Introduction

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Serotonin (5-HT) is a neuromodulator widely distributed in the central nervous system and has been implicated in a number of neurophysiological functions. The multiple actions of serotonin are mediated by at least 14 subtypes of membrane surface receptors, most of which are members of the G protein-coupled receptor superfamily (for review, see Barnes and Sharp, 1999).

The 5-HT_1B receptor was initially identified as a [³H]5-HT binding site with low affinity for spiperone in the rodent brain (Pedigo et al., 1981). Another [³H]5-HT binding site, designated the 5-HT_1D site, was found in other species, including human, dog, and guinea pig (Hoyer and Middlemiss, 1989). It had a distribution similar to that of the 5-HT_1B site in rodents, although the pharmacological profile was distinct. Subsequent molecular biological studies have demonstrated that the 5-HT_1D receptor found in higher species is a close homolog of the rodent 5-HT_1B receptor (Adham et al., 1992; Jin et al., 1992), leading to the recent reclassification of these receptors as the 5-HT_1B subtype (Hartig et al., 1996). On the other hand, the 5-HT_1D receptor, found in all mammalian species (Hamblin and Metcalf, 1991; Hamblin et al., 1992), is now termed the 5-HT_1D receptor (Hartig et al., 1996).

5-HT_1B receptors are expressed in many brain areas, including the basal ganglia and the midbrain raphe nuclei. 5-HT_1D receptors seem to be colocalized with 5-HT_1B receptors, although at much lower densities (Bruinvels et al., 1993). A unique feature of the expression pattern of 5-HT_1B receptors is the mismatch in the distribution of 5-HT_1B receptor mRNAs and 5-HT_1B binding sites, which suggests that 5-HT_1B receptors are expressed predominantly on nerve terminals of both 5-HT and non-5-HT neurons (Boschert et al., 1994). This anatomical distribution is consistent with the idea that 5-HT_1B receptors function both as 5-HT auto- and heteroreceptors to control the release of neurotransmitters.

The most selective ligands at 5-HT_1B and/or 5-HT_1D receptors are agonists. Thus, previous pharmacological studies were based on a combination of selective agonists and relatively nonselective antagonists. The recently developed selective 5-HT_1B/1D receptor antagonist GR 127935 has turned out to have a partial agonistic activity both in vitro and in vivo (Pauwels, 1997), compromising its usefulness as a probe for the functions of HT_1B/1D receptors.

In the present study, the 5-HT_1B receptor knockout mouse was used to re-evaluate the involvement of 5-HT_1B receptors in modulating the release of neurotransmitters in the central nervous system. To this end, synaptic potentials or currents were examined in three specific brain areas: the dorsal raphe, the ventral midbrain, and the nucleus accumbens. Each of these areas is known to have presynaptic 5-HT_1B receptors, which have been suggested to inhibit synaptic transmission. These include 5-HT-mediated slow IPSPs in the dorsal raphe (Pan and Williams, 1989a), GABAergic slow IPSPs in the ventral midbrain (Johnson et al., 1992; Cameron and Williams, 1994), and glutaminergic EPSCs in the nucleus accumbens (Muramatsu et al., 1998). Furthermore, the effects of cocaine, which elevates extracellular 5-HT concentration by uptake blockade, were investigated. The results indicate that activation of presynaptic 5-HT_1B receptors produces potent inhibition of the release of 5-HT, GABA, and glutamate in the central nervous system and unequivocally demonstrate the participation of 5-HT_1B receptors in the actions of cocaine.

	Materials and Methods

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Animals. All experiments were performed on tissues obtained from wild-type and 5-HT_1B knockout inbred mice (20-53 days old). Both wild-type and knockout mice were bred in the Portland VA Veterinary Medical Unit. Mice were maintained at 22 ± 1°C with a 12-h/12-h light/dark cycle. The background strain comprised a mixture of the 129/SvPas, 129/Sv-ter, and 129/SvEvTac substrains of 129 inbred mice (Phillips et al., 1999).

