SYNAPSE 6:309-320 (1990)
Dopamine Neuron Ontogeny: Electrophysiological Studies DAVID K. PITTS, ARTHUR S. FREEMAN, AND LOUIS A. CHIODO Laboratory of Neurophysiology, Center for Cell Biology, Sinai Research Institute, and The Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, Michigan 48235
KEY WORDS
Development, Nigrostriatal, Apomorphine, Quinpirole, Substantia nigra
ABSTRACT
The ontogeny of nigrostriatal dopamine (NSDA)neurons was examined with single-unit extracellular electrophysiologicalmethods. The physiological and pharmacological characteristics of 2-, 4-, and 5-week-old rat pup NSDA neurons were compared with those of adults (8-10 weeks old).Although the basal discharge rate, conduction velocity, and firing pattern of NSDA neurons from 4- and 5-week-old rats were similar to adults, the 2-week-old-rats differed significantly in all three of these physiological characteristics. The conduction velocity and basal discharge rate were found to be significantly lower in the 2-week-old pups relative to adults. In addition, there were significantly fewer bursting NSDA neurons in 2-week-oldsthan there were in adults. Two and 4-week-oldsexhibited significantly lower sensitivity to cumulative intravenous doses of apomorphine. In contrast, the sensitivity to cumulative intravenous doses of quinpirole was found to be similar across all age groups. It is evident that the pharmacologwal and physiological properties of NSDA neurons are in a dynamic state of fluxduring postnatal development. These electrophysiological findings are discussed in the context of the perinatal development of midbrain DA systems.
INTRODUCTION The anatomy, physiology, biochemistry, and pharmacology of dopamine (DA)-containingneurons have been extensively studied over the past few decades. Although the electrophysiological characteristics and neuropharmacology of single identified DA neurons have been reported by many investigators, the study of these neurons during early postnatal development has been largely overlooked. DA may, in fact, play a role in neuronal morphogenesis during ontogeny (Lankford et al., 1988; Moon-Edley and Herkenham, 1984; Tennyson et al., 1982, 1983). Behavioral, biochemical, and anatomical studies indicate that DA neurons undergo rapid morphological and functional changes during this period. Differentiation of DA neurons in the rat occurs over days 11-15 of estation (Lauder and Bloom, 1974). Catecholamine uorescense can be observed by day 13 of gestation indicatin the presence of DA in the neuronal perikarya and t eir processes (Olson and Seiger, 1972). Tyrosine hydroxylase activity precedes the apearance of catecholamine fluorescense and increases ramatically (a proximately four-fold)before birth (see Coyle and Axe rod, 1972; Lauder and Bloom, 1974; Olson and Seiger, 1972; Specht et al., 1981). Although most of the monoamine neurons and their axonal pathways are well developed at birth (with the possible exception of those originating in the hypothalamus; Seiger and Olson, 1973), postsynaptic synaptogenesis of the presumed terminal fields of nigrostriatal DA neurons continues in the striatum during postnatal
a
s
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0 1990 WILEY-LISS, INC.
i
development until there is the appearance of “adultlike” patterns of innervation by the end of the fourth postnatal week (Loizou, 1972; Olson and Seiger, 1972). During the early postnatal period (first week), the DA-containing fibers within the striatum are clustered together and form ‘(dopamineislands,” which, with the passage of time (weeks 2-41, give rise to a more “diffuse” or nonislandic pattern of innervation (Gerfen et al., 1987a,b; Graybiel, 1984a,b; Moon-Edley and Herkenham, 1984; Olsen and Seiger, 1972). These diffuse and islandic components of the DA innervation of the striatum persist into adulthood (although the diffuse component masks the islandic portion) and have been reported to arise from different DA neurons in the mesencephalon (Fuxe et al., 1979; Gerfen et al., 1987a,b; Graybiel et al., 1987). Developmental biochemical studies with rabbit brain slices have shown that both the neostriatum and cortex can concentrate [3Hldopnmine during late gestational and early postnatal development (Tennyson et al., 1972). The full capacity to concentrate dopamine is apparently not achieved in the rabbit until about postnatal days 30-45. Calcium-dependent depolarizationinduced DA release has been demonstrated as early as Received April 9,1990; accepted May 24,1990. Address re rint re uests to David K. Pitts, De artrnent of Pharmaceutical Sciences, Colpege of %harmacy and Allied Healt! Professions, Wayne State University, Detroit, MI 48202. This work was resented in part at the 18th Annual Meeting of the Society for Neuroscience in ?Poronto, Canada, November, 1988.
