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2 mM Ca²⁺ are used for convenience to improve the probability of release and therefore the rate of success during dual recordings. In addition, this Ca²⁺ concentration allows seeing the enhancer effect of the D₁-agonist on the IPSC₁ and the reduction caused by the D₂-agonists on the same response. Control STD-kinetics plot appeared almost at half the distance between both extremes (Fig. 5A). With lower Ca²⁺ concentrations, the number of failures increased and the time to collect the same data increased with it, making the experiments harder. However, the end result was the same in the experiments done with a lower Ca²⁺ concentration (1 mM), except that it was harder to see the reduction in IPSC₁ caused by the D₂-agonist because mean response was already reduced (although it was easier to see the enhancer D₁-action). Even with 2 mM Ca²⁺ it was harder to see the D₂-response. However, when using the population response, the strength of field stimulation was increased in the control to recruit more afferents and terminals to begin the experiment with an increased IPSC₁ (see Results). In those conditions, it was easy to see the D₂-agonist action. Thus, mean IPSC₁ amplitude in the controls for the D₂-agonist are larger than the controls used for the D₁-agonist (see Results). Therefore, each sample was compared with itself before and after treatment (Wilcoxon´s t test). In summary, high Ca²⁺ concentrations allow see better D₂-actions and low Ca²⁺ concentrations allow see better D₁-actions. But it was preferred to use the same concentration for both, to see both responses in the same conditions. The best concentration to see both responses was 2 mM.

Previously, it was shown that one can record GP®NSt connections after extensive lesions of GP neurons (1), that glutamate administered at the GP does not elicit synaptic responses in the striatum (1), that pallido-striatal synapses have different functional characteristics (2) and do not express D₁-class receptors (3), that changes in Ca²⁺-channels and dopamine signaling during development occur in parallel at both the somatodendritic and synaptic regions of SPNs; the latter being evaluated with field stimulation from the GP (4), and finally, that the most abundant muscarinic receptor at the somatodendritic region of SPNs is also located at the presynapsis (5). In addition, the present work shows that shape index parameters, STD kinetics, dopaminergic modulation of this kinetics, and amplitude vs. CV^-2 plots are virtually indistinguishable when comparing IPSCs evoked after field stimulation at the GP (GP®NSt) with unitary IPSCs recorded during dual recordings (SPN®SPN). Therefore, a great amount of evidence has accumulated supporting that the synapses that interconnect SPNs can be isolated after antidromic stimulation at the GP. This finding carries great importance for pharmacological analyses of these synapses because they bear several receptors important for basal ganglia function: canabinoids, purinergic, muscarinic, opiates, among others. They are modulated in a complex way by many transmitters. In addition, unitary data can receive strong support from population recordings (unitary connections are not exceptional but representative of many terminals doing the same thing). Hence, data from population and unitary connections can be pooled together to yield a strong statistical support to pharmacological studies. The pharmacology of these terminals is potentially important for the developing of drugs that alleviate motor disorders.

Dopaminergic D₂-class receptors (types D₂, D₃, D₄), have more affinity for dopamine than D₁-class receptors (types D₁ and D₅). K_i for the former is around 50 nM, and for the latter it goes to around 5 mM (6). This difference in affinity has led to propose that D₂-class receptor are responsible for maintaining a dopaminergic tone with low basal dopamine concentrations whereas D₁-class receptors are accountable for responding to sudden concentration peaks in dopamine (7, 8).

Among the most selective agonists for D₂-class receptors is quinelorane. However, it binds to both D₂ and D₃ type receptors. K_i for »341 nM for D₂ and »3.6 nM for D₃, with 100 times more affinity for the D₃-type receptor (9, 10). This means that a few D₃-type receptors (present in any of the SPNs populations) may produce a response as large as that produced by the more abundant D₂-type receptors. Therefore, its actions may not be so good to decide between co-localization or segregation between D₁ and D₂ receptor types.