Slice Preparation. The preparation of slices containing the dorsal raphe, the ventral midbrain, and the nucleus accumbens was as described previously (Pan et al., 1989; Cameron and Williams, 1994; Manzoni et al., 1998). Briefly, mice were anesthetized with halothane and killed. Brain slices (180-220 µm) were cut with a vibratome (Leica, Nussloch, Germany) in cold (4°C) physiological saline. For recordings from dorsal raphe neurons, coronal slices were taken at the level of the decussation of the cerebellar peduncle or where the aqueduct begins to open to the fourth ventricle. For recordings from midbrain dopamine neurons, horizontal slices were taken near the floor of the interpeduncular fossa. For recordings from medium spiny neurons, parasagittal slices containing the nucleus accumbens were cut. A single slice was placed in a recording chamber and superfused with warmed (35°C) physiological saline containing 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH₂PO₄, 1.2 mM MgCl₂, 2.4 mM CaCl₂, 11 mM glucose, 21.4 mM NaHCO₃, saturated with 95% O₂ and 5% CO₂.

Recordings in the Dorsal Raphe and the Ventral Midbrain. Cells were visualized with an upright microscope with infrared illumination, and whole-cell recordings of membrane potential were made with patch pipettes (1.8-2.5 M) containing 115 mM K gluconate, 20 mM KCl, 1.5 mM MgCl₂, 1 mM BAPTA, 2 mM MgATP, 0.2 mM GTP, 10 mM Na₂ phosphocreatine, buffered with 10 mM HEPES, pH 7.3, 285 mOsm/kg. An Axopatch 1D amplifier (Axon Instruments, Foster City, CA) was used to record the data, which were filtered at 1 kHz, digitized at 5 kHz, and collected on a personal computer using AxoGraph 4 or pCLAMP 6 (Axon Instruments).

Recordings in the Nucleus Accumbens. Whole-cell recordings of membrane current were made with patch pipettes (2-4 M) containing 128 mM KCl, 20 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 0.3 mM CaCl₂, 2 mM MgATP, 0.3 mM GTP, 0.2 mM cAMP, buffered with 10 HEPES, pH 7.3, 290 mOsm/kg. The holding potential was 70 mV. An Axopatch 200A amplifier (Axon Instruments) was used to record the data, which were filtered at 1 kHz, digitized at 5 kHz, and collected on a personal computer using ACQUIS-1 (Bio-Logic, Claix, France) or pCLAMP 6. To evoke EPSCs, extracellular stimuli (100 µs) were delivered every 30 s via bipolar tungsten stimulating electrodes at the prefrontal-accumbens border. The superfusion medium contained picrotoxin (100 µM) and strychnine (1 µM) to block GABA_A- and glycine-mediated synaptic currents, respectively. Two stimuli were applied at an interval of 50 ms to obtain a paired-pulse ratio, which was calculated by dividing the amplitude of the first EPSC by that of the second EPSC.

Drug Application. All drugs were applied by superfusion. Drugs used are as follows: 5-HT, cocaine, DAMGO, fenfluramine, picrotoxin, strychnine, and prazosin were from Sigma (St. Louis, MO); pindobind 5HT_1A, TFMPP, MK-801, and eticlopride were from RBI (Natick, MA); NBQX was from Tocris Cookson (Ballwin, MO); CGP35348 was a gift from Novartis (Basel, Germany); and GR 127935 was a gift from Glaxo Wellcome (Stevenage, UK).

Data Analysis. IPSP and EPSC amplitudes were measured, respectively, by averaging 20-ms and 2-ms windows around the peak and subtracting the average value obtained during 100-ms and 5-ms windows immediately before the stimuli. The time constants for the rising phase and decaying phase of the IPSP were obtained by fitting the IPSP trajectory with a double exponential function using AxoGraph 4. Data are expressed as mean ± S.E.M. Statistical significance was determined with unpaired Student's t test or Mann-Whitney U test. The difference was considered significant at P < .05.

	Results

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Dorsal Raphe Neurons. Whole-cell recordings of synaptic potentials were made at the resting membrane potential (55 to 65 mV) from dorsal raphe neurons in wild-type and 5-HT_1B knockout mice. All experiments were carried out in slices treated with NBQX (5 µM), MK-801 (50 µM), picrotoxin (100 µM), strychnine (1 µM), and prazosin (100 nM) (see Materials and Methods). A single electrical stimulus evoked a slow IPSP that was blocked by the selective 5-HT_1A receptor antagonist pindobind 5-HT_1A (1 µM) by 95 ± 3% and 100 ± 0% in wild-type and knockout mice, respectively (n = 3 in each group). This IPSP was reproducible throughout the duration of recordings (~3 h) with the application of stimuli at 60-s intervals. The presence of a 5-HT-mediated IPSP in the mouse dorsal raphe was expected from the experiments done in rats (Pan et al., 1989).