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day 18 of gestation in the rat (Nomura et al., 1981). The DA trans ort mechanism in the rat ap arently becomes functiona during late gestation (a ter day 18) and exhibits adult-like capacity in the striatum b the end of the first postnatal week (Yotsumoto anc! Nomura, 1981). Shaywitz et al. (1985) examined levels of monoamine transmitters and their metabolites in developing rat pups. Although whole-brain dopamine levels increased from postnatal days 1 2 4 2 (adult levels), homovanillic acid (HVA) progressively decreased in whole brain and cerebrospinal fluid over the same time period. In general, there is evidence for age-dependent differences in responses to drugs that affect catecholaminergic mechansims in many behavioral tests (e.g., spontaneous locomotor activity, stereotypic behaviors, catalepsy, and performance in avoidance tests). LDOPA has been shown to affect the level of spontaneous activity in an age-dependent manner: hyperactivity in l-day-old neonates and locomotor depression a t 21 days old (Kellogg and Lundborg, 1972). Hedner and Lundborg (1985)reported similar results with the dopamine agonist, (?)-3-PPP: no effects on locomotor activity in rats less than 14 days old and dose-dependent locomotor depression in 28-day-old rats. Shalaby and Spear (1980) reported that the suppression of locomotor movements from low-doseapomorphine did not appear until 35 days after birth (not seen at 7, 14, or 21 days after birth). These findings led Shalaby and Spear to suggest that DA autoreceptor function may not “mature”until postnatal week 5. In the present study, the physiological properties of identified nigrostriatal dopamine (NSDA)neurons were examined in the developing rat pup. In addition, the responsiveness of NSDA neurons to two different DA agonists, quinpirole (LY171555, a selective D2 DA receptor agonist) and apomorphine (a mixed DUD2 DA receptor agonist), were tested in order to assess the functional status of inhibitory DA somatodendritic autoreceptors (see Aghajanian, 1978; Bunney, 1979; Chiodo, 1988; for reviews).
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tered through a cannulated lateral tail vein. A 1 ml syringe was used to administer drugs intravenously in all experiments with the exce tion of the dose-response curves comparing adults an 2-week-old rat pups. In this case only 100 pl Hamilton syringes were used to administer cumulative doses of DA agonists in order to avoid volume loading (total drug and physiological saline volume administered approximately 200 pl) in the younger animals (the adults in these experiments also received cumulative agonist doses from a 100 p,1 Hamilton syringe). The skull was leveled between lambda and bregma, and a small burr hole was made over the recording site for the substantia nigra (for adults, from lambda, anterior 2.8-3.2 mm, lateral 1.8-2.2 mm; 6.5-7.5 mm ventral from the brain surface; Paxinos and Watson, 1986). The stereotaxic coordinates for younger animals were first approximated by reducing the anterior distance from lambda proportionally using the ratio: rat pup lambda to bregma distanceladult lambda to bregma distance. A concentric stimulating electrode (SNE-100, Rhodes Medical Instruments, Woodland Hills, CA) was placed in the dorsal caudate nucleus (for adults, from lambda, anterior 8.6 mm, lateral 3.2 mm; 4.5 mm ventral from the dura). The coordinates for rat pups were determined by a method similar to the one described above for the recording electrode and the osition of the stimulating electrode was verified histoogically (see below).
