Effect of 3-acetylpyridine on serotonin uptake and release from rat cerebelllar slices

European Journal of Pharmacology, 198 (1991) 7-14 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991003661

EJP 51869

Effect of 3-acetylpyridine on serotonin uptake and release from rat cerebellar slices C. Beas-Zhrate, A. Morales-Villagran, G. T a p i a - A r i z m e n d i 1 a n d A. Feria-Velasco 1 Laboratorio de Neuroquimica, Facultad de Ciencias Biol6gicas, Universidad de Guadalajara, and j Divisibn de Patologla Experimental, Unidad de Investigaci6n Biomddica de Occidente, L M. S.S. Apartado Postal 4-160, Guadalajara, Jalisco, Mdxico

Received 2 August 1990, revised MS received 18 February 1991, accepted 5 March 1991

The cerebellum receives indolaminergic fibers influencing Purkinje cell discharges. Data from our laboratories have demonstrated an endogenous release of serotonin (5-HT) and a Na+-dependent uptake and Ca2+-dependent release of [3H]5-HT from slices, homogenates and synaptosomal fractions of the rat cerebellar molecular layer. While the neurotransmitter produced by climbing fibers has been sought for in several studies and some of the classical transmitters have been ruled out, as yet this neurotransmitter is unknown. The aim of this work was to measure the 5-HT up~.ake and release from rat cerebellar slices, 6 h and 15 days after intraperitoneal injection of 3-acetylpyridine (3-AP) (75 mg/kg), harmaline (15 mg/kg) and nicotinamide (300 mg/kg). A histological study of medulla and cerebellar cortex in these animals showed destruction of neurons in the inferior olivary nuclei and changes in the granulation of the cortical molecular layer in the cerebellum. A significant reduction of the 5-HT content (1005[), 5-HT uptake (60%) and its Vma~ (60%) was seen on the 5th day, in cerebellar preparations obtained from rats injected with 3-AP. The Ca2+-dependent release of 5-HT from these preparations was found to be similar to the basal values, in spite of depolarizing stimuli with 53 mM KCI or veratrine (60/~g/ml). The results suggest that 5-HT could play an important role as neurotransmitter produced by some climbing fibers. 3-Acetylpyridine; 5-HT (5-hydroxytryptamine, serotonin); 5-HT uptake; 5-HT release; Cerebellar slices

1. Introduction Several studies have shown that the cerebellum receives a plexus of indolamine axons demonstrable by fluorescence (HiSkfelt and Fuxe, 1969), autoradiographic (Chan-Palay, 1977) and immunocytochemical (Bishop and Ho, 1983; 1985; 1986) techniques in various animal species. Ultrastructurally, three types of extrinsic non-myelinated serotoninergic afferents to the cerebellar cortex have been identified: type 1 axons ending as classical mossy fibers in glomeruli at the internal granular layer; type 2 axons as diffuse varicose and branching fibers in close apposition to Purkinje cell somata and dendrites of Golgi cells usually not establishing synaptic contacts; and type 3 axons, transverse to the molecular layer, bifurcating similarly to parallel fibers (Chan-Palay, 1975). Recent neurochemical data from our laboratories (Beas-Zhrate et al., 1984; Velhzquez et al., 1987) have

Correspondence to: C. Beas-Zhrate, Laboratorio de Neuroquimica, Facultad de Ciencias Biolbgicas, Universidad de Guadalajara, Apartado Postal 4-160, Guadalajar', Jalisco, M6xico.

demonstrated the endogenous release of serotonin (5HT) and Na+-dependent uptake and Ca2+-dependent release of [3H]5-HT from slices, homogenates and the synaptosomal fraction of the rat cerebellar molecular layer. The neurotransmitters released from climbing fibers still remains unknown. However, several studies aimed at its identification have eliminated some of the classical transmitter substances such as ~,-amino butyric acid (Rea et al., 1980; Toggenburger et al., 1983), taurine (Rea et al., 1980) glutamate (Nadi et al., 1977) and aspartate (Foster and Roberts, 1980; Rea et al., 1980). Specific destruction of olivocerebellar projections by a single intraperitoneal (i.p) injection of 3-acetylpyridine (3-AP), which acts as a nicotinamide antimetabolite (Coper, 1968), may provide a useful tool for the identification of the neurotransmitter released from the climbing fibers. We now approached the problem of identifying the climbing fiber transmitter by investigating the K ÷evoked Ca 2+-dependent release of [ 3H]5-HT, as well as its uptake system from cerebellar slices of animals which had received a single dose of 3-AP combined with harmaline and nicotinamide according to Llinfis et al. (1975).

