or the D3AODN for 3–6 d produced a marked attenuation in the ability of intravenously administered apomorphine to inhibit the spontaneous activity of nigral DAergic neurons. Representative examples of ratemeter recordings are shown in Figure 2. In cells recorded from untreated controls (Fig. 2 A) or ipsilateral to control oligodeoxynucleotide infusions (Fig.2 B), sequential intravenous injections of apomorphine produced marked inhibition of firing of antidromically identified nigrostriatal DAergic neurons before eventually producing complete suppression of spontaneous activity, typically by the time the cumulative dose reached 16 μg/kg. This inhibition could be readily reversed by subsequent administration of haloperidol (50–200 μg/kg, i.v.), as shown in Figure 2 A. In contrast, neurons ipsilateral to infusion of D2 (Fig. 2 B) or D3 (Fig. 2 C) AODN were far less susceptible to the inhibitory effects of apomorphine, and in some cases could not be completely inhibited even at a cumulative dose of 2048 μg/kg. Simultaneous infusion of both D2 and D3 AODN (Fig. 2 D) produced an even greater attenuation of the inhibitory response to apomorphine. The dose–response curves illustrated in Figure 2 E reveal that treatment with either AODN alone produced a dramatic shift to the right in the apomorphine dose–response relation, and that the effects of the D2 and D3 AODNs appeared to be additive (repeated measures F = 11.73; df = 3, 40;p < 0.05). Overall, the D2 AODN dose–response curve (p < 0.05), the D3 AODN dose–response curve (p < 0.05), and the D2 + D3 AODN dose–response curve (p < 0.05) were significantly different from the control dose–response curve. The D2 + D3 AODN dose–response curve also differed significantly from the D2 AODN curve (p < 0.05).
ED50s were estimated from the dose–response data. In the control groups, this was straightforward because all neurons were eventually completely inhibited by apomorphine, usually by the time the 16 μg/kg dose was reached. However, in the AODN-treated groups, many of the neurons could not be inhibited below 50% of baseline firing, even after the highest dose of apomorphine (1024 μg/kg). For these neurons, the ED50 was computed by fitting a log–probit curve to the data points and extrapolating. This was required for 6 of 14 D2 AODN-treated neurons, 5 of 12 D3AODN-treated neurons, and 4 of 8 neurons from animals treated with D2 + D3 AODN. The fact that so many AODN-treated neurons could not be completely inhibited, or even inhibited to 50% of baseline, generated considerable variability in their ED50 estimates, which resulted in very large SEMs. The values were: control = 10.05 ± 3.5 μg/kg; D2 AODN = 475.6 ± 253.3 μg/kg; D3AODN = 341.2 ± 248 μg/kg; D2 + D3AODN = 623.8 ± 297 μg/kg. The increased ED50s after AODN treatment were not attributable to increases in baseline firing rate (see below); there were no significant correlations between ED50 and baseline firing within any of the AODN treatment groups, or overall when the AODN groups were pooled. However, there was a significant correlation between firing rate and ED50 with the pooled control group when one outlier was eliminated (r = 0.62; F = 6.86; df = 1, 11;p < 0.05), as has been described previously (Tepper et al., 1982; White and Wang, 1984).
To begin to estimate the minimum time required for AODN treatment to cause a functional knockdown of DA autoreceptors, a number of rats were given supranigral D2 or D3 antisense infusions for 3 d and prepared for recording on the fourth day. The response of these rats to apomorphine was indistinguishable from that in rats that had been treated with the AODN for 6 d (data not shown). All other electrophysiological parameters (see below) were likewise identical in the 3 and 6 d treated groups. Therefore, in all subsequent data analyses, data from animals treated for 3 d and those treated for 6 d were pooled.