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Electrophysiology Single-unit extracellular recordings from spontaneously active DA neurons were made with glass micropiettes (1.5mm diameter, World Precision Instruments, kew Haven, CT). Each micropipette (1-2 pm tip diameter) was filled with a solution containing 1M NaCl and 1%pontamine sky blue dye. Electrode impedance typically measured 2.54.5 megohms at 135 Hz in physiological saline. Extracellular electrical signals were amplified by a high input im edance amplifier/window discriminator (Activational ystems, Inc., Warren, MI), with bandpass settings of 300 Hz and 3 KHz. The output of this amplifier was sent to a Hitachi oscilloscope, Grass audiomonitor, Fisher chart recorder, and an IBM MATERIALS AND METHODS XT computer through a Modular Instruments, Inc. Animals (Southeastern, PA), M-100 mainframe interface and Male Sprague-Dawley rats (Hilltop, Scottdale, PA) M-106 transient register. The analysis of interspike were housed in standard animal facilities for a mini- intervals was carried out with Modular Instruments, mum of 1 week prior to electrophysiological studies. Inc., software (modules S-200 and S-220). Two-week-old animals (14-15 days old) used in this DA neurons were initially identified by the wellstudy were delivered to the animal facility at an age of 7 established electrophysiological criteria for adult rats days with a lactating dam. Weanlings sent to the animal (Bunneyet al., 1973;for review, see Chiodo, 1988; Grace facility as 21-day-old males were used in the studies of and Bunney, 1983a,b). In addition, with the exception of 4-week-oldand 5-week-oldrat pups (28-29 days old and hemitransection experiments, nigrostriatal DA neu35-36 days old, respectively). Adults were defined as rons were identified by antidromic activation (constantrats 8-10 weeks old. Animals received food and water ad current square-wave pulses; 0.1-3.0 mA, 0.5 msec duralibitum. tion) from the caudate nucleus as previously described by Guyenet and Agha’anian (1978). Neurons were conGeneral sidered antidromical y activated when the following Rats were anesthetized with chloral hydrate (400 criteria were met: 1)one antidromic spike elicited per mgkg, i.p.), with additional supplements administered stimulus, 2) the latency for the stimulus-induced anas necessary. The animals were placed in a David Kopf tidromic spike was fixed 3) a stimulation frequency of stereotaxic a aratus and body temperature was main- up to 50 Hz was followed by the antidromic spike, and 4) tained a t 37Qk 1°C with a heating ad. In addition, collision between spontaneously occurring action poten2-week-old animals were covered wit a small terry- tials and stimulus-induced antidromic spikes occurred cloth blanket and intubated following a tracheotomy to (see Li ski, 1981). Conduction velocity was calculated facilitate spontaneous respiration. Drugs were adminis- for eac neuron by the quotient of the straight-line
8
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K
K
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DOPAMINENEURONONTOGENY
distance between the recording and stimulating electrodes and the measured latency (see Chiodo, 1988). In hemitransection experiments, an elliptically shaped wire knife (0.2 mm diameter wire) was passed through the coronal plane 4.5 mm anterior to lambda in 2-seek-old rat pups. This hemitransection separated the midbrain and forebrain and covered a reson approximately equivalent to that previously published for adults (Kelland et al., 1989b). The recording of single NSDA neuron activity was not begun for a minimum of 20 min following the transection. Histology Recording sites were marked by passing a -30 pA current through the pipette for 20 min to deposit a small amount of pontamine sky blue. Fifty micrometer serial frozen sections were stained with formal thionine and the recording and stimulating electrode sites were examined under a light microscope (see Chiodo, 1988). Statistics Group differences in dose-response (DR) curves were evaluated by an analysis of covariance with repeated measures (ANCOVA,BMDP Statistical Software, University of California Press). The dependent variable was discharge rate (spikeslsec), and the covariate was basal discharge rate. ANCOVA contrast analysis was used to compare each of the younger age groups to adult controls. Due to the intercorrelation among the burst parameters examined in this study, factor analysis (based on 112 adults) was used to divide these seven arameters into two subgroups (Table 111;see below). eparate multivariate analyses of variance (MANOVA) were used to examine the differences in each sub oup of parameters across the different age groups. %th the exception of within-burst interspike interval (rnsec)and firing rate (spikeshec), all the remaining variables in burst analysis were normalized by log transformation prior to statistical analysis. The application of most of these statistical procedures to the analysis of DR curves has previously been discussed in detail by Pitts et al. in conduction velocity using one-way test. A P value less than 0.05 was considered to be significant in all statistical tests.
t
Burst analysis Interspike intervals were collected on-line from a sample of 500 consecutive action potentials and saved in a binary file for later off-line burst analysis with the use of a program developed in our laboratory (BURSTAN). Criteria for the designation of a cell as a bursting neuron were as follows: 1)the occurrence of bursts, which are defined to begin with an interspike interval less than 80 msec and terminate with the first interspike interval greater than 160 msec, and 2) a minimum of two threespike bursts were encountered (Chiodo et al., 1984; Grace and Bunney, 1984). Drugs The following drugs were used in these studies: chloral hydrate (Sigma, St. Louis, MO), quinpirole HC1 (LY171555,Lilly), apomorphine HCl (Sigma, St. Louis, MO), SCH 23390 maleate (Schering, Bloomfield, NJ),
and haloperidol (MacNeil,Spring House, PA). All drugs with the exception of SCH 23390 were dissolved in physiological saline. SCH 23390 was dissolved in deionized water.