~aterials aiK! methods Harmaline, 3-acetylpyridine and nicotinamide were obtained from Sigma Chemicals Co. (St. Louis, MO), O-phthalaldehyde was from Calbiochem (San Diego, CA). [-~H]5-hydroxytryptamine (specific activity 26.3 Ci/mmol) was bought from Amersham, (England). The other chemicals were of reagent grade. Triton X-100, used to prepare scintillation liquid, was a gift from Rom and Haas (Sucursal Guadalajara, Jal., M6xico). 2.1. Treatment ,~4th 3-A P Destruction of olivo-cerebellar climbing fiber system in rats given 3-AP (Desclin and Escubi, 1974) was carried out according to modification of Llinfis et al. (1975). First, these rats received a single dose of 3-AP (75 mg/kg i.p.): then, after 3 h, harmaline (15 mg/kg) and, after another 90 min. nicotinamide (300 mg/kg). All substances were solubilized in physiological saline solution (PSS) and their pH ranged between 7.2 and 7.4. All compounds were administered i.p. in a final volume of 0.5 ml. Control rats received physiological saline solution (PSS) plus nicotinamide, or PSS, nicotinamide and harmaline instead of 3-AP. A recovery period of two weeks was allowed before the rats were killed and the endogenous 5-HT content, and uptake and release of [3H]5-HT from cerebellar slices were measured (chronic experiments). One group of rats received only 3-AP (75 mg/kg i.p.), and after 6 h, the animals were killed by decapitation, and the endogenous 5-HT content, [3H]5-HT uptake and release were then measured in cerebellar slices (acute experiments). 2.2. Histological assessment of lesions Some animals from each group were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and fixed by intracardial perfusion via the left ventricle. Phosphate buffer containing 0.3% sucrose, pH 7.2-7.4 with 300-320 mOsm/kg was used as washing solution and perfused for 3 min, at a pressure of 140 cm of water at room temperature according to procedure of Feria-Velasco and Karnovsky (1970). Perfusion was continued with a 1% glutaraldehyde and 3% paraformaldehyde solution diluted in phosphate buffer, pH 7.2-7.4 with 350-375 mOsm/kg for 15 min at the same pressure and temperature. The brain was carefully removed by craneotomy and immersed in the same glutaraldehyde-paraformaldehyde fixative for 3 h at room temperature. Two-millimetre slices of cerebellum, spinal cord, medulla oblongata and cerebral coronal sections were obtained, dehydrated and embedded in paraffin. Sections, 3 #m, obtained with a