No Compensatory Alterations in 5-HT_1B Knockout Mice. The time course of the IPSP in wild-type and knockout animals was analyzed by examining the first IPSP evoked by a pair of stimuli separated by 4 s. This IPSP (P₁) was fitted to a double exponential function to obtain time constants of the rising and decaying phases of IPSP (Fig. 1A). The rise time constant reflects the activation kinetics of the underlying potassium conductance, whereas the rate of 5-HT uptake by the serotonin transporter is the major determinant of the decay time constant (Pan et al., 1989). It was found that both of these time constants were not significantly different between wild-type and knockout mice (Fig. 1B). Furthermore, the peak amplitude of P₁, which should be determined by the rise and decay time constants, the amount of 5-HT released, and the sensitivity of postsynaptic 5-HT_1A receptor, was also similar in wild-type (13.6 ± 0.9 mV, 3.7-23.5 mV, n = 34) and knockout animals (13.4 ± 0.8 mV, 6.2-23.3 mV, n = 37, P > .8) (Fig. 1C). Thus, deletion of the presynaptic 5-HT_1B receptor caused no significant changes in the functioning of the serotonin transporter and the postsynaptic 5-HT_1A receptor, or in the presynaptic 5-HT release mechanism in the dorsal raphe.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Properties of 5-HT1A IPSPs in the dorsal raphe of wild-type and 5-HT1B knockout mice. A, representative traces of 5-HT1A IPSPs in wild-type and knockout mice. IPSPs were evoked by a pair of stimuli separated by 4 s to obtain the first (P1) and sond (P2) IPSPs. Dashed lines are the double exponential fit to P1. B, the time constants of the rising and decaying phases of IPSPs determined by the double exponential fit as in A. C, P1 and P2 amplitudes and the paired-pulse ratio (P1/P2). D, representative traces showing the effect of TFMPP (1 µM) in wild-type and knockout mice. Data are shown as means ± S.E.M. **P < .01; ***P < .001.

Presynaptic 5-HT_1B Receptor-Mediated Inhibition of 5-HT_1A IPSPs Is Absent in Knockout Mice. Application of TFMPP (1 µM), a moderately selective 5-HT_1B receptor agonist, reduced the P₁ amplitude to 20 ± 10% of control in wild-type mice (n = 3), whereas it slightly augmented the P₁ amplitude in knockout mice (107 ± 9% of control, n = 3, P < .005 versus wild-type) (Fig. 1D). TFMPP had no measurable effect on the baseline membrane potential nor did it block the hyperpolarization caused by application of the 5-HT₁ agonist 5-CT (100 nM; data not shown). Thus, it seemed that TFMPP selectively activated 5-HT_1B receptors without acting on 5-HT_1A receptors in this preparation. These data indicate that activation of presynaptic 5-HT_1B receptors potently inhibits evoked 5-HT release in the dorsal raphe.