Striatal-evoked inhibitory responses
To determine whether the reduction of the inhibitory response to apomorphine induced by the D2 or D3AODNs was specific to DAergic inhibitory responses rather than an attenuation of inhibitory responses in general, the responses of DAergic neurons to single-pulse stimulation of neostriatum (0.67 Hz; 100–500 μsec duration; 0.2–3 mA) were examined. Striatal stimulation typically produces inhibition in DAergic neurons (Tepper et al., 1990). DAergic neurons from AODN-treated animals displayed inhibitory responses to striatal stimulation that were indistinguishable from those in controls in terms of onset latency, duration, and magnitude of inhibition, as illustrated in Figure3.
Spontaneous activity was assessed by measuring spontaneous firing rate and calculating the coefficient of variation of the interspike interval. In addition, the pattern of firing for each neuron was categorized based on the autocorrelation histogram of the neuron, as described above. Neither D2, D3, nor D2 + D3 AODN significantly altered mean firing rate or the coefficient of variation of DAergic neurons, as shown in Figure 4, A and B. Similarly, the relative proportions of neurons firing in the pacemaker, random, or bursty modes were not affected by any antisense treatment, as shown in Figure 4 C.
Antidromic response properties
The neostriatal threshold stimulating current was used as an index of the excitability of the terminals of DAergic nigrostriatal neurons (Tepper et al., 1984a). The mean threshold current in AODN-treated neurons was significantly lower than in controls (F = 4.38; df = 3, 76; p < 0.05), indicating an increase in nigrostriatal terminal excitability. Post hoctests revealed significantly lower thresholds in DAergic neurons from D2 AODN-treated, D3 AODN-treated, and D2 + D3 AODN-treated rats than in controls (PLSD; p < 0.05). In addition, the proportion of antidromic spikes that consisted of the full spike [IS plus somatodendritic (SD) component] was calculated for each neuron as an index of somatodendritic excitability (Matsuda and Jinnai, 1980; Trent and Tepper, 1991). A significantly higher proportion of antidromic responses of neurons from AODN-treated rats consisted of a full spike than in control neurons (F = 3.49; df = 3, 61;p < 0.05), indicating a depolarization and increase in excitability of the somatodendritic membrane. As with antidromic excitability measurements, this could be attributed to a significantly higher proportion of full spike responses in each of the three AODN-treated groups compared with controls (PLSD; p < 0.05 for each comparison). These data are illustrated in Figure5.
Quantitative autoradiography was performed on selected sections from brains after supranigral infusion of D2 AODN, D3 AODN, D2 + D3 AODN, and random oligo infusion. Representative autoradiograms are shown in Figure6, and the quantitative analyses are summarized graphically in Figure 7. The specific binding of D2-class (3H-spiperone), D3(3H-7-OHDPAT), and D1-class receptors (3H-SCH-23390) in the substantia nigra on the treated side was compared with that on the control side by averaging the binding density over the pars compacta on either side of selected sections and dividing the average for the treated side by that for the control side. The decrease in 3H-spiperone binding on the D2AODN-treated side ranged from a low of 32.4% to a high of 76.1%. The mean decrease in 3H-spiperone binding was 48.9 ± 3.3% (t = 11.96; df = 1, 15; p < 0.05).
Although present at considerably lower levels than3H-spiperone binding, 3H-7-OH-DPAT binding clearly demonstrated the presence of D3 receptors in the substantia nigra (Fig. 6 E,F). Because the nonspecific binding of 3H-7-OH-DPAT was so low, the measurement of the change in D3 binding was made on total binding for 3H-7-OH-DPAT rather than on specific binding, as was the case for 3H-spiperone and3H-SCH-23390 binding, both of which showed considerable nonspecific binding. There was no significant decrease in3H-7-OH-DPAT binding after D2 AODN treatment (see Fig. 6 E). Conversely, D3 AODN did not significantly decrease 3H-spiperone binding in substantia nigra (−4.3 ± 2.8%; see Fig. 6 G,H) but did decrease 3H-7-OH-DPAT binding by 44.6 ± 5.8% (t = 7.17; df = 1, 12; p < 0.05; see Fig. 6 F). Combined treatment with D2and D3 AODNs decreased 3H-spiperone binding by 56.2 + 4.7% (t = 11.06; df = 1, 11;p < 0.05) and 3H-7-OH-DPAT binding by 49.8 + 6.2% (t = 7.53; df = 1, 11; p< 0.05). In contrast, there was no appreciable change in D1-class (3H-SCH23390) binding in substantia nigra after D2, D3, or combined D2 + D3 AODN infusion. Thus, the effects of the D2and D3 AODNs were specific to the receptors for which they were designed, and were localized to the substantia nigra and immediately surrounding regions on the ipsilateral side.