In contrast, SKF-81297 is considered a "full agonist" with a K_i of about 2.2 nM for D₁-class receptors; it does not distinguish between D₁ and D₅ types. It has an EC₅₀ of about 18 nM to stimulate adenylyl cyclase. D₂-class receptors have a K_i about 10 mM for SKF-81297 (11). Note that this difference in affinity is the reverse of that seen for dopamine when comparing affinities between D₁ and D₂ class receptors. In this sense, and for physiological experiments, dopamine may be considered a D₂-class selective agonist. Note also that the experimental Results of the present work show that only about 50% of the synapses responded to SKF-81297. This is the number expected for receptor segregation at the level of the terminals. This result suggests that D₁-agonists are better fit to assess receptor segregation.

Age of Wistar rats ranged from 19-22 postnatal days (PD). Slices were perfused (3 ml/min) with an extracellular saline containing (in mM): 126 NaCl, 3 KCl, 1 MgCl₂, 2 CaCl₂, 26 NaHCO₃, and 10 glucose (pH 7.4 with NaOH, 298 mOsm/l with glucose; saturated with 95% O₂ and 5% CO₂; 25-27°C. 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline disodium salt (CNQX) (10 mM) and D-(-)-2-amino-5 phosphonovaleric acid (AP5) (50 mM) were added to block L-a-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA)-kainate and N-methyl-D-aspartate (NMDA) receptors respectively (Fig. 1D). Tight-seal whole cell recordings were used to record inhibitory postsynaptic currents (IPSCs) with the help of an Axoclamp 2B and/or Axopatch 200B amplifiers (Axon Instruments, Foster City, CA). All traces shown are the average of 4-min recordings (24 traces) taken when the amplitude had been stabilized for a given condition. A small hyperpolarizing voltage command (10 mV) was constantly given during the experiment to monitor input conductance. The relative importance of pallidostriatal transmission on SPNs was assessed by comparing the effects of glutamate applications on the synaptic activity of the recorded SPN (fig. 2B in ref. 1). The intracellular saline for postsynaptic medium spiny neurons contained (in mM): 72 KH₂PO₄, 36 KCl, 2 MgCl₂, 10 Hepes, 1.1 EGTA, 0.2 Na₂ATP, 0.2 Na₃GTP, 5 QX-314, and 1% biocytin, pH 7.2, 275 mOsm/l for a theoretical E_Cl^- = -30 mV. All experiments were performed at 25-27°C (room temperature). Series resistances ranged from 5 to 20 MW and were partially compensated (60-90%). All recordings were filtered at 1-3 kHz and digitized with an AT-MIO-6040E, a DAQ (NI-DAQ) board (National Instruments, Austin, TX) and a PC clone. On-line data acquisition used custom programs made in the LabVIEW environment (National Instruments). Neurons were visualized under infrared differential interference contrast (IR-DIC) microscopy using a Nikon Eclipse E600FN microscope equipped with a CCD camera (CCD-100, Dage-MTl Inc., Michigan City, IN) and a 40X long-working-distance water immersion objective.

The field stimulating electrode was a sharp (pencil shape) concentric bipolar tungsten electrode (12 mm at the tip; 50 ± 8 kW d.c. resistance) (FHC Inc., Bowdoinham, ME) located at the globus pallidus (GP) and attached to an isolation unit (Digitimer LTD, Hertfordshire, UK) connected to the AT-MIO card (1). Distance between recording and stimulating electrode in all configurations was 0.5-1 mm. Single stimuli or train of 10 stimuli (1-3 msec, 1-60 V; 1-500 mA) were delivered at 10, 20, and 50 Hz, every 10 sec (Fig. 4A; GP®NSt). If threshold to evoke an IPSC was reached, with an apparently stable response for several minutes, the stimulation intensity was then set at 1.5-2.0 the value of the threshold and administration of agonists and antagonists begun.