Persistent Effect of 5-HT on the Presynaptic 5-HT_1B Receptor. The effect of 5-HT released by the first stimulus on presynaptic 5-HT_1B receptors outlasted the duration of the postsynaptic 5-HT_1A receptor-mediated IPSP. After 4 s, the 5-HT_1A IPSP produced by the first stimulus had recovered but the second stimulus caused a much smaller IPSP (Fig. 1A). We next examined whether this differential kinetics of presynaptic versus postsynaptic effects of synaptically released 5-HT could also be observed for exogenously applied 5-HT. Superfusion of 5-HT (1 µM) caused a membrane hyperpolarization of 16.1 ± 3.2 mV (n = 4) and 17.6 ± 2.4 mV (n = 3) in wild-type and knockout mice, respectively (P > .7) (Fig. 2B), further confirming the similar sensitivity of the postsynaptic 5-HT_1A receptor in both animals. In wild-type mice, 5-HT (1 µM) almost completely abolished the IPSP (7 ± 3% of control, n = 4) (Fig. 2B). After washout of 5-HT, the membrane hyperpolarization recovered rapidly (~3 min). However, inhibition of the IPSP recovered much more slowly over the time course of ~10 min. Thus, a significant reduction in the IPSP amplitude was observed even when the membrane potential had completely recovered (Fig. 2A), indicating that 5-HT-induced inhibition of the IPSP was not simply caused by occlusion of the IPSP by the membrane hyperpolarization. In contrast, in the same experiments with knockout mice, the IPSP was largely occluded but not abolished. In the presence of 5-HT (1 µM), the IPSP was still 31 ± 6% of control (n = 3, P < .05 versus wild-type) (Fig. 2D). In addition, after washout of 5-HT, the membrane hyperpolarization and IPSP inhibition recovered over a similar time course (Fig. 2, C and D), suggesting that postsynaptic occlusion was mainly responsible for the IPSP inhibition. We observed a small inhibition of IPSP transiently remaining after hyperpolarization had subsided in one cell from knockout mice, which may reflect a presynaptic effect of 5-HT not mediated by 5-HT_1B receptors. Taken together, these results show that the effect of 5-HT on presynaptic 5-HT_1B receptors is more persistent than that on postsynaptic 5-HT_1A receptors.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   The effects of exogenous 5-HT on the membrane potential and IPSPs in the dorsal raphe. A and C, representative traces of IPSPs illustrating the effects of 5-HT (1 µM) in wild-type (A) and 5-HT1B knockout (C) mice. Traces of IPSPs immediately before application of 5-HT (Control), in the presence of 5-HT (5-HT), and at the time when the membrane potential had recovered to control level after washout of 5-HT (Wash) are shown. The baseline membrane potential before the stimulus is not subtracted in these traces to depict the hyperpolarization induced by 5-HT. B and D, summary graphs illustrating the time course of the effects of 5-HT (1 µM) in wild-type (n = 4; B) and knockout (n = 3; D) mice. The baseline membrane potential before the stimulus is plotted after subtraction of the mean membrane potential obtained during the 5-min period before application of 5-HT, and the IPSP amplitude is normalized to the mean amplitude obtained during the same 5-min period. Data are expressed as mean ± S.E.M.

The Effects of 5-HT Uptake Inhibition by Cocaine. Application of cocaine increased the decay time constant of IPSP, resulting from the blockade of 5-HT uptake (Fig. 3A and B). This action was similar in wild-type and knockout mice, indicating that the sensitivity of the serotonin transporter was not different. However, cocaine affected the peak amplitude of the IPSP differently in two groups of animals (Fig. 3, A and C). Superfusion of cocaine (1 µM) increased the P₁ amplitude to 128 ± 8% and 152 ± 29% in wild-type and knockout animals, respectively (n = 5 each, P > .4). Increasing the concentration of cocaine from 1 µM to 10 µM caused a reduction in the P₁ amplitude to 88 ± 13% of control in wild-type mice (n = 5), whereas the P₁ amplitude remained at the same level in knockout mice (148 ± 19% of control, n = 5, P < .05 versus wild-type). Under the conditions of these experiments, cocaine had no effect on the membrane potential in either group of animals. These results indicate that an increase in the ambient concentration of 5-HT produced by cocaine acts selectively on presynaptic 5-HT_1B receptors to inhibit 5-HT release.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   The effects of cocaine on IPSPs in the dorsal raphe. A, representative traces of IPSPs illustrating the effects of cocaine in wild-type and 5-HT1B knockout mice. B, a summary bar graph showing the effects of cocaine on the time constants of the rising and decaying phases of IPSP. Sequential application of increasing concentrations of cocaine (1 µM and 10 µM) was made in each cell. The time constants are normalized to those obtained in the absence of cocaine. C, a summary bar graph depicting the effects of cocaine on the P1 amplitude. The P1 amplitude is normalized to that obtained in the absence of cocaine. Data are expressed as mean ± S.E.M. *P < .05.

