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Evidence for different exocytosis pathways in dendritic and terminal dopamine release in vivo
Brain Research 950 (2002) 245–253 www.elsevier.com / locate / bres
Research report
Evidence for different exocytosis pathways in dendritic and terminal dopamine release in vivo Filip Bergquist*, Haydeh Shahabi Niazi, Hans Nissbrandt ¨ ¨ University, Box 431, SE 405 30 Goteborg , Sweden Department of Pharmacology, Institute of Physiology and Pharmacology, Goteborg Accepted 4 June 2002
Abstract Although dendritic release was first proposed in the 1970s, the mechanism of release is still subject to debate. We have used in vivo microdialysis to study the acute effects of botulinum toxin A, B and tetanus toxin injected in the substantia nigra or striatum of freely moving rats. Spontaneous and evoked dopamine release decreased in both regions after treatment with the SNAP-25 (synaptosomeassociated protein of 25 kDa) cleaving protease botulinum toxin A (1000 mouse lethal doses, MLD). Tetanus toxin (4000 MLD) did not significantly change spontaneous or evoked dopamine release in striatum or in the substantia nigra. Another synaptobrevin cleaving protease, botulinum toxin B, inhibited release in the striatum by 55% but did not affect dopamine release when injected in the substantia nigra. The results indicate that both terminal and somatodendritic dopamine release need intact SNAP-25 to occur, but somatodendritic dopamine release in contrast to terminal release depends on a botulinum toxin B resistant pathway. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitter systems and channels Keywords: Dopamine release; Exocytosis; SNAP-25; Striatum; Substantia nigra; Synaptobrevin
1. Introduction The characteristic symptoms of Parkinson’s disease have been attributed to the loss of dopaminergic terminals in the striatal region. Dopamine can, however, also be released from the dopaminergic soma and dendrites in the reticulate part of substantia nigra [13,18,30] and is in the position to influence neurotransmission also in this region [2,10,26,29]. The reticulate part of substantia nigra is one of the major output relay nuclei, and therefore somatodendritically released dopamine could theoretically modulate motor control also at this level. Somatodendritic dopamine release in rat have properties typical for vesicular neurotransmitter release in that it is sodium channel dependent (blocked by tetrodotoxin) [14,37] and calcium dependent [4,9,10,40], also reserpine, an inhibitor of vesicular monoamine transport, significantly reduces dendritic dopamine stores and release [5,21]. Somatodendritic dopamine re*Corresponding author. Tel.: 146-31-773-3425; fax: 146-31-811-792. E-mail address: [email protected] (F. Bergquist).
lease does however have different characteristics compared to terminal release in regard to for example the calcium sensitivity [4,6]. Also, histological investigations have only shown a scarce occurrence of vesicular structures in the soma and dendrites of dopaminergic neurones [20,31]. Consequently other possibilities for release have been proposed, such as release from smooth endoplasmatic reticulum [15,28,31], or extravesicular release via reversal of the dopamine carrier [14,17] (for review, see Ref. [25]). Investigations of the molecular mechanisms of vesicular release have identified three proteins: syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptobrevin (also known as VAMP, vesicle associated membrane protein), as the minimal machinery for membrane fusion [39], although several other proteins may have regulatory functions (for review, see Ref. [19]). Synaptobrevin, syntaxin and SNAP-25 are remarkably homologous proteins in different organisms but there are some different isoforms which may to some extent account for the specificity of intracellular membrane trafficking [27,38]. The clostridial neurotoxins botulinum toxin A–G
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03047-0
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and tetanus toxin are metalloproteases that specifically cleave SNAP-25 (botulinum toxin A, C and E), synaptobrevin (botulinum toxin B, D, F and G) or syntaxin (botulinum toxin C) (for review, see Ref. [33]). The present study aimed at comparing the effects of synaptobrevin specific protease and SNAP-25 specific protease on dopamine release in the reticular part of substantia nigra and in the striatum. The method of in vivo microdialysis in rat brain was combined with acute microinjections of clostridial toxins. We show that spontaneous and evoked somatodendritic and terminal dopamine releases are both disrupted by treatment with botulinum toxin A, but that botulinum toxin B only inhibits terminal release. This is the most direct evidence of exocytosis from dopaminergic dendrites in substantia nigra this far, but the findings also indicate a fundamental difference between terminal and somatodendritic release mechanisms.
2.3. Microdialysis probes and surgery
2. Materials and methods
2.4. Microdialysis
The experimental design was approved by the local ¨ ethical committee in Goteborg, and carried out in accordance with the European Communities Council Directive of 24 November 1986.
2.4.1. Acute experiments On the day of the experiment the animals were connected to a swivelled perfusion system and the microdialysis probes were perfused with the modified Ringer solution at 2.0 ml / min. After 1 h of adaptation and equilibration, four consecutive 40 ml samples were collected and analysed for basal levels of dopamine, 3,4dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), with high performance liquid chromatography followed by electrochemical detection. During fraction five, toxin or vehicle were slowly (2 ml in 15 min) injected manually through stiff PEP-tubing directly connected to the injection capillary. Microinjections were made with botulinum toxin A, B, tetanus toxin or with the two different vehicles. After
2.1. Animals Male SD rats (280–320 g) from B&K Universal AB (Sollentuna, Sweden) were housed four per cage under controlled environmental conditions (26 8C, 60–65% humidity, light 05:00–19:00 h, dark 19:00–05:00 h) for 1 week before the experiments. Food and tap water were allowed ad lib. Experimental procedures were carried out during daytime.
To be able to locally administer substances of as large molecular weight as botulinum and tetanus toxin we modified an I-shaped microdialysis probe by attaching a small diameter fused silica capillary (inner diameter 90 mm) adjacent to the dialysis membrane (Fig. 1). Deadspace in the injection capillary was calculated to approximately 0.2 ml. A dialysis membrane with a 20-kDa cut-off was used and the active part was 2 mm long regardless of whether the probe was placed in the substantia nigra or in the striatum. Before implantation the probes were perfused and washed with 70% ethanol and then with modified Ringer solution. The injection capillary was washed and filled with modified Ringer solution and all tubings were then sealed. The animals were stereotactically implanted with microdialysis probes as previously described [4] and allowed to recover single-housed for 48 h before the experiment.
2.2. Drugs For surgical anaesthesia ketamine and xylazin were used. Botulinum toxin A and B (Sigma-Aldrich Sweden AB) were dispensed in vials and frozen. At the day of experiment the stem solution (1 mg / ml in buffered saline) was diluted with modified Ringer solution containing 140 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl 2 and 1.0 mM Mg Cl 2 to a final concentration of 0.5 mg / ml (approximately 500 MLD/ ml). Tetanus holotoxin (Alomone, Jerusalem, Israel) was dissolved in 0.9% NaCl with 0.1% bovine serum albumin (BSA) on the day of experiment to a final concentration of 0.05 mg / ml (approximately 2000 MLD/ ml). Vehicle solutions were prepared in the same ways but without toxin. L-Glutamic acid HCl (Sigma-Aldrich Sweden AB) and tetrodotoxin (Alomone, Jerusalem, Israel) was dissolved in the modified Ringer solution, and pH was adjusted to 7.4 immediately before use.
Fig. 1. Combined microdialysis and microinjection probe. Schematic drawing of a microdialysis probe modified by the attachment of a fused silica capillary allowing microinjections of large molecular weight substances such as clostridial toxins.