Twenty to thirty consecutive 20-Hz trains of IPSCs, evoked at a frequency of 0.1 Hz, were used to perform variance-mean analyses in which mean amplitudes of evoked IPSCs (corrected for basal lines from previous IPSCs) were plotted against their peak variances (12, 13). Then, a parabola of the form:

)

was fitted with a Marquart algorithm; where y represents IPSC variance (ordinates), x represents IPSC mean amplitude (abscissa) and A and B are free parameters. Basically, parameter A indicates the initial slope of the parabola and parameter B depends on the width of the parabola. From this fit, a weighted average of the quantal amplitude, Qw, was obtained:

1+CV²) (2)

where CV is the coefficient of variation of the IPSC amplitude at an individual release site. In addition, the approximate number of release sites (N) and the average probability of release across release sites (assuming a binomial distribution) can be approximated by (Eqs. 4 and 5):

)

)

Previous reports (4, 14) have shown that this analysis hardly distinguishes between N and P when the number of contacts is small (by definition in pair recordings). Both quantal variability and a discrepancy between functional release sites and anatomical active zones impede a precise distinction. For example, if a release site was almost silent (low P) in the control and then its P increases much after the treatment, it may bee seen as an increase in N or P or both (14). However, independent measurements can be used to confirm an increase in P [e.g., (4)]

However, the initial slope (A) is very reliable (4, 15, 16) and it would be essentially the same in both cases: with a change in N or P. Both N and P indicate a presynaptic change, whereas a change in Q most probably indicates a postsynaptic change. This makes the analysis useful to distinguish between preand postsynaptic changes even if N and P cannot be distinguished precisely. Therefore, to fit the parabolas in Fig. 5B, we chose to fix N to the control value, and then, quantify all changes as changes in P.

For comparing different samples the Kruskal-Wallis test was used. Post hoc pairwise comparison then used the Dunn´s test (17). Given the intrinsic variance (quantal variability) of the synaptic responses when only a few terminals are involved, it was important to see if changes seen in the responsive cases were significant. Therefore, the same sample of responsive cases was compared before and after a given treatment with the Wilcoxon's t test (17). But because of this same variability, we also checked for significant changes in variance in each responsive case to see if the treatment induced significant changes. Thus, variances were compared before and after treatments with the F test. Large variances are related with intermediate release probabilities and lower variances with relatively low or high release probabilities (13). Thus, significant changes in the coefficient of variation (CV) for samples of responsive cases were also searched. Changes in CV are indicative of presynaptic actions. CV is inversely related to quantal content (m) according to: m = (1-p)/CV^-2 (18). Therefore, changes in CV were searched in samples of responsive cases to yield independent evidence of significant presynaptic modulation in these cases. P < 0.05 was considered significant in all tests.

Neurons were filled with biocytin during recording (1, 15, 16). Slices with a single or two filled neurons (paired recordings) were taken into consideration for immunocytochemistry. A combination of intracellular labeling and substance P (SP), enkephalin (ENK, Leu, or Met) or parvalbumin (PV) immunocytochemistry, was used in each occasion to identify the recorded neurons. Slices containing injected neurons were fixed overnight in 4% paraformaldehyde and 1% picric acid in 0.1 M PBS at pH = 7.4. The slices were then infiltrated with 30% sucrose and cut on a vibratome into 40 mm parasagittal sections. The sections were incubated 4-6 h in PBS solution containing 0.2n Triton X-100 and avidin conjugated with Cy3 (1:200 dissolved in PBS, Zymed Laboratories, San Francisco, CA), to label the recorded neuron. Briefly, sections were rinsed in PBS and incubated for 18-24 h at 4°C with a primary rabbit antibody against ENK or SP (diluted 1:200) (Peninsula Labs, San Carlos, CA) or 36 h at 4°C with a mouse monoclonal antibody against parvalbumin (anti-PV; 1:2000, Sigma-Aldrich dissolved in PBS containing 0.25% Triton-X). After rinsing in PBS containing 1% albumin, sections were reincubated for 1 h in a dark room with secondary antibodies conjugated to FITC (diluted 1:100). Sections were mounted in an anti-quenching media (Vectashield, Vector Laboratories) and examined under a confocal microscope (MRC- 1024; Bio-Rad, Natford, UK) equipped with a krypton-argon mixed-gas laser. Two laser lines emitting at 490 and 560 nm were used for exciting FITC and Cy3, respectively. Immunostained cells were studied either on single confocal images or on reconstructed sections made by projecting z-series of three to four consecutive confocal images 10 mm apart and collected throughout the thickness of the section. The background noise was reduced averaging three to six images. Digitized images were transferred to a personal computer (Confocal Assistant, T. C. Brelje).

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