There was apparently no retrograde transport of the D2 AODN back to the neostriatum with subsequent postsynaptic receptor loss after supranigral infusion of DA receptor AODNs, because3H-spiperone binding in the neostriatum did not differ significantly between the control and treated sides, despite the large differences that were seen in substantia nigra (Fig.6 I,J).
Tyrosine hydroxylase immunocytochemistry
To determine whether the electrophysiological changes seen after AODN infusion could be attributable to nonspecific toxic effects on DAergic neurons, DAergic neurons in the ventral mesencephalon were visualized directly by TH immunocytochemistry. As can be seen in Figure8, after a 6 d infusion of D2 AODN, there was no apparent loss of TH immunoreactive neurons ipsilateral to the AODN infusion, demonstrating that the D2 AODN did not produce nonspecific damage to DAergic neurons. Although no attempt was made to quantify these data, it is interesting that the DAergic neurons ipsilateral to the infusion appear to stain more intensely than those on the contralateral side.
Behavioral observations during AODN treatment
The behavior of rats receiving unilateral intranigral infusion of D2 and/or D3 AODN was, for the most part, unremarkable. However, AODN-treated rats exhibited a modest contralateral postural deviation during rest, and intermittent or continuous spontaneous contralateral rotations at a relatively low rate, as shown in Figure 9. Both the contralateral postural deviation and the spontaneous contralateral rotation were evident by 24 hr after the start of the infusion, and became maximal after 3 d of infusion. Repeated measures ANOVA revealed a significant increase in rotations among both AODN-treated groups (F = 6.10; df = 3, 26; p < 0.05). A subsequent factorial ANOVA revealed that all treated groups differed from the random oligodeoxynucleotide-treated controls on all days of treatment (p < 0.05), and that the combined D2 + D3 AODN-treated group also differed significantly (p < 0.05) from either the D2 AODN-treated or the D3 AODN-treated groups on days 2, 3, and 5, suggesting an additivity in the effects of D2 and D3 receptor knockdown on spontaneous rotation. These asymmetric postural and locomotor behaviors were never observed in either saline- or random oligodeoxynucleotide-infused rats, and suggest an increased DA release in the ipsilateral striatum as a result of D2 and/or D3 autoreceptor knockdown. When challenged with the DA agonist apomorphine, no additional rotational behavior was induced in these rats, but the postural and locomotor asymmetries were almost completely abolished, as shown in Figure 9 B.
Specificity and extent of in vivo DA receptor AODN knockdown
The autoradiographic data demonstrate that the receptor knockdown produced by local infusion of DA receptor AODNs into the brain is relatively localized around the infusion site on the ipsilateral side. This is consistent with data showing that the maximal spread of anS-oligodeoxynucleotide of similar size injected intracerebrally was between 0.7 and 1.3 mm (Sklarczyk and Kaczmarek, 1995). Furthermore, D2 AODN reduced specifically3H-spiperone (D2) binding in the substantia nigra without affecting D3 binding, and D3 AODN reduced specifically 3H-7OH DPAT (D3) binding without affecting D2 binding.