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the injection seven consecutive 40-ml samples were collected and analysed. Then a short pulse of 5 min with perfusion media containing 275 mM KCl was given to study effects on dopamine release evoked by high osmolarity and depolarisation. In one set of experiments, microdialysis probes without injection cannulas were implanted in the substantia nigra and used to administer a 20-min pulse of 0.1 mM L-glutamic acid. This pulse was given in the first sampling fraction following the collection of baseline samples and the perfusion then continued for another hour. Tetrodotoxin was perfused in nigral probes at the concentration 1 mM after baseline sampling. All animals were sacrificed immediately after the experiment and the location of the microdialysis probe was verified macroscopically after slicing the brain with a vibratome. Animals with probe locations outside the target area or with intracerebral haemorrhage were excluded.
2.4.2. ‘ No net flux’ experiment: On day 2 after the implantation a ‘no net flux’ determination of extracellular dopamine concentrations was performed. After a stable baseline of dopamine concentrations was obtained, the perfusion medium was changed to one containing approximately 10 nM dopamine. When dopamine concentrations were stable again in the dialysate, this was repeated with a perfusion media containing approximately 20 nM dopamine. Lower dopamine concentrations (1 and 2 nM) were used when the ‘no net flux’ procedure was performed in substantia nigra. The inlet and outlet concentrations were determined and the net change in dialysate dopamine concentration was calculated. The perfusion media contained 3.5 mM reduced glutathione to prevent the added dopamine from autooxidation, and the pH was adjusted to 7.4 throughout the experiment. In seven animals with striatal probes, the ‘no net flux’ measurement was followed by the injection of 1 mg botulinum toxin A, or sham vehicle. In these animals the ‘no net flux’ determination of extracellular dopamine concentration was repeated on the following day to evaluate the effects of the treatment.
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0.45 V oxidising potential with a Decade detector (Antec Leyden, Leiden, Netherlands) and the metabolites at 0.6 V with a Waters 460 detector (Millipore Waters, Milford, MA, USA). The resulting currents were analysed with Dionex Chromeleon software (Dionex, Sunnyvale, CA, USA).
2.6. Calculations and statistics To compensate for the different recovery in individual probes and to facilitate comparison between the two brain regions statistics were calculated with relative values expressed as percent of baseline concentration. The three last samples before injection were used to calculate the baseline concentration. The extracellular dopamine concentrations determined by the ‘no net flux’ measurements were calculated by linear regression of the net change in dialysate concentration as a function of inlet dopamine concentration followed by an extrapolation to find the ‘point of no net flux’ which corresponds to equal concentrations in dialysate and tissue extracellular fluid (Fig. 6). Statistic tests were performed using GraphPad Prism 3 software. Comparisons of the acute effects of botulinum toxins and the corresponding vehicle on spontaneous dopamine release were made with one-way analysis of variance using the average of the three last measurements from each treatment followed by a Bonferroni post-hoc test. Because of significantly different variances the evoked release was evaluated with the nonparametric Kruskal–Wallis test followed by a Dunnet multiple comparison post-hoc test. The perfusion with L-glutamic acid was evaluated with one-way analysis of variance followed by a Dunnet multiple comparison test when appropriate, and the effect of tetrodotoxin was evaluated with Student’s t-test, comparing the last baseline concentrations with the concentration of fraction two during tetrodotoxin perfusion.
3. Results
2.5. Biochemical assay of the dialysate The dialysate was split in two and injected using a cooled autoinjector (CMA Microdialysis AB, Solna, Sweden). For the analysis of DOPAC, HVA and 5-HIAA, 12 ml of the dialysate was injected on a reverse phase column (Nucleosil 3m C 18 120A; Phenomenex, Torrance, CA, USA), mobile phase containing 7.5% methanol, 40 mM citric acid, 10 mM K 2 HPO 4 and 12 mM EDTA) and for the analysis of dopamine 26 ml was injected on another reverse phase column (Prodigy 3m ODS(3) 100A, Phenomenex, Torrance, CA, USA), with the mobile phase containing 30% methanol, 3.0 mM citric acid, 10 mM sodium citrate, 0.275 mM 1-decane sulfonic acid and 12 mM EDTA. Dopamine was electrochemically detected at
The basal dopamine concentration measured in the dialysate from substantia nigra was 0.43860.039 nM (n5 39, mean6S.E.M.). In striatum the basal dialysate concentration was 5.7560.44 nM (n528, mean6S.E.M.). There was no significant difference in baseline dopamine concentrations between the different treatment groups (Table 1). Based on the ‘no net flux’ experiments the extracellular dopamine concentrations in SN (see Fig. 6) were estimated to 2.6160.35 nM (n55, mean6S.E.M.) and in the striatum to 25.463,0 nM (n514, mean6S.E.M.). To establish that the dopamine concentration in the nigral dialysate reflects active release, a group of four animals were subjected to nigral treatment with tetrodotoxin (1 mM). This treatment lowered the
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Table 1 Baseline dopamine concentrations (nM, mean6S.E.M.) in the dialysates from substantia nigra and striatum stratified to the different treatment groups Treatment group
Dopamine (nM, mean6S.E.M.) Substantia nigra
Striatum
Ringer vehicle Botulinum toxin A Botulinum toxin B BSA / NaCl vehicle Tetanus toxin L-glutamic acid
0.5660.09 0.2960.12 0.5160.10 0.4460.08 0.4160.05 0.4260.11
6.6060.34 4.9261.14 4.4060.74 6.0960.54 6.7460.79 N.D.
One-way ANOVA
F[5,36]51.04, P50.41
F[4,31]51.32, P50.21
One-way ANOVA does not reveal any significant differences between the treatment groups within the measurements from nigral or striatal probes. N.D.5not determined.
dopamine concentrations to 15%61% of baseline within 40 min (P,0.0001). After the injection of 1 mg botulinum toxin A dopamine levels progressively declined for 1.5–2.0 h and then reached a plateau level. The pattern was strikingly similar in the two brain regions striatum and substantia nigra (Fig. 2). Dopamine release induced by a 5-min pulse with 275 mM KCl in Ringer was significantly inhibited both in the striatum and the substantia nigra as compared to vehicle treatment (Fig. 4A). The injection of 1 mg botulinum toxin B in the striatum resulted in decreased dopamine concentrations in the dialysate, but when the same amount of toxin was injected in the substantia nigra there was no effect on dopamine concentrations in the dialysate as compared to vehicle (Fig. 2). Similarly, KCl-evoked release was impaired in the striatum but was not significantly impaired in the substantia nigra (Fig. 4A). Tetanus toxin, which like botulinum toxin B cleaves synaptobrevin II but using a different binding site, did not affect spontaneous or evoked dopamine release in the striatum or substantia nigra. (Figs. 3 and 4B). The concentrations of the dopamine metabolites DOPAC and HVA and the serotonin metabolite 5-HIAA were also investigated after the injection of vehicles or toxins. Evidently in striatum botulinum toxin A and B induced significant increases in DOPAC but not in HVA (Fig. 5). In the substantia nigra no increases were observed in DOPAC or HVA levels (Fig. 5). 5-HIAA concentrations decreased significantly in striatum after botulinum toxin A treatment. There was also a decrease in 5-HIAA after tetanus treatment, but due to a larger variability the difference compared to vehicle treatment was not significant. Some of the ‘no net flux’ experiments were followed by the injection of botulinum toxin A or its vehicle into the striatum and the ‘no net flux’ experiment was repeated 24 h later (Fig. 6A). As expected the extracellular dopamine concentration was decreased on the following day in the toxin treated (1.561.5 nM, mean6S.E.M.), but not in the
Fig. 2. Acute effects of botulinum toxins A and B on spontaneous dopamine release. The effect of an acute injection of 1.0 mg botulinum toxin A (Bot A) or B (Bot B) in striatum (A) or in the substantia nigra (B) compared with vehicle injections. Dopamine concentration is expressed as percent of baseline mean6S.E.M. The number of independent experiments with injections in striatum were n55 (Bot A), n55 (Bot B) and n55 (vehicle). The corresponding number of experiments with injections in substantia nigra were n58 (Bot A), n57 (Bot B) and n55 (vehicle). Statistics were calculated using one-way analysis of variance using the mean of the three last measurements after the injection (Striatum: F580.05, R 2 50.9390, P,0.0001; substantia nigra: F586.26, R 2 50.8961, P,0.0001). The test was followed by a Bonferroni’s multiple comparison test comparing vehicle with each toxin treatment in striatum and in substantia nigra. *** P,0.001 compared with vehicle treatment.