rotating microtome were stained with hematoxylin-eosin and cresyl-violet for examination under light microscopy. 2.3. Sfice preparation The rats were killed by decapitation. The cerebella were immediately dissected out over ice and cut sagitally with a TC-2 Smith-Farquhar tissue chopper. The slices were weighed and immediately transferred to a vial containing freshly oxygenated (95% 02-5% CO2), Krebs-bicarbonate buffer, pH 7.2 (mM: 110 NaCI, 3 KCI, 2 CaCI 2, 1.2 MgCI 2, 1.2 Na2HPO 4, 25 NaHCO 3 and 10 glucose). 2.4. Uptake and release experiments For measuring the accunmlation of [3H]5-HT, slices were preincubated in Krebs-bicarbonate medium containing 1 mM ascorbic acid and pargyline (0.03 mM) at 37°C, the radiolabelled amine was added after 5 min at a final concentration of 0.5 /.tM, and incubation continued for another 5 min. The slices were washed twice by filtration with 10 ml of the same medium at room temperature. The filters were transferred to scintillation vials and the radioactivity was determined in a Packard 3390 liquid scintillation spectrometer. Appropriate control samples were incubated at 4°C in each experiment and the radioactivity accumulated under these conditions was subtracted from the values obtained at 37°C. The results were expressed as pmol of [3H]5-HT accumulated/mg protein per 5 min. In the release experiments, both baseline efflux and stimulated release of [3H]5-HT were measured as previously described (Beas-Zfirate et al., 1984). After loading with [3H]5-HT, slices were plated onto a glass fiber filter and washed six times with 2 ml of Krebs-bicarbonate medium containing 1 mM EGTA; each wash lasted 30 s and the solutions were drained by vacuum. After the sixth wash, the slices received a 30 s pulse with 2 ml of one of the following solutions: (1) Ca z +-free medium containing 1 mM EGTA (baseline efflux); (2) Ca2+-free medium containing 1 mM EGTA and 53 mM KCI (NaCI reduced to 53 mM); (3) medium containing 2 mM CaCI 2 and 53 mM KCI; (4) medium containing 1 mM EGTA and veratrine (60 #g/ml) or (5) medium containing 2 mM CaCl 2 and veratrine (60/.tg/ml). All operations were carried out at room temperature and the radioactivity in the effluents and in the filters was determined at the end of each experiment. Effluents and filters were transferred to separate scintillation vials and measured in a Packard 3390 liquid scintillation spectrometer. In previous experiments (Beas-Zhrate et al., 1984), it was shown that the baseline efflux of [3H]5-HT with Ca2+-free medium, EGTA and 3 mM KCI, in cerebellar

slices was not statistically different from the efflux with normal medium containing 2 mM CaCI and 3 mM KCI (n = 10 experiments in duplicate). Radiolabelled amine efflux was expressed as a percentage of the total radioactivity present on the filters immediately before the experimental pulse.

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2.5. 5 - H T concentrations and statistics

To determine the 5-HT content in cerebellum and inferior olivary nucleus, the tissue samples were weighed and homogenized in 10 volumes of butanol-acid and a pellet was removed by centrifugation at 10 000 × g. The

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0~ ~Se Fig. 1. Inferior olivary nucleus. (A) Normal aspect of nerve cells in the inferior olivary nucleus of a rat injected with PSS and nicotinamide, and killed 15 days later. Nissl stain (×240). (B) Normal aspect of nerve cells in the inferior olivary nucleus of a rat injected with PSS, harmaline and nicotinarnide, and killed 15 days later. Nissl stain (× 240). (C) Inferior olivary nucleus of a rat injected with 3-AP and harmaline, and killed 6 h after 3-AP injection. There is severe damage and destruction of most nerve cells (arrows). Nissl stain (×240). (D) Inferior olivary nucleus of a rat injected with 3-AP, l-,armaline and nicotinamide, and killed 15 days after 3-AP injection. The very tew nerve cells depicted appear hyperchromatic and damaged (arrows). The tissue elements of the nucleus are mostly represented by glial cells and nerve fibers. Nissl stain ( x 240).

10 supernatant was used for 5-HT extraction and quantification by a spectrofluorometric m e t h o d according to Curzon and G r e e n (1970). The statistical significance of differences between the mean values for the groups was determined with Student's t-test (Snedecor and Cochran, 1967).

3, Results 3.1. Morphological data N o significant morphological alterations were observed under light microscopy in sections from cervical spinal cord a n d in the brain coronal sections corresponding to the five groups of animals studied. Both control groups corresponding to animals not injected with 3-AP showed the n o r m a l a r r a n g e m e n t of neurons and glial cells in the inferior olivary nucleus (fig. 1A and B), and no microscopic changes were seen in tissue elements of the cerebellum, particularly those of the cortical molecular layer (fig. 2A). All animals injected with 3-AP h a d severe lesions in the inferior olivary nucleus. W h e n the animals received 3-AP followed by harmaline and PSS a n d were killed 6 h after the 3-AP injection, most nerve cells a p p e a r e d shrunken, hyperchromatic, with pycnotic nuclei or with cariolysis, a n d vacuolated cytoplasm. Tissue disorganization was evident (fig. 1C). W h e n the animals received 3-AP, followed by harmaline or PSS a n d nicotinamide, and were killed 15 days after 3-AP injection, almost no