Supranigral infusion of D2 receptor AODN did not affect striatal 3H-spiperone binding. This observation is particularly important because it indicates that there is no retrograde knockdown of DA receptors. This is presumably because there is no uptake of the AODNs by striatonigral nerve terminals in substantia nigra. Thus, local infusion of AODNs can be used to selectively inactivate presynaptic or postsynaptic DA receptors, something that has not been possible by the use of conventional receptor antagonists or transgenic knockouts.
The maximal extent of the receptor loss, estimated from autoradiography, was always less than 100%. However, because the pars compacta is a relatively poorly defined structure and because the areas included in the autoradiographic comparisons unavoidably contained regions of low DA receptor density, averaging over the entire pars compacta would tend to underestimate the maximal knockdown in the most affected areas. In addition, 3H-spiperone can also label D3 receptors (Sokoloff et al., 1990; Bouthenet et al., 1991). D2 AODN does not affect D3 binding, and thus some of the residual 3H-spiperone binding after D2 AODN infusion is likely attributable to D3receptors. Finally, because there is no retrograde uptake of D2 AODN, any D2-class receptors that may exist on nigral afferents would also be unaffected, and might contribute to the remaining 3H-spiperone binding. Thus, it is difficult to specify the upper limit of the knockdown potential of AODNs infused into the brain, but our data suggest that in the best cases there is an extensive reduction in DA receptors.
There were no obvious toxic effects of D2 AODN. The substantia nigra on the infused side appeared indistinguishable from its contralateral control in Nissl-stained sections, and TH immunocytochemistry failed to reveal any loss of DAergic neurons. Indeed, if there was any difference at all, the TH immunostaining appeared to be darker on the treated side than on the contralateral control. This may reflect a disinhibition of TH synthesis caused by the loss of D2 somatodendritic autoreceptors (Gauda and Gerfen, 1995).
Effects of DA receptor AODN treatment on apomorphine-induced inhibition of firing
Treatment with D2 or D3 AODN produced equivalent shifts to the right in the apomorphine dose–response curve. Combined treatment with both AODNs produced a significantly greater attenuation of the inhibitory effect of apomorphine. Although there was significant variability in the degree of attenuation, it is important that virtually all of the cells that were treated with either D2 or D3 AODN exhibited significantly less inhibition than control neurons. Thus, the variability did not arise because some neurons were essentially unaffected by D2 or D3 AODN treatment while others showed a near-complete blockade of apomorphine inhibition to one or the other AODN, which would suggest that some DA neurons express only D2receptors, whereas others express only D3 receptors. Rather, the data suggest that virtually all neurons tested expressed both D2 and D3 somatodendritic autoreceptors.
The inhibition of nigrostriatal neurons in response to systemic administration of apomorphine at low doses has been shown to be attributable to a local action on somatodendritic autoreceptors (Akaoka et al., 1992; Pucak and Grace, 1994). Although it has long been known that DAergic neurons possess an autoreceptor of the D2 family (Groves et al., 1975; Aghajanian and Bunney, 1977; Lacey et al., 1987), these data provide direct electrophysiological evidence that DAergic neurons express both D2 and D3 somatodendritic autoreceptors, and that both participate in the inhibition of firing caused by DA agonists.
The decrease in spontaneous activity after administration of higher doses of apomorphine that persisted in many neurons from D2or D3 AODN-treated animals could have resulted from incomplete knockdown of D2 or D3 autoreceptors. However, some of the inhibition resulting from systemic administration of other DAergic agonists (e.g., amphetamine) results from effects on forebrain structures (Bunney and Aghajanian, 1978; Sasaki et al., 1990), and whereas low doses of apomorphine (<16 μg/kg, i.v.) affect only DAergic neurons, higher doses can affect neostriatal neurons (Skirboll et al., 1979). Thus, it may be that a significant component of the inhibition that persists at high doses after AODN treatment is attributable to long-loop postsynaptic effects rather than local autoreceptor stimulation.