vehicle treated animals (25.566.2 nM, mean6S.E.M.). The linear regression slopes, which indicate the in vivo recovery, changed significantly after the injection of botulinum toxin and after the sham injection. There were however no significant differences between the pre-injection slopes or between the post-injection slopes. This shows that the decrease in extracellular dopamine concentration is a toxin-specific effect whereas the change in recovery seems to be caused mainly by the trauma invoked by the injection. To find out whether the somatodendritic dopamine release in substantia nigra is stimulated by glutamate, six animals where treated with a 20-min pulse with 0.1 mM L-glutamic acid in the perfusate. This treatment did not change the dialysate concentration of dopamine (F51.344, R 2 50.2118, P50.26) but induced an increase in DOPAC and HVA concentrations (F55.214, R 2 50.4834, P5
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Fig. 3. Acute effects of tetanus toxin on spontaneous dopamine release. The effect of the acute injection of 0.1 mg tetanus toxin (Tet tox) in striatum (A) or in the substantia nigra (B) compared with vehicle injections. Dopamine concentration is expressed as percent of baseline mean6S.E.M. The number of independent experiments with injections in striatum were n56 (Tet tox) and n55 (vehicle). The corresponding number of experiments with injections in substantia nigra were n54 (Tet tox) and n54 (vehicle). Statistics were calculated using the two-tailed t-test comparing the average of the three last measurement with toxin or vehicle (Striatum: t50.8398, df59, P50.4228; substantia nigra: t5 0.7328, df511, P50.4790).
0.0003 and F54.235, R 2 50.4383, P50.0015, respectively). The maximum increases were seen 20 min after the 20-min pulse and reached 153% of baseline DOPAC concentration (P,0.001) and 161% of baseline HVA concentration (P,0.001).
4. Discussion It has previously been demonstrated that dopamine can be released in quanta from the somata of cultured dopaminergic cells and from dopaminergic cell somata in rat brain slices [23,34]. Those findings indicate a vesicular origin of dopamine release from the somata of dopaminergic cells. That kind of direct measurements of single events are, to our knowledge, not feasible to perform on as delicate structures as dendrites. In a recent study, Falkenburger et al. [17] concluded that dopamine can be released from the dendritic field by reversal of the dopamine uptake transporter (DAT) following increased glutamate release induced by stimulation of nucleus subthalamicus. This concept of reverse carrier mediated release finds support
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Fig. 4. Effects of botulinum and tetanus toxin on evoked dopamine release. The effects of botulinum toxin A or B on evoked dopamine release is depicted in (A), and the effects of tetanus toxin in (B). Five minutes of hyperosmolar and depolarising perfusion with 275 mM KCl in Ringer (total osmolarity 0.840 mol / l), was made in fraction 8 after toxin or vehicle injection. The bars indicate the dopamine concentration in fraction 8 expressed as percent of baseline, mean6S.E.M. Data were grouped according to the different treatments and analysed with Kruskal– Wallis nonparametric test (P50.0002) followed by a Dunnet multiple comparison test comparing toxin with vehicle treatment in each region. Botulinum toxin A (Bot A) inhibited KCl-evoked dopamine release in the striatum (P,0.05) and in the substantia nigra (P,0.01) as compared with vehicle treatment. Botulinum toxin B (Bot B) decreased KCl-evoked release in striatum (P,0.01) but was without significant effect in substantia nigra. It can be noted that in the following fraction 9, dopamine levels practically returned to the same levels as just before KCl-treatment, indicating that the effects from the KCl-perfusion were short term and reversible (data not shown). * P,0.05, ** P,0.01 compared with vehicle treatment. No significant effects were seen with tetanus treatment.
especially in studies of calcium independent release [1,14], but the physiological relevance is unclear. In contrast the present study presents further evidence for that the spontaneous release of dopamine from dendrites is mainly mediated by exocytosis. This has also been suggested in previous studies, although less directly [9,36]. There are nevertheless some alternative explanations to the effects of botulinum toxin A and B that must be considered. A possible reason for the decrease in dopamine release seen in substantia nigra after botulinum toxin A treatment could be a reduced excitatory input to the dopaminergic neurones. Although this explanation is difficult to exclude, there is a large body of evidence speaking against it. An excitatory influence of glutamate on nigral dopamine release is for example contradicted by the negative results of L-glutamate perfusion found in this study, and recent findings show that nigral dopamine release is not decreased by treatment with NMDA- or AMPA-antagonists [7,8]. It has also been reported that
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Fig. 5. The effect of botulinum toxin A or B or tetanus toxin injections on the concentrations of metabolites. Botulinum toxin A (Bot A) and botulinum toxin B (Bot B) significantly increased DOPAC concentration in the striatum but not in the substantia nigra (One-way analysis of variance, F55.149, R 2 50.4597, P,0.0001, followed by Bonferroni’s multiple comparison test, Bot A vs. vehicle: P,0.001, Bot B vs. vehicle: P,0.01.) A significant decrease in 5-HIAA was seen in the striatum after Bot A treatment (One-way analysis of variance F53,190, R 2 50,3319, P,0.0001, followed by Bonferroni’s multiple comparison test, Bot A vs. vehicle: P,0.05). No significant changes were seen in HVA concentrations. * P,0.05, ** P,0.01 and *** P,0.001 compared to the corresponding vehicle.