RESULTS Figure 1A illustrates the waveform of an NSDA neuron recorded from a 4-week-old rat up. A typical positive-negative wide action potentia with a prominent initial segment-somatodendritic (IS-SD) break in the rising phase can be seen. Also depicted in Figure 1A is an antidromic s ike recorded from this same neuron elicited by stimu ation of the caudate nucleus (0.5msec duration, 2.0 mA). Note that only the initial se component of the waveform is present in the anti romic spike as is typical for midbrain DA neurons in adults. A larger percentage of antidromic spikes in 2-week-oldrat pups (a proximately 50%; Fig. 1B; 0.5 msec duration, 0.8 mAf appeared to be full IS-SD spikes than was observed in adults (none observed in this study). Table I lists the antidromic conduction velocities and mean discharge rates of NSDA neurons determined studied. The conduction velocity for found to be significantly lower 5-week-olds, and adults. Thus, despite the smaller brain size of the 2-week-old pups, a significantly lower mean conduction velocity resulted in a mean latenc that was not significantly different from that in adu ts (adults = 11.8 -+ 0.4 msec; 2-weekolds = 11.6 2 0.5 msec). The overall mean discharge rate of NSDA neurons of 2-week-old rats was also found to be significantly lower than in adults (Table I>. Figure 2 shows typical interspike interval (ISI)histograms from three nonbursting (left column) and three bursting (right column) NSDA neurons. In Table I1 neurons from each age group studied were classified according to the extent of burst activity observed: single (mostly single spikes), doublets (most burst activity represented by doublet pattern), and bursts (most burst activity represented by bursts of three or more spikes). In 2-week-old rat pups, significantly fewer bursting cells were observed relative to adults (Table TI; bursting cells vs. nonbursting cells in adults and 2-week-olds; x2 = 10.51, P < 0.005). Only five of a total of 44 cells examined in 2-week-oldswere found to be burstin cells. In addition, many of the neurons from 2-wee -olds found in the single category exhibited very regular firing patterns, with little variation in interspike interval (e.g., see nonbursting cell in Figure 2). A more detailed analysis of the discharge pattern of the cells classified in the burst category is shown in Table 111.The bursting cells for all four age groups were examined for differences among the seven listed burst parameters (Table 111). This analysis indicated that there were no significant differences in these burst parameters among the different age groups. However, there may be insufficient statistical power to detect differences among these burst parameters due to the small number of bursting cells in younger age groups. There appears to be a trend towards lower firing rates and burst lengths in ounger animals. The responses of N DA neurons from a 4-week-old rat pup (lower panel) and an adult to cumulative doses of intravenous apomorphine (APO)are shown in Figure 3. Despite the higher basal firing rate of the NSDA neuron
f
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rt
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a
H
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D.K. PITTS ET AL
A
Y
1 msec
T
3 msec
B
v
200 pv
1 msecl
3 msecl
200 p
I
i
Fig. 1. Action potentials recorded from NSDA neurons in a 4-week-old (A) and a 2-week-old (B) rat pup are illustrated on the left. Note the presence of a prominent intial segment-somatodendritic IS-SD break in the rising phase of the positive-negative action poten-
tial. The antidromic spikes recorded from these same neurons were elicited by stimulation ofthe caudate nucleus (A: 0.5msec duration, 2.0 mA; B: 0.5 msec duration, 0.8 mA) are shown on the right. Arrows indicate the stimulus artifacts.
from the adult animal, it was clearly more sensitive to the inhibitory effects of apomorphine on spontaneous discharge than the NSDA neuron from the 4-week-old pup. In both cases, subsequent administration of haloperidol reversed the inhibitory effects of apomorphine.