Dopamine Neurons in the Ventral Midbrain. A train of 10 extracellular stimuli was applied to evoke synaptic potentials mediated by GABA_B receptors in midbrain dopamine neurons. The membrane potential was maintained at 55 to 65 mV to stop the spontaneous firing and GABA_B IPSPs were isolated pharmacologically (see Materials and Methods). IPSPs thus recorded were completely blocked by the GABA_B receptor antagonist CGP35348 (100 µM) in both wild-type and knockout mice (n = 3 each). The average amplitude of the IPSP was similar in wild-type (10.2 ± 1.1 mV, 4.6-22.1 mV, n = 18) and knockout mice (10.5 ± 0.8 mV, 5.2-17.8 mV, n = 20, P > .8).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   5-HT1B receptor-mediated inhibition of GABAB IPSPs in midbrain dopamine neurons. A, representative traces of GABAB IPSPs showing the effect of 5-HT (10 µM) in wild-type and 5-HT1B knockout mice. IPSPs were evoked by a train of 10 stimuli (70 Hz). B, A summary bar graph of the inhibition of GABAB IPSPs by 5-HT (10 µM, 30 µM) and DAMGO (300 nM). C, representative traces of IPSPs depicting the effect of cocaine (1 µM) in wild-type and knockout mice. D, a summary bar graph of the inhibition of IPSPs by cocaine (1 µM) and fenfluramine (10 µM). Data are shown as means ± SEM. *P < .05; **P < .01; ***P < .001.

Medium Spiny Neurons in the Nucleus Accumbens. Whole-cell recordings were made at a holding potential of 70 mV from medium spiny neurons in the nucleus accumbens. Glutamate EPSCs elicited by focal extracellular stimuli were isolated by blocking IPSCs with receptor antagonists (see Materials and Methods). The 5-HT_1B receptor agonist TFMPP (1 µM) inhibited EPSCs in wild-type mice (Fig. 5, A and B). This inhibition was associated with an increase in the paired-pulse ratio (151 ± 41% of control, n = 5), consistent with a presynaptic locus of action. Superfusion with fenfluramine (10 µM) also caused a reduction in the EPSC amplitude (Fig. 5B). Both of these drugs failed to affect EPSCs in knockout mice. These results show that activation of presynaptic 5-HT_1B receptors inhibits glutamate release and that this can be achieved by the endogenous 5-HT.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   5-HT1B receptor-mediated inhibition of glutamate EPSCs in the nucleus accumbens. A, Representative traces of EPSCs illustrating the effect of TFMPP (1 µM) in wild-type and 5-HT1B knockout mice. EPSCs were evoked by a pair of stimuli separated by 45 ms. B, a summary bar graph showing the inhibition of EPSCs by TFMPP (1 µM) and fenfluramine (10 µM). C, a summary bar graph depicting the effect of cocaine on EPSCs. Sequential application of increasing concentrations of cocaine (1 µM and 10 µM) was made in each cell. The EPSC amplitude is normalized to that obtained in the absence of cocaine. Data are expressed as mean ± S.E.M. *P < .05.

	Discussion

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Using the 5-HT_1B receptor knockout mouse, this study demonstrates that the presynaptic 5-HT_1B receptor functions both as an autoreceptor to inhibit 5-HT release in the dorsal raphe and as a heteroreceptor to inhibit the release of GABA and glutamate in the ventral midbrain and the nucleus accumbens, respectively. Endogenous 5-HT, released either electrically, spontaneously or pharmacologically, can activate presynaptic 5-HT_1B receptors on all of these three types of terminals (i.e., serotonergic, GABAergic, and glutaminergic). Furthermore, the cocaine-induced inhibition of the 5-HT-mediated IPSP in the dorsal raphe and the GABAergic IPSP in the ventral midbrain was mediated by the activation of presynaptic 5-HT_1B receptors. In contrast, presynaptic 5-HT_1B receptors were not involved in the effects of cocaine on the glutaminergic transmission in the nucleus accumbens.

Presynaptic 5-HT_1B Receptor as an Autoreceptor in the Dorsal Raphe. 5-HT_1A IPSPs exhibited paired-pulse depression when two IPSPs were elicited 4 s apart. The magnitude of paired-pulse depression was reduced significantly in knockout mice, indicating a significant contribution of the activation of presynaptic 5-HT_1B autoreceptors. Paired-pulse depression of slow synaptic potentials has been described for 5-HT_1A IPSPs in the nucleus prepositus hypoglossi (Bobker and Williams, 1990) and for GABA_B IPSPs in the ventral midbrain and the CA1 region of the hippocampus (Davies et al., 1990; Cameron and Williams, 1994). In all these cases, neurotransmitters released by the first stimulus were suggested to activate presynaptic autoreceptors to suppress their own release. Our data with 5-HT_1B receptor knockout mice provides the most compelling evidence for the involvement of presynaptic autoreceptors in this form of frequency-dependent fatigue of the slow synaptic transmission.