Time course of DA receptor AODN knockdown
Neither the apomorphine dose–response curves nor any of the other electrophysiological properties of DAergic neurons differed between animals treated with D2 AODN for 3 or 6 d, suggesting that the functional knockdown is near maximal by 3 d. This is consistent with our observations that AODN-treated animals began to exhibit spontaneous rotation after only 24 hr of treatment that reached a maximum at 3 d. Previous studies of the rate of DA receptor turnover based on the recovery of 3H-spiperone binding after irreversible receptor alkylation showed that the estimatedt 1/2 for D2-class receptor turnover depends on a number of factors, including age, DA content, and the precise methods used to inactivate the receptors (McKernan and Campbell, 1982; Hall et al., 1983; Leff et al., 1984; Norman et al., 1987), and can range from 8 hr to more than 6 d. Our electrophysiological and behavioral data favor the lower range of these turnover estimates.
Effects of DA receptor AODN treatment on spontaneous activity and somatodendritic excitability
Despite the marked effects of D2 and/or D3AODN on the inhibition produced by apomorphine, neither AODN altered the spontaneous firing rate or pattern of nigrostriatal neurons. It is possible that compensatory changes in DAergic neurons or in their afferents masked the increase in spontaneous firing rate that would be predicted on the basis of the self-inhibition hypothesis. However, this explanation seems unlikely because of the rather short times involved (3 d) and because other related properties, including the response to apomorphine and the terminal and somatodendritic excitability, were markedly altered.
Changes in antidromic invasion of the somatodendritic region seen in extracellular recordings have been shown to correspond to changes in the level of membrane polarization of the somatodendritic region (Matsuda and Jinnai, 1980). Using this as an index of somatodendritic excitability, we have proposed previously that the physiological role of DAergic somatodendritic autoreceptors is more subtle than that of directly controlling the firing rate of the neuron as a whole (Trent and Tepper, 1991). In the present study, although D2 and/or D3 knockdown failed to increase the firing rate, the proportion of striatal-evoked antidromic responses that consisted of IS–SD spikes more than doubled. This was not attributable to the decreased threshold current in the treated cells (which might lead to a decrease in orthodromic inhibition from striatum and hence increase the excitability of the somatodendritic region; see Grace and Bunney, 1985;Grace, 1987; Trent and Tepper, 1991), because there was no correlation between the IS–SD percentage and the antidromic threshold current within any treatment group. Rather, the change appears attributable to a reduction in a tonic depression of somatodendritic excitability mediated by D2 and D3 autoreceptors interacting with endogenous DA. These data provide another example of an uncoupling of events in the somatodendritic and initial segment regions of the DAergic neuron (Grace, 1990; Hausser et al., 1995) and reinforce our previous suggestion (Trent and Tepper, 1990) that, at least in anesthetized rats, somatodendritic autoreceptors may not function to control the overall firing rate of the cell directly, but rather function to modulate somatodendritic excitability in a more subtle and local way.
Effects of DA receptor AODN treatment on antidromic thresholds
Axon terminal autoreceptors have been shown to modulate the stimulus-evoked release of DA in a number of different paradigmsin vitro and in vivo (for review, see Starke et al., 1989). The threshold current has been used as an index of the excitability of DAergic nerve terminals and has been shown to vary depending on the extent of terminal autoreceptor stimulation; autoreceptor antagonists reduce the threshold current, whereas autoreceptor agonists increase threshold (Tepper et al., 1984a,b, 1987). In the present experiments, threshold currents of nigrostriatal neurons that were recorded ipsilateral to nigral infusion of D2 or D3 AODN were significantly lower than those in controls, demonstrating that DAergic neurons possess both functional D2 and D3 autoreceptors at the axon terminals, as well as in the somatodendritic region. The identification of a D2 terminal autoreceptor is consistent with a previous study using D2 AODN knockdown (Silvia et al., 1994), and the finding that there is also a D3 terminal autoreceptor is consistent with claims from biochemical studies (Meller et al., 1993; Nissbrandt et al., 1995). In vivo, terminal autoreceptors are sufficiently occupied by endogenous DA that administration of antagonists (Tepper et al., 1984a) or AODN knockdown significantly decreases the inhibitory tone on the terminal, resulting in increased terminal excitability and, presumably, increased DA release. It is likely this effect that is responsible for the spontaneous contralateral rotation observed during AODN treatment. These data also demonstrate that it is possible to knock out receptors on axon terminals of neurons that originate in the infusion site but extend several millimeters away.