Fig. 6. ‘No net flux’ estimate of extracellular dopamine concentration in striatum and substantia nigra. The graphs plot the net change in dopamine concentration in the dialysate ([DA] in 2[DA] out ) versus input concentration [DA] in . The intercept of the plot and the horizontal dashed line (at [DA] in 2[DA] out 50) is marked with an open circle and represents the point of no net flux, where the dopamine concentration added to the dialysate equals the dopamine concentration outside the dialysis membrane. (A) The mean ‘no net flux’ regression plots before and 24 h after vehicle injections (n54) or 1 mg botulinum toxin A injections (n53) in the striatum. The regression plots before (thin dashed line) and after (thin solid line) vehicle injections have significantly different slopes (0.3460.11 and 0.1960.02, respectively, P50.045), as have the regression plots before (bold dashed line) and after (bold solid line) botulinum toxin A injection (0.3960.11 and 0.1660.01, respectively, P50.037). There is no significant difference between the two pre-injection slopes (P50.69) or between the two post injections slopes (P50.31). (B) A typical example of a ‘no net flux’ determination of extracellular dopamine concentration in the substantia nigra of an untreated rat. Measurements from the substantia nigra were made in five different animals, and the extracellular dopamine concentration could be determined by linear regression with an average accuracy (95% confidence interval) of 60.4 nM. The extracellular concentrations of dopamine in the untreated striatum were determined in 14 animals with an average accuracy of 62.6 nM (not shown). The mean concentrations are given in Section 3.
nigral injections with the acetylcholinesterase inhibitor fasciculin, which will amplify cholinergic transmission, does not affect nigral dopamine tissue concentrations [11]. An absence of cholinergic influence on spontaneous nigral dopamine is also supported by microdialysis investigations
from our laboratory, which do not show inhibitory effects of nicotine antagonists (unpublished findings). Furthermore, it is likely that the very strong stimulus used here to evoke release is enough to cause a direct stimulation of the dopaminergic cells. The observed decrease in evoked
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nigral dopamine release after botulinum toxin A treatment, therefore, argues against substantial influences from other known or unknown excitatory transmitters. An indirect presynaptic explanation of the botulinum toxin effects is not likely in the striatum considering that the firing pattern of the dopaminergic neuron will not be directly influenced by altered excitatory or inhibitory inflow in the striatum. Although loop-mediated effects are always possible when microdialysis is performed in the intact brain, it is our opinion that inhibitory effects of the magnitude seen here are very unlikely to be mediated that way. Another possible explanation of decreases in dialysate transmitter concentrations is a decrease in in vivo recovery. The ‘no net flux’ experiments performed before and after botulinum toxin A treatment in the striatum serve to demonstrate that the toxin-induced decreases in dialysate dopamine indeed represent a decrease in extracellular dopamine concentrations. It can, however, also be concluded from those experiments that the injection leads to a decreased in vivo recovery of dopamine as judged by the ‘no net flux’ slopes (Fig. 6). This effect was identical in sham- and toxin-treated animals and therefore shows that botulinum toxin A does not change the in vivo recovery of dopamine. The observed change must instead be ascribed to the trauma caused by the injection as such, and it appears safe to assume that the toxin does not cause unspecific cell-death in the used preparation. An important disadvantage with performing ‘no net flux’ experiments 24 h after the botulinum toxin injection is the risk for adverse effects. All the animals that were used in these experiments displayed inhibited ability to move. The number of animals used was therefore kept at a minimum, and the experimental design was not repeated in the substantia nigra. Interestingly, botulinum toxin B effectively inhibited terminal but not somatodendritic release. To exhibit full toxicity both botulinum toxin A and B need to be activated by a reducing agent. Because reducing agents such as glutathione are very abundant in the brain and the levels are not dramatically different in the striatum compared to the substantia nigra [24] differences in activation hardly explain the effects seen here. Another possibility, which cannot be entirely excluded is that the uptake and internalisation of toxin differs in terminal and dendritic regions. The toxicity and neuronal selectivity of clostridial toxins depend largely on the existence of the appropriate surface receptors on the nerve cells. To our knowledge a difference in uptake within the same cell type has not been described, but theoretically this may be the case. The clear effects seen with botulinum toxin B on dopamine release in the striatum do however favour the most straightforward explanation: that the lack of effects in substantia nigra involves a difference in the exocytosis machinery rather than a difference in uptake and activation of the toxin. If somatodendritic dopamine release originates from the rather scarce population of dense core vesicles or small synaptic vesicles that can be observed in dopaminergic
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dendrites [31] the present results predict that these vesicles do not contain the same vesicular SNAP-receptor component as vesicles in the terminal region. It would therefore be of interest to determine whether vesicles in dendrites and vesicles in terminals can express different synaptobrevin isoforms. The alternatively proposed release mechanism, that dopamine is released directly from tubulovesicular structures, is not excluded by our findings, but the few observed vesicles may just as well constitute a small but active releasable pool of vesicles in equilibrium with budding and fusing vesicles from the smooth endoplasmatic reticulum [35]. An advantage with the microdialysis model with awake rats is that somatodendritic dopamine release resulting from a nearly normal physiological activity in the substantia nigra can be detected without adding a releaseevoking stimuli. It is however not known to what degree the release depends on afferent inputs as compared to the intrinsic activity of the releasing neurones. A difference in the influence of afferents on the release between the striatum and the substantia nigra could lead to a different sensitivity to botulinum toxin B in the two regions, because spontaneous and evoked neurotransmitter release can be differently sensitive to clostridial toxins [22]. The fact that botulinum toxin B was without effect on KClevoked release in substantia nigra but not in striatum should however rule out this possibility in the present experiments. In our hands tetanus toxin did not interfere with dopamine release in striatum or substantia nigra. This is most likely due to poor uptake of tetanus toxin in dopaminergic cells and is in accordance with other investigators who have either seen no effects, small effects or only late effects on dopamine levels after tetanus injections [3,41]. In contrast even smaller doses of tetanus toxin, when injected in substantia nigra, have very rapid effects on GABA-release as measured by striatal-evoked inhibition of substantia nigra neurones [12]. This fact could indicate that the spontaneous dopamine release in the substantia nigra is not tonically inhibited by GABA releasing afferents to any larger extent when measured by microdialysis, but contrasts with the stimulatory effects on nigral dopamine release seen after treatment with GABAantagonists [7,8]. After botulinum toxin A or B injections into the striatum an increase in DOPAC was detected. This was not the case in the substantia nigra. Similar responses in the two brain regions have previously been reported both in microdialysis and in biochemical experiments after dopamine-depleting regimens such as reserpine treatment [15,21]. The small effects on DOPAC in substantia nigra after reserpine treatment has been attributed to a higher dopamine turnover rate in substantia nigra, or to different catabolic pathways [16]. The same reasons can tentatively account for the findings after botulinum toxin A treatment. This study also presents the first estimate of extracellular
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dopamine concentrations in substantia nigra based on the ‘no net flux’ method. This method bypasses the problem with varying in vivo microdialysis recovery in different brain areas depending on for example the rate of turnover, or of uptake [32]. The ‘no net flux’ estimates indicate tenfold higher dopamine concentrations in the striatum as compared to the substantia nigra, which is in line with present and previous microdialysis and biochemistry data. One should however keep in mind that the extracellular concentrations obtained with microdialysis is a crude overall measure and will not reflect local variations in the tissue, but only an average concentration, possibly better correlated to the density of release sites, then to the actual effects of the release. The target receptors could still be subject to similar concentrations of dopamine in the two regions if they are located at similar distances from the release sites. Therefore, the low extracellular dopamine concentrations in substantia nigra do not exclude a physiological relevance of the release. It can be concluded that somatodendritic dopamine release in vivo as measured by microdialysis is strongly reduced by the SNAP-25 cleaving protease botulinum toxin A, but in contrast to terminal dopamine release, not by the synaptobrevin II cleaving protease botulinum toxin B. This difference between somatodendritic and terminal release indicates a botulinum toxin B-resistant pathway for somatodendritic dopamine release, possibly due to a different synaptobrevin isoform in the dendrites.
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16]
Acknowledgements
[17]
¨ The study was supported by Goteborg Medical Society and the Swedish Research Council, Grant No. 12208.