Cumulative dose-response (DR) curves for the inhibitory effects of apomorphine on NSDA neurons from 4-week-old, 5-week-old, and adult rats are shown in Figure 4. Only the DR curve from the 4-week-old rat pups was significantly different from adults (F1,34 =
313
DOPAMINENEURONONTOGENY TABLE I. DA neuron conduction velocities at different postnatal ages'
Discharge rate C.V. (m/sec) n
2 Weeks
4 Weeks
5 Weeks
Adult
3.36*
3.97 * 0.55 0.38 * 0.02
* 4.15 0.29 0.54
f 0.29
f 0.14 0.48**
t 0.02
0.56 ?t0.02
f 0.03 30
25
40
4.58
44
'ANOVA, followed by Dunnett's t test for comparisonto adult values. 'P
Dopamine Neuron Ontogeny: Electrophysiological Studies DAVID K. PITTS, ARTHUR S. FREEMAN, AND LOUIS A. CHIODO Laboratory of Neurophysiology, Center for Cell Biology, Sinai Research Institute, and The Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, Michigan 48235
KEY WORDS
Development, Nigrostriatal, Apomorphine, Quinpirole, Substantia nigra
ABSTRACT
The ontogeny of nigrostriatal dopamine (NSDA)neurons was examined with single-unit extracellular electrophysiologicalmethods. The physiological and pharmacological characteristics of 2-, 4-, and 5-week-old rat pup NSDA neurons were compared with those of adults (8-10 weeks old).Although the basal discharge rate, conduction velocity, and firing pattern of NSDA neurons from 4- and 5-week-old rats were similar to adults, the 2-week-old-rats differed significantly in all three of these physiological characteristics. The conduction velocity and basal discharge rate were found to be significantly lower in the 2-week-old pups relative to adults. In addition, there were significantly fewer bursting NSDA neurons in 2-week-oldsthan there were in adults. Two and 4-week-oldsexhibited significantly lower sensitivity to cumulative intravenous doses of apomorphine. In contrast, the sensitivity to cumulative intravenous doses of quinpirole was found to be similar across all age groups. It is evident that the pharmacologwal and physiological properties of NSDA neurons are in a dynamic state of fluxduring postnatal development. These electrophysiological findings are discussed in the context of the perinatal development of midbrain DA systems.
INTRODUCTION The anatomy, physiology, biochemistry, and pharmacology of dopamine (DA)-containingneurons have been extensively studied over the past few decades. Although the electrophysiological characteristics and neuropharmacology of single identified DA neurons have been reported by many investigators, the study of these neurons during early postnatal development has been largely overlooked. DA may, in fact, play a role in neuronal morphogenesis during ontogeny (Lankford et al., 1988; Moon-Edley and Herkenham, 1984; Tennyson et al., 1982, 1983). Behavioral, biochemical, and anatomical studies indicate that DA neurons undergo rapid morphological and functional changes during this period. Differentiation of DA neurons in the rat occurs over days 11-15 of estation (Lauder and Bloom, 1974). Catecholamine uorescense can be observed by day 13 of gestation indicatin the presence of DA in the neuronal perikarya and t eir processes (Olson and Seiger, 1972). Tyrosine hydroxylase activity precedes the apearance of catecholamine fluorescense and increases ramatically (a proximately four-fold)before birth (see Coyle and Axe rod, 1972; Lauder and Bloom, 1974; Olson and Seiger, 1972; Specht et al., 1981). Although most of the monoamine neurons and their axonal pathways are well developed at birth (with the possible exception of those originating in the hypothalamus; Seiger and Olson, 1973), postsynaptic synaptogenesis of the presumed terminal fields of nigrostriatal DA neurons continues in the striatum during postnatal
a
s
P
0 1990 WILEY-LISS, INC.
i
development until there is the appearance of “adultlike” patterns of innervation by the end of the fourth postnatal week (Loizou, 1972; Olson and Seiger, 1972). During the early postnatal period (first week), the DA-containing fibers within the striatum are clustered together and form ‘(dopamineislands,” which, with the passage of time (weeks 2-41, give rise to a more “diffuse” or nonislandic pattern of innervation (Gerfen et al., 1987a,b; Graybiel, 1984a,b; Moon-Edley and Herkenham, 1984; Olsen and Seiger, 1972). These diffuse and islandic components of the DA innervation of the striatum persist into adulthood (although the diffuse component masks the islandic portion) and have been reported to arise from different DA neurons in the mesencephalon (Fuxe et al., 1979; Gerfen et al., 1987a,b; Graybiel et al., 1987). Developmental biochemical studies with rabbit brain slices have shown that both the neostriatum and cortex can concentrate [3Hldopnmine during late gestational and early postnatal development (Tennyson et al., 1972). The full capacity to concentrate dopamine is apparently not achieved in the rabbit until about postnatal days 30-45. Calcium-dependent depolarizationinduced DA release has been demonstrated as early as Received April 9,1990; accepted May 24,1990. Address re rint re uests to David K. Pitts, De artrnent of Pharmaceutical Sciences, Colpege of %harmacy and Allied Healt! Professions, Wayne State University, Detroit, MI 48202. This work was resented in part at the 18th Annual Meeting of the Society for Neuroscience in ?Poronto, Canada, November, 1988.