Relative roles of D2 and D3 autoreceptors on substantia nigra neurons
The attenuating effects of D2 AODN on apomorphine-induced inhibition of nigrostriatal neurons and the increases in terminal and somatodendritic excitability were expected based on the large amount of D2 mRNA and D2-class binding present in substantia nigra. However, that D3 AODN was as effective as D2 AODN, and that the effects of D2 and D3 AODN treatment were additive were somewhat surprising given recent reports that there is only a low level of D3 mRNA present in the mesencephalon and that D3 mRNA was below detectable levels on many DAergic nigral neurons (Diaz et al., 1995). However, the difficulty in detecting D3 mRNA in the midbrain may simply reflect the relative overabundance of D2 mRNA and protein relative to that of D3 mRNA (Griffon et al., 1995). Our autoradiographic data suggest the presence of D3 binding in substantia nigra, albeit at relatively low levels. It may be that lower, but still physiologically functional, levels of D3 receptors are present on most or all mesencephalic DAergic neurons, but these are associated with lower levels of mRNA that require more sensitive PCR or related techniques for detection (Sokoloff et al., 1990; Valerio et al., 1994) and that are missed by in situ hybridization. A recent study using transfection of D2 and D3receptors into a DAergic mesencephalic clonal line showed that D3 receptors were more than twice as potent at inhibiting DA release than D2 receptors, despite the fact thatB max of the D2 receptors was three times greater than that of the D3 receptors (Tang et al., 1994), perhaps indicating that the receptor coupling mechanism(s) is more efficient for D3 receptors. Nevertheless, the fact that both D2 and D3 AODNs attenuated autoreceptor function at both the terminal and the somatodendritic regions of the neuron more or less equally suggests that the normal electrophysiological response attributed to somatodendritic and terminal autoreceptor stimulation may require coactivation of both D2 and D3 receptors.
In vivo knockdown of specific DA receptors by injection of AODNs into discrete brain regions is a valuable technique for assessing the functional roles and sites of action of different DA receptor subtypes for which selective ligands do not yet exist. The lack of retrograde knockdown suggests that this technique offers a method for selective inactivation of either pre- or postsynaptic receptors, something that has not previously been possible. The data presented here demonstrate that DAergic neurons possess functional D2 and D3 autoreceptors at both their axon terminal and somatodendritic regions. Both types of terminal autoreceptors modulate terminal excitability and presumably the release of DA from the nerve terminals and/or its synthesis, whereas somatodendritic D2 and D3 autoreceptors play a role in modulating the excitability of local dendritic regions.
This research was supported by the Johnson & Johnson Discovery Research Fund, the Hoechst-Celanese Innovative Research Award, and National Institute for Mental Health Grants MH-52450 and MH-52383. We thank Dr. Ming Zhang for work on the development of the D2 and D3 oligodeoxynucleotides and for assistance with the receptor autoradiography, Dr. P. Celada for help with the TH immunocytochemistry, L. Taylor for performing the ED50calculations, and T. Koós for comments on this manuscript.
Correspondence should be addressed to Dr. James M. Tepper, Center for Molecular and Behavioral Neuroscience, Aidekman Research Center, Program in Cellular and Molecular Biodynamics, Rutgers University, University Heights, 197 University Avenue, Newark, NJ 07102.