[18]
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Research report
Evidence for different exocytosis pathways in dendritic and terminal dopamine release in vivo Filip Bergquist*, Haydeh Shahabi Niazi, Hans Nissbrandt ¨ ¨ University, Box 431, SE 405 30 Goteborg , Sweden Department of Pharmacology, Institute of Physiology and Pharmacology, Goteborg Accepted 4 June 2002
Abstract Although dendritic release was first proposed in the 1970s, the mechanism of release is still subject to debate. We have used in vivo microdialysis to study the acute effects of botulinum toxin A, B and tetanus toxin injected in the substantia nigra or striatum of freely moving rats. Spontaneous and evoked dopamine release decreased in both regions after treatment with the SNAP-25 (synaptosomeassociated protein of 25 kDa) cleaving protease botulinum toxin A (1000 mouse lethal doses, MLD). Tetanus toxin (4000 MLD) did not significantly change spontaneous or evoked dopamine release in striatum or in the substantia nigra. Another synaptobrevin cleaving protease, botulinum toxin B, inhibited release in the striatum by 55% but did not affect dopamine release when injected in the substantia nigra. The results indicate that both terminal and somatodendritic dopamine release need intact SNAP-25 to occur, but somatodendritic dopamine release in contrast to terminal release depends on a botulinum toxin B resistant pathway. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotransmitter systems and channels Keywords: Dopamine release; Exocytosis; SNAP-25; Striatum; Substantia nigra; Synaptobrevin
1. Introduction The characteristic symptoms of Parkinson’s disease have been attributed to the loss of dopaminergic terminals in the striatal region. Dopamine can, however, also be released from the dopaminergic soma and dendrites in the reticulate part of substantia nigra [13,18,30] and is in the position to influence neurotransmission also in this region [2,10,26,29]. The reticulate part of substantia nigra is one of the major output relay nuclei, and therefore somatodendritically released dopamine could theoretically modulate motor control also at this level. Somatodendritic dopamine release in rat have properties typical for vesicular neurotransmitter release in that it is sodium channel dependent (blocked by tetrodotoxin) [14,37] and calcium dependent [4,9,10,40], also reserpine, an inhibitor of vesicular monoamine transport, significantly reduces dendritic dopamine stores and release [5,21]. Somatodendritic dopamine re*Corresponding author. Tel.: 146-31-773-3425; fax: 146-31-811-792. E-mail address: [email protected] (F. Bergquist).
lease does however have different characteristics compared to terminal release in regard to for example the calcium sensitivity [4,6]. Also, histological investigations have only shown a scarce occurrence of vesicular structures in the soma and dendrites of dopaminergic neurones [20,31]. Consequently other possibilities for release have been proposed, such as release from smooth endoplasmatic reticulum [15,28,31], or extravesicular release via reversal of the dopamine carrier [14,17] (for review, see Ref. [25]). Investigations of the molecular mechanisms of vesicular release have identified three proteins: syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptobrevin (also known as VAMP, vesicle associated membrane protein), as the minimal machinery for membrane fusion [39], although several other proteins may have regulatory functions (for review, see Ref. [19]). Synaptobrevin, syntaxin and SNAP-25 are remarkably homologous proteins in different organisms but there are some different isoforms which may to some extent account for the specificity of intracellular membrane trafficking [27,38]. The clostridial neurotoxins botulinum toxin A–G
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03047-0
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and tetanus toxin are metalloproteases that specifically cleave SNAP-25 (botulinum toxin A, C and E), synaptobrevin (botulinum toxin B, D, F and G) or syntaxin (botulinum toxin C) (for review, see Ref. [33]). The present study aimed at comparing the effects of synaptobrevin specific protease and SNAP-25 specific protease on dopamine release in the reticular part of substantia nigra and in the striatum. The method of in vivo microdialysis in rat brain was combined with acute microinjections of clostridial toxins. We show that spontaneous and evoked somatodendritic and terminal dopamine releases are both disrupted by treatment with botulinum toxin A, but that botulinum toxin B only inhibits terminal release. This is the most direct evidence of exocytosis from dopaminergic dendrites in substantia nigra this far, but the findings also indicate a fundamental difference between terminal and somatodendritic release mechanisms.
2.3. Microdialysis probes and surgery
2. Materials and methods
2.4. Microdialysis
The experimental design was approved by the local ¨ ethical committee in Goteborg, and carried out in accordance with the European Communities Council Directive of 24 November 1986.
2.4.1. Acute experiments On the day of the experiment the animals were connected to a swivelled perfusion system and the microdialysis probes were perfused with the modified Ringer solution at 2.0 ml / min. After 1 h of adaptation and equilibration, four consecutive 40 ml samples were collected and analysed for basal levels of dopamine, 3,4dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), with high performance liquid chromatography followed by electrochemical detection. During fraction five, toxin or vehicle were slowly (2 ml in 15 min) injected manually through stiff PEP-tubing directly connected to the injection capillary. Microinjections were made with botulinum toxin A, B, tetanus toxin or with the two different vehicles. After
2.1. Animals Male SD rats (280–320 g) from B&K Universal AB (Sollentuna, Sweden) were housed four per cage under controlled environmental conditions (26 8C, 60–65% humidity, light 05:00–19:00 h, dark 19:00–05:00 h) for 1 week before the experiments. Food and tap water were allowed ad lib. Experimental procedures were carried out during daytime.
To be able to locally administer substances of as large molecular weight as botulinum and tetanus toxin we modified an I-shaped microdialysis probe by attaching a small diameter fused silica capillary (inner diameter 90 mm) adjacent to the dialysis membrane (Fig. 1). Deadspace in the injection capillary was calculated to approximately 0.2 ml. A dialysis membrane with a 20-kDa cut-off was used and the active part was 2 mm long regardless of whether the probe was placed in the substantia nigra or in the striatum. Before implantation the probes were perfused and washed with 70% ethanol and then with modified Ringer solution. The injection capillary was washed and filled with modified Ringer solution and all tubings were then sealed. The animals were stereotactically implanted with microdialysis probes as previously described [4] and allowed to recover single-housed for 48 h before the experiment.
2.2. Drugs For surgical anaesthesia ketamine and xylazin were used. Botulinum toxin A and B (Sigma-Aldrich Sweden AB) were dispensed in vials and frozen. At the day of experiment the stem solution (1 mg / ml in buffered saline) was diluted with modified Ringer solution containing 140 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl 2 and 1.0 mM Mg Cl 2 to a final concentration of 0.5 mg / ml (approximately 500 MLD/ ml). Tetanus holotoxin (Alomone, Jerusalem, Israel) was dissolved in 0.9% NaCl with 0.1% bovine serum albumin (BSA) on the day of experiment to a final concentration of 0.05 mg / ml (approximately 2000 MLD/ ml). Vehicle solutions were prepared in the same ways but without toxin. L-Glutamic acid HCl (Sigma-Aldrich Sweden AB) and tetrodotoxin (Alomone, Jerusalem, Israel) was dissolved in the modified Ringer solution, and pH was adjusted to 7.4 immediately before use.
Fig. 1. Combined microdialysis and microinjection probe. Schematic drawing of a microdialysis probe modified by the attachment of a fused silica capillary allowing microinjections of large molecular weight substances such as clostridial toxins.