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D.K. PITTS ET AL
day 18 of gestation in the rat (Nomura et al., 1981). The DA trans ort mechanism in the rat ap arently becomes functiona during late gestation (a ter day 18) and exhibits adult-like capacity in the striatum b the end of the first postnatal week (Yotsumoto anc! Nomura, 1981). Shaywitz et al. (1985) examined levels of monoamine transmitters and their metabolites in developing rat pups. Although whole-brain dopamine levels increased from postnatal days 1 2 4 2 (adult levels), homovanillic acid (HVA) progressively decreased in whole brain and cerebrospinal fluid over the same time period. In general, there is evidence for age-dependent differences in responses to drugs that affect catecholaminergic mechansims in many behavioral tests (e.g., spontaneous locomotor activity, stereotypic behaviors, catalepsy, and performance in avoidance tests). LDOPA has been shown to affect the level of spontaneous activity in an age-dependent manner: hyperactivity in l-day-old neonates and locomotor depression a t 21 days old (Kellogg and Lundborg, 1972). Hedner and Lundborg (1985)reported similar results with the dopamine agonist, (?)-3-PPP: no effects on locomotor activity in rats less than 14 days old and dose-dependent locomotor depression in 28-day-old rats. Shalaby and Spear (1980) reported that the suppression of locomotor movements from low-doseapomorphine did not appear until 35 days after birth (not seen at 7, 14, or 21 days after birth). These findings led Shalaby and Spear to suggest that DA autoreceptor function may not “mature”until postnatal week 5. In the present study, the physiological properties of identified nigrostriatal dopamine (NSDA)neurons were examined in the developing rat pup. In addition, the responsiveness of NSDA neurons to two different DA agonists, quinpirole (LY171555, a selective D2 DA receptor agonist) and apomorphine (a mixed DUD2 DA receptor agonist), were tested in order to assess the functional status of inhibitory DA somatodendritic autoreceptors (see Aghajanian, 1978; Bunney, 1979; Chiodo, 1988; for reviews).
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tered through a cannulated lateral tail vein. A 1 ml syringe was used to administer drugs intravenously in all experiments with the exce tion of the dose-response curves comparing adults an 2-week-old rat pups. In this case only 100 pl Hamilton syringes were used to administer cumulative doses of DA agonists in order to avoid volume loading (total drug and physiological saline volume administered approximately 200 pl) in the younger animals (the adults in these experiments also received cumulative agonist doses from a 100 p,1 Hamilton syringe). The skull was leveled between lambda and bregma, and a small burr hole was made over the recording site for the substantia nigra (for adults, from lambda, anterior 2.8-3.2 mm, lateral 1.8-2.2 mm; 6.5-7.5 mm ventral from the brain surface; Paxinos and Watson, 1986). The stereotaxic coordinates for younger animals were first approximated by reducing the anterior distance from lambda proportionally using the ratio: rat pup lambda to bregma distanceladult lambda to bregma distance. A concentric stimulating electrode (SNE-100, Rhodes Medical Instruments, Woodland Hills, CA) was placed in the dorsal caudate nucleus (for adults, from lambda, anterior 8.6 mm, lateral 3.2 mm; 4.5 mm ventral from the dura). The coordinates for rat pups were determined by a method similar to the one described above for the recording electrode and the osition of the stimulating electrode was verified histoogically (see below).