TABLE 1 Effect of 3-AP on 5-HT levels in cerebellum and olivary nucleus. 5-HT levels in whole cerebellum and olivary nucleus at 1 and 5 days after 3-AP administration. The results are expressed as ~g/g of fresh tissue and represent the means + S.E.M. of five experiments in duplicate. n.d. = 5-HT was not detected in this group. Cerebellum Olivary nuclei

Control

3-AP (1 day)

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1.125 + 0.030

0.991+ 0.064

3-AP (5 days) a

n.d. 0.614 + 0.070 b

"~P < 0.02 significantly different with respect to control group; b p < 0.001 significantly different with respect to control group.

nerve cells were identified in the region c o r r e s p o n d i n g to the inferior olivary nucleus, p r e d o m i n a t i n g nerve fibers and glial cells (fig. 1D). N o microscopic changes were seen in the cortical molecular layer of the cerebellum in animals injected with 3-AP and killed 6 h later. However, the animals treated with 3-AP a n d killed after 15 days showed an evident change in the g r a n u l a r pattern of the cerebellar molecular layer (fig. 2B).

3.2. 5-HT Content 5 - H T concentration was measured in all groups that received the various treatments. However, the results showed that only 3-AP produced an i m p o r t a n t reduction in the 5 - H T content, corresponding to 32 a n d 100% on the first and fifth d a y after its administration, re-

Fig. 2. Cerebellar cortex. (A) Normal aspect of a portion of cerebellar cortex from a rat injected w,~th PSS, harmaline and nicotinamide and killed 15 days later. M = molecular layer; P = Purkinje cells; G = granular layer. Nissl stain ( × 240). (B) Portion of cerebellar cortex of a rat injected with 3-AP, harmaline and nicotinamide and killed 15 days after 3-AP injection. An evident change in the pattern of granulation is seen in the molecular layer (M) and some Purkinje cells appear hyperchromatic (open arrow). Nissl stain ( x 240).

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spectively, compared to the control groups (table 1). Only a 45% reduction was found in the olivary nuclei on the fifth day after 3-AP treatment (table 1). On the other hand, the groups receiving harmaline or nicotinamide either alone or combined, had no significant changes in 5-HT levels in either the olivary nucleus or the cerebellar cortex (data not shown).

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Fig. 4. Calcium-dependent release of ['H]5-HT from slices of cerebellar cortex under different experimental conditions of stimulation with 53 mM KCI ( t K ÷ ) or 60 # g / m l veratrine (V), after chronic and acute treatment with 3-AP. P S S = physiological saline solution; H = harmaline; N = nicotinamide; 3 - A P - - 3 - a c e t y i p y r i d i n e ; [3H]5-HT = tritium-labelled serotonin; ~,K + = 3 m M KCI; Ca + + = calcium. The data represent the means + S.E.M. of 10 experiments in duplicate. * P < 0.05; @ P < 0.001; N.S. = non-significant. All statistical comparisons are made against basal

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TABLE 2 Kinetics." of [~HI5-HT uptake in cerebellar slices after 3-AP. Active transport of [~H]5-HT under different experimental conditions. K m and ~,~, ~ere obtained using a range of [aH]5-HT concentrations of 0.05-0.9 ~tM and the values represent the means of five experiments in duplicate. H = harmaline, N = nicotinamide; PSS = physiological saline solution; 3-AP = 3-acetylpyridine; 5-HT = serotonin. Experimental conditions

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HT uptake, as compared to that in the group treated with harmaline, nicotinamide or PSS (table 2). Nicotinamide alone produced no changes in uptake of [3H]5-HT in the group injected with PSS. This group was used as control in all experiments. 3.4. [~H]5-HT release with 3-,4P and harmafine treatttlelll