Presynaptic 5-HT_1B Receptor as a Heteroreceptor in the Ventral Midbrain and the Nucleus Accumbens. 5-HT, the 5-HT₁ agonist TFMPP, or the 5-HT releasing agent fenfluramine suppressed GABAergic and glutaminergic transmission in the ventral midbrain and the nucleus accumbens, respectively, only in wild-type mice. These results confirm previous pharmacological studies reporting the involvement of presynaptic 5-HT_1B receptors in these effects (Johnson et al., 1992; Cameron and Williams, 1994; Muramatsu et al., 1998). The ventral midbrain and the nucleus accumbens are known to receive substantial 5-HT innervation from the median and dorsal raphe (Parent et al., 1981). Moreover, ultrastructural studies have found evidence for nonjunctional communications made by these 5-HT terminals in the ventral midbrain and the nucleus accumbens (Hervé et al., 1987; Van Bockstaele and Pickel, 1993). Thus, endogenous 5-HT may well diffuse extrasynaptically to act on presynaptic 5-HT_1B receptors expressed at non-5-HT terminals. Indeed, cocaine inhibited GABA_B IPSPs in midbrain dopamine neurons from wild-type mice but not from knockout mice, showing that facilitating the diffusion of endogenous 5-HT by uptake blockade can induce activation of presynaptic 5-HT_1B receptors at GABAergic terminals in the ventral midbrain. A similar conclusion was reached on a pharmacological basis in a previous study done in slices from guinea pig (Cameron and Williams, 1994). In the nucleus accumbens, on the other hand, cocaine produced a comparable inhibition of glutaminergic EPSCs in both wild-type and knockout mice, excluding the involvement of 5-HT_1B receptors. This is in accord with the previous report showing that cocaine suppresses glutaminergic transmission in the nucleus accumbens through an increase in endogenous extracellular dopamine levels and subsequent activation of presynaptic dopamine receptors (Nicola et al., 1996). It should be noted that a relatively high concentration (10 µM) was required to obtain this inhibitory effect of cocaine in the nucleus accumbens compared with the concentration of cocaine (1 µM) that caused inhibition of GABAergic transmission in the midbrain. This is consistent with the known lower affinity of cocaine for the dopamine transporter than for the serotonin transporter (Ritz et al., 1987).

Significance. Studies on the functions of 5-HT_1B receptors have been confounded by the lack of a potent and selective antagonist. GR 127935, a benzanilide compound, was originally developed as a highly selective HT_1B/1D receptor antagonist (Skingle et al., 1993). However, accumulating evidence has demonstrated that it has a considerable intrinsic activity at HT_1B/1D receptors both in vitro and in vivo (Pauwels, 1997). In line with this, GR 127935 (1 µM) inhibited GABA_B IPSPs and occluded the effect of 5-HT in midbrain dopamine neurons (data not shown). On the other hand, the 5-HT_1B receptor knockout mice have been used successfully to probe the functions of 5-HT_1B receptors in behavior and neuropsychiatric disorders (Scearce-Levie et al., 1999). Although some researchers have suggested that developmental compensations are responsible for the increased vulnerability to cocaine in 5-HT_1B receptor knockout mice (Rocha et al., 1998; Shippenberg et al., 2000), our results show that 5-HT_1B receptors are indeed responsible for the effects of cocaine on synaptic transmission in the ventral midbrain and the dorsal raphe, two brain areas believed to be important for cocaine sensitization (Parsons and Justice, 1993). Furthermore, deletion of 5-HT_1B receptors seemed to induce no compensatory changes in the properties of synaptic transmission recorded with brain slices. Thus, the impact of ablation of the 5-HT_1B receptor protein could be unambiguously detected when the assay was performed at the site of its expression (i.e., the presynaptic terminal).