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Electrophysiology Single-unit extracellular recordings from spontaneously active DA neurons were made with glass micropiettes (1.5mm diameter, World Precision Instruments, kew Haven, CT). Each micropipette (1-2 pm tip diameter) was filled with a solution containing 1M NaCl and 1%pontamine sky blue dye. Electrode impedance typically measured 2.54.5 megohms at 135 Hz in physiological saline. Extracellular electrical signals were amplified by a high input im edance amplifier/window discriminator (Activational ystems, Inc., Warren, MI), with bandpass settings of 300 Hz and 3 KHz. The output of this amplifier was sent to a Hitachi oscilloscope, Grass audiomonitor, Fisher chart recorder, and an IBM MATERIALS AND METHODS XT computer through a Modular Instruments, Inc. Animals (Southeastern, PA), M-100 mainframe interface and Male Sprague-Dawley rats (Hilltop, Scottdale, PA) M-106 transient register. The analysis of interspike were housed in standard animal facilities for a mini- intervals was carried out with Modular Instruments, mum of 1 week prior to electrophysiological studies. Inc., software (modules S-200 and S-220). Two-week-old animals (14-15 days old) used in this DA neurons were initially identified by the wellstudy were delivered to the animal facility at an age of 7 established electrophysiological criteria for adult rats days with a lactating dam. Weanlings sent to the animal (Bunneyet al., 1973;for review, see Chiodo, 1988; Grace facility as 21-day-old males were used in the studies of and Bunney, 1983a,b). In addition, with the exception of 4-week-oldand 5-week-oldrat pups (28-29 days old and hemitransection experiments, nigrostriatal DA neu35-36 days old, respectively). Adults were defined as rons were identified by antidromic activation (constantrats 8-10 weeks old. Animals received food and water ad current square-wave pulses; 0.1-3.0 mA, 0.5 msec duralibitum. tion) from the caudate nucleus as previously described by Guyenet and Agha’anian (1978). Neurons were conGeneral sidered antidromical y activated when the following Rats were anesthetized with chloral hydrate (400 criteria were met: 1)one antidromic spike elicited per mgkg, i.p.), with additional supplements administered stimulus, 2) the latency for the stimulus-induced anas necessary. The animals were placed in a David Kopf tidromic spike was fixed 3) a stimulation frequency of stereotaxic a aratus and body temperature was main- up to 50 Hz was followed by the antidromic spike, and 4) tained a t 37Qk 1°C with a heating ad. In addition, collision between spontaneously occurring action poten2-week-old animals were covered wit a small terry- tials and stimulus-induced antidromic spikes occurred cloth blanket and intubated following a tracheotomy to (see Li ski, 1981). Conduction velocity was calculated facilitate spontaneous respiration. Drugs were adminis- for eac neuron by the quotient of the straight-line
8
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K
K
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DOPAMINENEURONONTOGENY
distance between the recording and stimulating electrodes and the measured latency (see Chiodo, 1988). In hemitransection experiments, an elliptically shaped wire knife (0.2 mm diameter wire) was passed through the coronal plane 4.5 mm anterior to lambda in 2-seek-old rat pups. This hemitransection separated the midbrain and forebrain and covered a reson approximately equivalent to that previously published for adults (Kelland et al., 1989b). The recording of single NSDA neuron activity was not begun for a minimum of 20 min following the transection. Histology Recording sites were marked by passing a -30 pA current through the pipette for 20 min to deposit a small amount of pontamine sky blue. Fifty micrometer serial frozen sections were stained with formal thionine and the recording and stimulating electrode sites were examined under a light microscope (see Chiodo, 1988). Statistics Group differences in dose-response (DR) curves were evaluated by an analysis of covariance with repeated measures (ANCOVA,BMDP Statistical Software, University of California Press). The dependent variable was discharge rate (spikeslsec), and the covariate was basal discharge rate. ANCOVA contrast analysis was used to compare each of the younger age groups to adult controls. Due to the intercorrelation among the burst parameters examined in this study, factor analysis (based on 112 adults) was used to divide these seven arameters into two subgroups (Table 111;see below). eparate multivariate analyses of variance (MANOVA) were used to examine the differences in each sub oup of parameters across the different age groups. %th the exception of within-burst interspike interval (rnsec)and firing rate (spikeshec), all the remaining variables in burst analysis were normalized by log transformation prior to statistical analysis. The application of most of these statistical procedures to the analysis of DR curves has previously been discussed in detail by Pitts et al. in conduction velocity using one-way test. A P value less than 0.05 was considered to be significant in all statistical tests.
t
Burst analysis Interspike intervals were collected on-line from a sample of 500 consecutive action potentials and saved in a binary file for later off-line burst analysis with the use of a program developed in our laboratory (BURSTAN). Criteria for the designation of a cell as a bursting neuron were as follows: 1)the occurrence of bursts, which are defined to begin with an interspike interval less than 80 msec and terminate with the first interspike interval greater than 160 msec, and 2) a minimum of two threespike bursts were encountered (Chiodo et al., 1984; Grace and Bunney, 1984). Drugs The following drugs were used in these studies: chloral hydrate (Sigma, St. Louis, MO), quinpirole HC1 (LY171555,Lilly), apomorphine HCl (Sigma, St. Louis, MO), SCH 23390 maleate (Schering, Bloomfield, NJ),
and haloperidol (MacNeil,Spring House, PA). All drugs with the exception of SCH 23390 were dissolved in physiological saline. SCH 23390 was dissolved in deionized water.