Figure 4 shows the percentage of [3H]5-HT released from cerebeilar slices in the various experimental conditions, considering the total amount of [3H]5-HT accumulated in the slices before the medium was changed for stimulation as 100%. A significant increase, by 23% (P < 0.05), of [3H]5-HT release from the basal efflux (fig. 4) was observed when depolarizing stimuli (53 mM KC! and veratrine) were applied to the cerebellar slices. The group treated with PSS, harmaline and nicotinamide showed an increase of 70% in the Ca2+-dependent and K+-evoked [3H]5.HT release when compared with the basal efflux of [ 3 H ] 5 HT (fig. 4). In contrast, the Ca2+-dependent release of [3H]5-HT with both types of depolarizing stimuli (53 mM KCI and veratrine) was completely abolished in animals given acute or chronic 3-AP (fig. 4).

4. Discussion

Although the inferior olivary nucleus and accessory olives in the medulla oblongata are the gray matter areas mainly affected by the systemic administration of 3-AP (Denk et al., 1968; Desclin, 1974; Desclin and Escubi, 1974; Anderson and Flumerfelt, 1984), other brainstem nuclei also show degenerative changes with loss of neurons (Desclin and Escubi, 1974). These nuclei include some of the reticular formation, as well as a few raphe nuclei which pi'oduce 5-HT as neurotransmitter. It is not well known which substances are released as neurotransmitters by the climbing fibers; or whether there is only one transmitter released by all climbing

fiber terminals. Most of the axons contributing to form climbing fibers in the various regions of the cerebellar cortex come mainly from the olivary complex in the medulla (Anderson and Flumerfelt, 1980; Desclin and Escubi, 1975). Various authors have demonstrated serotoninergic nerve fibers in the rat cerebellar cortex by means of biochemical (Palkovits et al., 1974; Velfizquez et al., 1987; Beas-Zfirate et al., 1984), autoradiographic (Chan-Palay, 1975; Chan-Palay et al., 1977), histochemical (HiSkfelt and Fuxe, 1969) and immunohistochemical (Bishop and Ho, 1983; 1985; 1986; Takeuchi et al., 1982a) techniques. Several nuclei located in the medulla and pons, including some in the raphe area, project to the cerebellum (Chan-Palay, 1977; Kimoto et al., 1978; Shinnar et al., 1973), which produce and release 5-HT as neurotransmitter (Chan-Palay, 1975; 1977; Shinnar et al., 1973). However, it is important to consider that some raphe nuclei do not produce 5-HT (Bowker et al., 1981; Chan-Palay et al., 1978; Htikfelt et al., 1978; 1979; Johansson et al., 1981; Takeuchi et al., 1982a; Voogd, 1964) and it is also important to note that the reticular formation projecting to the cerebellum (Kimoto et al., 1978) has serotoninergic neurons (Bishop and Ho, 1984; DahlstriSm and Fuxe, 1964; Steinbusch, 1981; Takeuchi et al., 1982b). Among these nuclei are the reticularis pontis oralis, the paragigantocellularis and the gigantocellularis (Bishop and Ho, 1983). Although serotoninergic fibers are distributed in all layers of the cerebellar cortex, most of these terminals are found in the molecular layer (Takeuchi et al., 1982a). In the present work with neurochemical techniques aimed to evaluate [3H]5-HT uptake by terminals and with Ca 2+-dependent [ 3H]5-HT release experiments, we also obtained information regarding the existence of serotoninergic terminals in the cerebellum, as part of the climbing fiber system. Moreover, the results presented further support the assumption that most of the serotoninergic fibers are distributed in the cerebellar molecular layer. Although the histological preparations showed that the main lesions induced by 3-AP were located in the olivary complex, as demonstrated by several authors (Desclin and Escubi, 1975; Tapia-Arizmendi and Feria-Velasco, 1980), it cannot be ruled out that some small cell groups of the ponto-medullary reticular formation and some nerve cells of raphe nuclei could also have been damaged. Biochemical data obtained in the present work showed an important reduction in 5-HT concentration, in both cerebellum and olivary nuclei. There was also a considerable reduction in uptake, uptake kinetics and 5-HT release from cerebellar subcellular fractions obtained from animals injected with 3-AP. However, 5-HT has been demonstrated by immuno-