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the injection seven consecutive 40-ml samples were collected and analysed. Then a short pulse of 5 min with perfusion media containing 275 mM KCl was given to study effects on dopamine release evoked by high osmolarity and depolarisation. In one set of experiments, microdialysis probes without injection cannulas were implanted in the substantia nigra and used to administer a 20-min pulse of 0.1 mM L-glutamic acid. This pulse was given in the first sampling fraction following the collection of baseline samples and the perfusion then continued for another hour. Tetrodotoxin was perfused in nigral probes at the concentration 1 mM after baseline sampling. All animals were sacrificed immediately after the experiment and the location of the microdialysis probe was verified macroscopically after slicing the brain with a vibratome. Animals with probe locations outside the target area or with intracerebral haemorrhage were excluded.
2.4.2. ‘ No net flux’ experiment: On day 2 after the implantation a ‘no net flux’ determination of extracellular dopamine concentrations was performed. After a stable baseline of dopamine concentrations was obtained, the perfusion medium was changed to one containing approximately 10 nM dopamine. When dopamine concentrations were stable again in the dialysate, this was repeated with a perfusion media containing approximately 20 nM dopamine. Lower dopamine concentrations (1 and 2 nM) were used when the ‘no net flux’ procedure was performed in substantia nigra. The inlet and outlet concentrations were determined and the net change in dialysate dopamine concentration was calculated. The perfusion media contained 3.5 mM reduced glutathione to prevent the added dopamine from autooxidation, and the pH was adjusted to 7.4 throughout the experiment. In seven animals with striatal probes, the ‘no net flux’ measurement was followed by the injection of 1 mg botulinum toxin A, or sham vehicle. In these animals the ‘no net flux’ determination of extracellular dopamine concentration was repeated on the following day to evaluate the effects of the treatment.
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0.45 V oxidising potential with a Decade detector (Antec Leyden, Leiden, Netherlands) and the metabolites at 0.6 V with a Waters 460 detector (Millipore Waters, Milford, MA, USA). The resulting currents were analysed with Dionex Chromeleon software (Dionex, Sunnyvale, CA, USA).
2.6. Calculations and statistics To compensate for the different recovery in individual probes and to facilitate comparison between the two brain regions statistics were calculated with relative values expressed as percent of baseline concentration. The three last samples before injection were used to calculate the baseline concentration. The extracellular dopamine concentrations determined by the ‘no net flux’ measurements were calculated by linear regression of the net change in dialysate concentration as a function of inlet dopamine concentration followed by an extrapolation to find the ‘point of no net flux’ which corresponds to equal concentrations in dialysate and tissue extracellular fluid (Fig. 6). Statistic tests were performed using GraphPad Prism 3 software. Comparisons of the acute effects of botulinum toxins and the corresponding vehicle on spontaneous dopamine release were made with one-way analysis of variance using the average of the three last measurements from each treatment followed by a Bonferroni post-hoc test. Because of significantly different variances the evoked release was evaluated with the nonparametric Kruskal–Wallis test followed by a Dunnet multiple comparison post-hoc test. The perfusion with L-glutamic acid was evaluated with one-way analysis of variance followed by a Dunnet multiple comparison test when appropriate, and the effect of tetrodotoxin was evaluated with Student’s t-test, comparing the last baseline concentrations with the concentration of fraction two during tetrodotoxin perfusion.
3. Results
2.5. Biochemical assay of the dialysate The dialysate was split in two and injected using a cooled autoinjector (CMA Microdialysis AB, Solna, Sweden). For the analysis of DOPAC, HVA and 5-HIAA, 12 ml of the dialysate was injected on a reverse phase column (Nucleosil 3m C 18 120A; Phenomenex, Torrance, CA, USA), mobile phase containing 7.5% methanol, 40 mM citric acid, 10 mM K 2 HPO 4 and 12 mM EDTA) and for the analysis of dopamine 26 ml was injected on another reverse phase column (Prodigy 3m ODS(3) 100A, Phenomenex, Torrance, CA, USA), with the mobile phase containing 30% methanol, 3.0 mM citric acid, 10 mM sodium citrate, 0.275 mM 1-decane sulfonic acid and 12 mM EDTA. Dopamine was electrochemically detected at
The basal dopamine concentration measured in the dialysate from substantia nigra was 0.43860.039 nM (n5 39, mean6S.E.M.). In striatum the basal dialysate concentration was 5.7560.44 nM (n528, mean6S.E.M.). There was no significant difference in baseline dopamine concentrations between the different treatment groups (Table 1). Based on the ‘no net flux’ experiments the extracellular dopamine concentrations in SN (see Fig. 6) were estimated to 2.6160.35 nM (n55, mean6S.E.M.) and in the striatum to 25.463,0 nM (n514, mean6S.E.M.). To establish that the dopamine concentration in the nigral dialysate reflects active release, a group of four animals were subjected to nigral treatment with tetrodotoxin (1 mM). This treatment lowered the
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Table 1 Baseline dopamine concentrations (nM, mean6S.E.M.) in the dialysates from substantia nigra and striatum stratified to the different treatment groups Treatment group
Dopamine (nM, mean6S.E.M.) Substantia nigra
Striatum
Ringer vehicle Botulinum toxin A Botulinum toxin B BSA / NaCl vehicle Tetanus toxin L-glutamic acid
0.5660.09 0.2960.12 0.5160.10 0.4460.08 0.4160.05 0.4260.11
6.6060.34 4.9261.14 4.4060.74 6.0960.54 6.7460.79 N.D.
One-way ANOVA
F[5,36]51.04, P50.41
F[4,31]51.32, P50.21
One-way ANOVA does not reveal any significant differences between the treatment groups within the measurements from nigral or striatal probes. N.D.5not determined.
dopamine concentrations to 15%61% of baseline within 40 min (P,0.0001). After the injection of 1 mg botulinum toxin A dopamine levels progressively declined for 1.5–2.0 h and then reached a plateau level. The pattern was strikingly similar in the two brain regions striatum and substantia nigra (Fig. 2). Dopamine release induced by a 5-min pulse with 275 mM KCl in Ringer was significantly inhibited both in the striatum and the substantia nigra as compared to vehicle treatment (Fig. 4A). The injection of 1 mg botulinum toxin B in the striatum resulted in decreased dopamine concentrations in the dialysate, but when the same amount of toxin was injected in the substantia nigra there was no effect on dopamine concentrations in the dialysate as compared to vehicle (Fig. 2). Similarly, KCl-evoked release was impaired in the striatum but was not significantly impaired in the substantia nigra (Fig. 4A). Tetanus toxin, which like botulinum toxin B cleaves synaptobrevin II but using a different binding site, did not affect spontaneous or evoked dopamine release in the striatum or substantia nigra. (Figs. 3 and 4B). The concentrations of the dopamine metabolites DOPAC and HVA and the serotonin metabolite 5-HIAA were also investigated after the injection of vehicles or toxins. Evidently in striatum botulinum toxin A and B induced significant increases in DOPAC but not in HVA (Fig. 5). In the substantia nigra no increases were observed in DOPAC or HVA levels (Fig. 5). 5-HIAA concentrations decreased significantly in striatum after botulinum toxin A treatment. There was also a decrease in 5-HIAA after tetanus treatment, but due to a larger variability the difference compared to vehicle treatment was not significant. Some of the ‘no net flux’ experiments were followed by the injection of botulinum toxin A or its vehicle into the striatum and the ‘no net flux’ experiment was repeated 24 h later (Fig. 6A). As expected the extracellular dopamine concentration was decreased on the following day in the toxin treated (1.561.5 nM, mean6S.E.M.), but not in the
Fig. 2. Acute effects of botulinum toxins A and B on spontaneous dopamine release. The effect of an acute injection of 1.0 mg botulinum toxin A (Bot A) or B (Bot B) in striatum (A) or in the substantia nigra (B) compared with vehicle injections. Dopamine concentration is expressed as percent of baseline mean6S.E.M. The number of independent experiments with injections in striatum were n55 (Bot A), n55 (Bot B) and n55 (vehicle). The corresponding number of experiments with injections in substantia nigra were n58 (Bot A), n57 (Bot B) and n55 (vehicle). Statistics were calculated using one-way analysis of variance using the mean of the three last measurements after the injection (Striatum: F580.05, R 2 50.9390, P,0.0001; substantia nigra: F586.26, R 2 50.8961, P,0.0001). The test was followed by a Bonferroni’s multiple comparison test comparing vehicle with each toxin treatment in striatum and in substantia nigra. *** P,0.001 compared with vehicle treatment.