RESULTS Figure 1A illustrates the waveform of an NSDA neuron recorded from a 4-week-old rat up. A typical positive-negative wide action potentia with a prominent initial segment-somatodendritic (IS-SD) break in the rising phase can be seen. Also depicted in Figure 1A is an antidromic s ike recorded from this same neuron elicited by stimu ation of the caudate nucleus (0.5msec duration, 2.0 mA). Note that only the initial se component of the waveform is present in the anti romic spike as is typical for midbrain DA neurons in adults. A larger percentage of antidromic spikes in 2-week-oldrat pups (a proximately 50%; Fig. 1B; 0.5 msec duration, 0.8 mAf appeared to be full IS-SD spikes than was observed in adults (none observed in this study). Table I lists the antidromic conduction velocities and mean discharge rates of NSDA neurons determined studied. The conduction velocity for found to be significantly lower 5-week-olds, and adults. Thus, despite the smaller brain size of the 2-week-old pups, a significantly lower mean conduction velocity resulted in a mean latenc that was not significantly different from that in adu ts (adults = 11.8 -+ 0.4 msec; 2-weekolds = 11.6 2 0.5 msec). The overall mean discharge rate of NSDA neurons of 2-week-old rats was also found to be significantly lower than in adults (Table I>. Figure 2 shows typical interspike interval (ISI)histograms from three nonbursting (left column) and three bursting (right column) NSDA neurons. In Table I1 neurons from each age group studied were classified according to the extent of burst activity observed: single (mostly single spikes), doublets (most burst activity represented by doublet pattern), and bursts (most burst activity represented by bursts of three or more spikes). In 2-week-old rat pups, significantly fewer bursting cells were observed relative to adults (Table TI; bursting cells vs. nonbursting cells in adults and 2-week-olds; x2 = 10.51, P < 0.005). Only five of a total of 44 cells examined in 2-week-oldswere found to be burstin cells. In addition, many of the neurons from 2-wee -olds found in the single category exhibited very regular firing patterns, with little variation in interspike interval (e.g., see nonbursting cell in Figure 2). A more detailed analysis of the discharge pattern of the cells classified in the burst category is shown in Table 111.The bursting cells for all four age groups were examined for differences among the seven listed burst parameters (Table 111). This analysis indicated that there were no significant differences in these burst parameters among the different age groups. However, there may be insufficient statistical power to detect differences among these burst parameters due to the small number of bursting cells in younger age groups. There appears to be a trend towards lower firing rates and burst lengths in ounger animals. The responses of N DA neurons from a 4-week-old rat pup (lower panel) and an adult to cumulative doses of intravenous apomorphine (APO)are shown in Figure 3. Despite the higher basal firing rate of the NSDA neuron
f
P
rt
7
a
H
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A
Y
1 msec
T
3 msec
B
v
200 pv
1 msecl
3 msecl
200 p
I
i
Fig. 1. Action potentials recorded from NSDA neurons in a 4-week-old (A) and a 2-week-old (B) rat pup are illustrated on the left. Note the presence of a prominent intial segment-somatodendritic IS-SD break in the rising phase of the positive-negative action poten-
tial. The antidromic spikes recorded from these same neurons were elicited by stimulation ofthe caudate nucleus (A: 0.5msec duration, 2.0 mA; B: 0.5 msec duration, 0.8 mA) are shown on the right. Arrows indicate the stimulus artifacts.
from the adult animal, it was clearly more sensitive to the inhibitory effects of apomorphine on spontaneous discharge than the NSDA neuron from the 4-week-old pup. In both cases, subsequent administration of haloperidol reversed the inhibitory effects of apomorphine.
Cumulative dose-response (DR) curves for the inhibitory effects of apomorphine on NSDA neurons from 4-week-old, 5-week-old, and adult rats are shown in Figure 4. Only the DR curve from the 4-week-old rat pups was significantly different from adults (F1,34 =
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DOPAMINENEURONONTOGENY TABLE I. DA neuron conduction velocities at different postnatal ages'
Discharge rate C.V. (m/sec) n
2 Weeks
4 Weeks
5 Weeks
Adult
3.36*
3.97 * 0.55 0.38 * 0.02
* 4.15 0.29 0.54
f 0.29
f 0.14 0.48**
t 0.02
0.56 ?t0.02
f 0.03 30
25
40
4.58
44
'ANOVA, followed by Dunnett's t test for comparisonto adult values. 'P