histochemistry (Steinbusch, 1981) and fluorescence histochemical techniques (Fuxe and Ljundgren, 1965) in nerve terminals ending at the olivary cells (Toggenburger et al., 1983), but not in the somata of these neurons. Based on these data, the reduction in indolaminergic transmission observed in the cerebellum as an effect of 3-AP administration could hardly be explained only by destruction of olivary cells. It is possible that some nerve cells in some reticular nuclei such as nucleus magnocellularis, nucleus gigantocellularis and nucleus paragigantocellularis could have been damaged due to the 3-AP effect, thus explaining the biochemical findings regarding 5-HT efflux. The reduction of 5-HT concentration in the olivary region of the medulla can :. cxplained by the dense indolaminergic innervation of :.~ltvary cells c o m i n g from the p a l l i d u m raphe nucleus (Toggenburger et al., 1983). Whether 3-AP, after destruction of olivary cells, induces retrograde trans-synaptic lesion of serotoninergic fibers, or whether some of these terminals come from 3-AP sensitive raphe nerve cells innervating the olivary nucleus, cannot be explained by the data now presented. The results obtained for the group of harmalinetreated animals showed a reduction in 5-HT uptake in cerebellum, as well as a decreased Vma~, simultaneously with a significant increase of 5-HT release in the cerebellum. Together these results could indicate an important decrease in the n u m b e r of cerebellar indolaminergic fibers. However, it m a y not have been the case in the present experiments, as an increase in the 5-HT release as an effect of h a r m a l i n e in cerebellum was detected, and apparently affected its transport dynamics in only a few serotoninergic fibers, since the affinity for 5-HT was unchanged, and the Vmax only decreased by 30%. This point finds support from the fact that h a r m a l i n e induces an increase in the physiological activity of climbing fibers, manifested by a higher discharge frequency of the cerebellar Purkinje cells (1-2 to 10-12 c y c l e s / s ) (SjiSlund et al., 1977). It is clear that some climbing fibers produce 5-HT, releasing it at the cerebellar molecular layer. Other climbing fibers apparently produce aspartate or taurine (Rea et al., 1980; Toggenburger et al., 1983). Some controversy still remains regarding the possibility that glutamate is a neurotran~mitter in climbing fibers, as suggested by some authors (Toggenburger et al., 1983; Guidotti et al., 1975). When 3-AP was combined with harmaline the uptake and release of some transmitter a m i n o acids such as glutamate, glycine and G A B A were only partially modified (Foster and Roberts, 1980; Toggenburger et al., 1983). These data suggest that these a m i n o acids play an insignificant role as neurotransmitter released by climbing fibers, mostly originating in the inferior olivary nucleus. Furthermore, the present results indicate that 5-HT is a neuro~ransmitter released

by some climbing fibers probably originating in the neighbourhood of the inferior olivary nucleus, or in other serotoninergic nerve cell complexes projecting their fibers to the cerebellar cortex as indolaminergic climbing fibers. It is known that 5-HT reduces the frequency of discharge of Purkinje cells when it is applied microiontophoretically (Strahlendorf and Strahlendorf, 1985). O n the other hand, when this indoleamine is applied for longer time intervals, it increases the spontaneous discharges of Purkinje cells (Strahlendorf and Strahlendorf, 1989; Batini et al., 1987). Whether the indolaminergic endings contact directly to Purkinje cells, or whether this innervation is mediated by Golgi type !i interneurons cannot be ascertained from the present work and needs to be further investigated. Studies with biochemical procedures to investigate the 5-HT turnover in conditions of surgical lesions of middle or inferior cerebellar peduncles, with thorough correlated morphological examination of brainstem nuclei, could help to explain the role of 5-HT as neurotransmitter in the cerebellar climbing fiber system.

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