vehicle treated animals (25.566.2 nM, mean6S.E.M.). The linear regression slopes, which indicate the in vivo recovery, changed significantly after the injection of botulinum toxin and after the sham injection. There were however no significant differences between the pre-injection slopes or between the post-injection slopes. This shows that the decrease in extracellular dopamine concentration is a toxin-specific effect whereas the change in recovery seems to be caused mainly by the trauma invoked by the injection. To find out whether the somatodendritic dopamine release in substantia nigra is stimulated by glutamate, six animals where treated with a 20-min pulse with 0.1 mM L-glutamic acid in the perfusate. This treatment did not change the dialysate concentration of dopamine (F51.344, R 2 50.2118, P50.26) but induced an increase in DOPAC and HVA concentrations (F55.214, R 2 50.4834, P5
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Fig. 3. Acute effects of tetanus toxin on spontaneous dopamine release. The effect of the acute injection of 0.1 mg tetanus toxin (Tet tox) in striatum (A) or in the substantia nigra (B) compared with vehicle injections. Dopamine concentration is expressed as percent of baseline mean6S.E.M. The number of independent experiments with injections in striatum were n56 (Tet tox) and n55 (vehicle). The corresponding number of experiments with injections in substantia nigra were n54 (Tet tox) and n54 (vehicle). Statistics were calculated using the two-tailed t-test comparing the average of the three last measurement with toxin or vehicle (Striatum: t50.8398, df59, P50.4228; substantia nigra: t5 0.7328, df511, P50.4790).
0.0003 and F54.235, R 2 50.4383, P50.0015, respectively). The maximum increases were seen 20 min after the 20-min pulse and reached 153% of baseline DOPAC concentration (P,0.001) and 161% of baseline HVA concentration (P,0.001).
4. Discussion It has previously been demonstrated that dopamine can be released in quanta from the somata of cultured dopaminergic cells and from dopaminergic cell somata in rat brain slices [23,34]. Those findings indicate a vesicular origin of dopamine release from the somata of dopaminergic cells. That kind of direct measurements of single events are, to our knowledge, not feasible to perform on as delicate structures as dendrites. In a recent study, Falkenburger et al. [17] concluded that dopamine can be released from the dendritic field by reversal of the dopamine uptake transporter (DAT) following increased glutamate release induced by stimulation of nucleus subthalamicus. This concept of reverse carrier mediated release finds support
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Fig. 4. Effects of botulinum and tetanus toxin on evoked dopamine release. The effects of botulinum toxin A or B on evoked dopamine release is depicted in (A), and the effects of tetanus toxin in (B). Five minutes of hyperosmolar and depolarising perfusion with 275 mM KCl in Ringer (total osmolarity 0.840 mol / l), was made in fraction 8 after toxin or vehicle injection. The bars indicate the dopamine concentration in fraction 8 expressed as percent of baseline, mean6S.E.M. Data were grouped according to the different treatments and analysed with Kruskal– Wallis nonparametric test (P50.0002) followed by a Dunnet multiple comparison test comparing toxin with vehicle treatment in each region. Botulinum toxin A (Bot A) inhibited KCl-evoked dopamine release in the striatum (P,0.05) and in the substantia nigra (P,0.01) as compared with vehicle treatment. Botulinum toxin B (Bot B) decreased KCl-evoked release in striatum (P,0.01) but was without significant effect in substantia nigra. It can be noted that in the following fraction 9, dopamine levels practically returned to the same levels as just before KCl-treatment, indicating that the effects from the KCl-perfusion were short term and reversible (data not shown). * P,0.05, ** P,0.01 compared with vehicle treatment. No significant effects were seen with tetanus treatment.
especially in studies of calcium independent release [1,14], but the physiological relevance is unclear. In contrast the present study presents further evidence for that the spontaneous release of dopamine from dendrites is mainly mediated by exocytosis. This has also been suggested in previous studies, although less directly [9,36]. There are nevertheless some alternative explanations to the effects of botulinum toxin A and B that must be considered. A possible reason for the decrease in dopamine release seen in substantia nigra after botulinum toxin A treatment could be a reduced excitatory input to the dopaminergic neurones. Although this explanation is difficult to exclude, there is a large body of evidence speaking against it. An excitatory influence of glutamate on nigral dopamine release is for example contradicted by the negative results of L-glutamate perfusion found in this study, and recent findings show that nigral dopamine release is not decreased by treatment with NMDA- or AMPA-antagonists [7,8]. It has also been reported that
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Fig. 5. The effect of botulinum toxin A or B or tetanus toxin injections on the concentrations of metabolites. Botulinum toxin A (Bot A) and botulinum toxin B (Bot B) significantly increased DOPAC concentration in the striatum but not in the substantia nigra (One-way analysis of variance, F55.149, R 2 50.4597, P,0.0001, followed by Bonferroni’s multiple comparison test, Bot A vs. vehicle: P,0.001, Bot B vs. vehicle: P,0.01.) A significant decrease in 5-HIAA was seen in the striatum after Bot A treatment (One-way analysis of variance F53,190, R 2 50,3319, P,0.0001, followed by Bonferroni’s multiple comparison test, Bot A vs. vehicle: P,0.05). No significant changes were seen in HVA concentrations. * P,0.05, ** P,0.01 and *** P,0.001 compared to the corresponding vehicle.
Fig. 6. ‘No net flux’ estimate of extracellular dopamine concentration in striatum and substantia nigra. The graphs plot the net change in dopamine concentration in the dialysate ([DA] in 2[DA] out ) versus input concentration [DA] in . The intercept of the plot and the horizontal dashed line (at [DA] in 2[DA] out 50) is marked with an open circle and represents the point of no net flux, where the dopamine concentration added to the dialysate equals the dopamine concentration outside the dialysis membrane. (A) The mean ‘no net flux’ regression plots before and 24 h after vehicle injections (n54) or 1 mg botulinum toxin A injections (n53) in the striatum. The regression plots before (thin dashed line) and after (thin solid line) vehicle injections have significantly different slopes (0.3460.11 and 0.1960.02, respectively, P50.045), as have the regression plots before (bold dashed line) and after (bold solid line) botulinum toxin A injection (0.3960.11 and 0.1660.01, respectively, P50.037). There is no significant difference between the two pre-injection slopes (P50.69) or between the two post injections slopes (P50.31). (B) A typical example of a ‘no net flux’ determination of extracellular dopamine concentration in the substantia nigra of an untreated rat. Measurements from the substantia nigra were made in five different animals, and the extracellular dopamine concentration could be determined by linear regression with an average accuracy (95% confidence interval) of 60.4 nM. The extracellular concentrations of dopamine in the untreated striatum were determined in 14 animals with an average accuracy of 62.6 nM (not shown). The mean concentrations are given in Section 3.
nigral injections with the acetylcholinesterase inhibitor fasciculin, which will amplify cholinergic transmission, does not affect nigral dopamine tissue concentrations [11]. An absence of cholinergic influence on spontaneous nigral dopamine is also supported by microdialysis investigations
from our laboratory, which do not show inhibitory effects of nicotine antagonists (unpublished findings). Furthermore, it is likely that the very strong stimulus used here to evoke release is enough to cause a direct stimulation of the dopaminergic cells. The observed decrease in evoked
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nigral dopamine release after botulinum toxin A treatment, therefore, argues against substantial influences from other known or unknown excitatory transmitters. An indirect presynaptic explanation of the botulinum toxin effects is not likely in the striatum considering that the firing pattern of the dopaminergic neuron will not be directly influenced by altered excitatory or inhibitory inflow in the striatum. Although loop-mediated effects are always possible when microdialysis is performed in the intact brain, it is our opinion that inhibitory effects of the magnitude seen here are very unlikely to be mediated that way. Another possible explanation of decreases in dialysate transmitter concentrations is a decrease in in vivo recovery. The ‘no net flux’ experiments performed before and after botulinum toxin A treatment in the striatum serve to demonstrate that the toxin-induced decreases in dialysate dopamine indeed represent a decrease in extracellular dopamine concentrations. It can, however, also be concluded from those experiments that the injection leads to a decreased in vivo recovery of dopamine as judged by the ‘no net flux’ slopes (Fig. 6). This effect was identical in sham- and toxin-treated animals and therefore shows that botulinum toxin A does not change the in vivo recovery of dopamine. The observed change must instead be ascribed to the trauma caused by the injection as such, and it appears safe to assume that the toxin does not cause unspecific cell-death in the used preparation. An important disadvantage with performing ‘no net flux’ experiments 24 h after the botulinum toxin injection is the risk for adverse effects. All the animals that were used in these experiments displayed inhibited ability to move. The number of animals used was therefore kept at a minimum, and the experimental design was not repeated in the substantia nigra. Interestingly, botulinum toxin B effectively inhibited terminal but not somatodendritic release. To exhibit full toxicity both botulinum toxin A and B need to be activated by a reducing agent. Because reducing agents such as glutathione are very abundant in the brain and the levels are not dramatically different in the striatum compared to the substantia nigra [24] differences in activation hardly explain the effects seen here. Another possibility, which cannot be entirely excluded is that the uptake and internalisation of toxin differs in terminal and dendritic regions. The toxicity and neuronal selectivity of clostridial toxins depend largely on the existence of the appropriate surface receptors on the nerve cells. To our knowledge a difference in uptake within the same cell type has not been described, but theoretically this may be the case. The clear effects seen with botulinum toxin B on dopamine release in the striatum do however favour the most straightforward explanation: that the lack of effects in substantia nigra involves a difference in the exocytosis machinery rather than a difference in uptake and activation of the toxin. If somatodendritic dopamine release originates from the rather scarce population of dense core vesicles or small synaptic vesicles that can be observed in dopaminergic
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dendrites [31] the present results predict that these vesicles do not contain the same vesicular SNAP-receptor component as vesicles in the terminal region. It would therefore be of interest to determine whether vesicles in dendrites and vesicles in terminals can express different synaptobrevin isoforms. The alternatively proposed release mechanism, that dopamine is released directly from tubulovesicular structures, is not excluded by our findings, but the few observed vesicles may just as well constitute a small but active releasable pool of vesicles in equilibrium with budding and fusing vesicles from the smooth endoplasmatic reticulum [35]. An advantage with the microdialysis model with awake rats is that somatodendritic dopamine release resulting from a nearly normal physiological activity in the substantia nigra can be detected without adding a releaseevoking stimuli. It is however not known to what degree the release depends on afferent inputs as compared to the intrinsic activity of the releasing neurones. A difference in the influence of afferents on the release between the striatum and the substantia nigra could lead to a different sensitivity to botulinum toxin B in the two regions, because spontaneous and evoked neurotransmitter release can be differently sensitive to clostridial toxins [22]. The fact that botulinum toxin B was without effect on KClevoked release in substantia nigra but not in striatum should however rule out this possibility in the present experiments. In our hands tetanus toxin did not interfere with dopamine release in striatum or substantia nigra. This is most likely due to poor uptake of tetanus toxin in dopaminergic cells and is in accordance with other investigators who have either seen no effects, small effects or only late effects on dopamine levels after tetanus injections [3,41]. In contrast even smaller doses of tetanus toxin, when injected in substantia nigra, have very rapid effects on GABA-release as measured by striatal-evoked inhibition of substantia nigra neurones [12]. This fact could indicate that the spontaneous dopamine release in the substantia nigra is not tonically inhibited by GABA releasing afferents to any larger extent when measured by microdialysis, but contrasts with the stimulatory effects on nigral dopamine release seen after treatment with GABAantagonists [7,8]. After botulinum toxin A or B injections into the striatum an increase in DOPAC was detected. This was not the case in the substantia nigra. Similar responses in the two brain regions have previously been reported both in microdialysis and in biochemical experiments after dopamine-depleting regimens such as reserpine treatment [15,21]. The small effects on DOPAC in substantia nigra after reserpine treatment has been attributed to a higher dopamine turnover rate in substantia nigra, or to different catabolic pathways [16]. The same reasons can tentatively account for the findings after botulinum toxin A treatment. This study also presents the first estimate of extracellular
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dopamine concentrations in substantia nigra based on the ‘no net flux’ method. This method bypasses the problem with varying in vivo microdialysis recovery in different brain areas depending on for example the rate of turnover, or of uptake [32]. The ‘no net flux’ estimates indicate tenfold higher dopamine concentrations in the striatum as compared to the substantia nigra, which is in line with present and previous microdialysis and biochemistry data. One should however keep in mind that the extracellular concentrations obtained with microdialysis is a crude overall measure and will not reflect local variations in the tissue, but only an average concentration, possibly better correlated to the density of release sites, then to the actual effects of the release. The target receptors could still be subject to similar concentrations of dopamine in the two regions if they are located at similar distances from the release sites. Therefore, the low extracellular dopamine concentrations in substantia nigra do not exclude a physiological relevance of the release. It can be concluded that somatodendritic dopamine release in vivo as measured by microdialysis is strongly reduced by the SNAP-25 cleaving protease botulinum toxin A, but in contrast to terminal dopamine release, not by the synaptobrevin II cleaving protease botulinum toxin B. This difference between somatodendritic and terminal release indicates a botulinum toxin B-resistant pathway for somatodendritic dopamine release, possibly due to a different synaptobrevin isoform in the dendrites.
[8]
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[12]
[13]
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Acknowledgements
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¨ The study was supported by Goteborg Medical Society and the Swedish Research Council, Grant No. 12208.
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