Neuronal cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor agonist WIN 55,212-2

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Neuronal cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor agonist WIN 55,212-2 Patricia Tagliaferro a , Alberto Javier Ramos a , Emmanuel S. Onaivi b , Sergio Gustavo Evrard a , Javier Lujilde a , Alicia Brusco a,⁎ a Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis”, Facultad de Medicina, Universidad de Buenos Aires, er Paraguay 2155, 3 piso, (C1121ABG), Buenos Aires, Argentina b Department of Biology, William Paterson University, Wayne, NJ 07470, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

Cannabinoid CB1 receptors are the most abundant G-protein-coupled receptors in the brain.

Accepted 21 December 2005

Its presynaptic location suggests a role for cannabinoids in modulating the release of

Available online 29 March 2006

neurotransmitters from axon terminals by retrograde signaling. The neuroprotective effects of cannabinoid agonists in animal models of ischemia, seizures, hypoxia, Multiple Sclerosis,

Keywords:

Huntington and Parkinson disease have been demonstrated in several reports. The

Cannabinoid receptor

proposed mechanism for the neuroprotection ranges from antioxidant effects, reduction

Neurofilament

of microglial activation and anti-inflammatory reaction to receptor-mediated reduction of

MAP-2

glutamate release. In the present work, we analyzed the morphological changes induced by

GFAP

a chronic treatment with the synthetic cannabinoid receptor agonist, WIN 55,212-2, in four

Synaptophysin

brain regions where the CB1 cannabinoid receptor is present in high density: the CA1

Synaptic plasticity

hippocampal area, corpus striatum, cerebellum and frontal cortex. After a twice-daily treatment for 14 days with the cannabinoid receptor agonist (3 mg/kg sc, each dose) to male Wistar rats (150–170 g), the expression of neurofilaments (Nf-160 and Nf-200), microtubuleassociated protein-2 (MAP-2), synaptophysin (Syn) and glial fibrillary acidic protein (GFAP) was studied by immunohistochemistry and digital image analysis. Ultrastructural study of the synapses was done using electron microscopy. After the treatment, a significant increase in the expression of neuronal cytoskeletal proteins (Nf-160, Nf-200, MAP-2) was observed, but we did not find changes in the expression of GFAP, the main astroglial cytoskeletal protein. In cerebellum, there was an increase in Syn expression and in the number of synaptic vesicles, while, in the hippocampus, an increase in the Syn expression and in the thickness of the postsynaptic densities was observed. The results obtained from these studies provide evidences on the absence of astroglial reaction and a sprouting phenomena induced by the WIN treatment that might be a key contributor to the long-term neuroprotective effects observed after cannabinoid treatments in different models of central nervous system (CNS) injury reported in the literature. © 2005 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +54 11 4637 0923. E-mail address: [email protected] (A. Brusco). 0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.089

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Abbreviations: AcB, acetate buffer CA1, CA1 hippocampal area CNS, central nervous system DMSO, dimethylsulfoxyde FCx, frontal cortex GABA, gamma amino butyric acid GFAP, glial fibrillar acidic protein MAP-2, microtubule-associated protein-2 Nf-160, 160 kDa neurofilaments Nf-200, 200 kDa neurofilaments PBS, phosphate buffer saline ROD, relative optical density Syn, synaptophysin Strt, corpus striatum StrtM, striatal matrix StrtP, striatal patches

1.

Introduction

The presence of CB1 receptors in the central nervous system (CNS) has been largely characterized (see for review Ameri, 1999), and, recently, CB2 receptors have been also demonstrated in the brainstem (Van Sickle et al., 2005; Gong et al., 2005, in press; Onaivi et al., 2006). CB1 receptors are expressed in neurons of specific brain regions showing the highest density in the basal ganglia, cerebellum, hippocampal CA1 pyramidal cell layer and dentate gyrus and cortical layers I and IV (Dove Pettit et al., 1998). The widespread distribution of the cannabinoid receptors in the brain is well correlated with the cannabinoid effects on memory, perception and control of movement (Ameri, 1999). Physiological, pharmacological and high-resolution anatomical studies provided evidence that the major physiological effect of cannabinoids is the regulation of neurotransmitter release via activation of presynaptic CB1 receptors located on distinct types of axon terminals throughout the brain (Alger, 2002; Freund et al., 2003; Pertwee, 1997). The endogenous ligands for CB receptors are the endocannabinoids anandamide (N-arachidonyl-ethanolamine) (Devane et al., 1992), 2-arachidonylglycerol (Mechoulam et al., 1995; Martin et al., 1999; Sugiura et al., 1995), 2-arachidonylglyceryl ether (Hanus et al., 2001) and virodhamine (Porter et al., 2002). The administration of endocannabinoids to experimental animals produces several of the pharmacological and behavioral actions associated with exogenous cannabinoids (Onaivi et al., 1995; Salzet et al., 2000; Onaivi et al., 2002), although the chemical structure is different (Salzet et al., 2000). The neuroprotective properties of synthetic cannabinoids and phytocannabinoids have been largely demonstrated in the last years in vitro and in vivo in animal models of ischemia, seizures, hypoxia, Multiple Sclerosis, Huntington and Parkinson disease (see for example Alger, 2004; De Lago et al., 2005; Jackson et al., 2005; Lastres-Becker et al., 2005; Khaspekov et al., 2004); furthermore, the use of these type of cannabinoids to improve memory and cognitive deficits in Alzheimer's disease has been proposed (Grotenhermen, 2005). The molecular mechanisms proposed for these wide effects

on different models of injury range from antioxidant properties and anti-inflammatory effects to inhibition of glutamate release (and excitotoxicity), reduction of calcium influx, activation of the phosphatidylinositol 3-kinase/protein kinase B pathway, induction of phosphorylation of extracellular regulated kinases (ERK), activation of NF-κB transcription factors and neurotrophins release (see for review Van der Stelt and Di Marzo, 2005). The cannabinoids effects in synaptic plasticity (Kim and Thayer, 2001) were proposed since cannabimetic drugs are known to inhibit adenylyl cyclase and impair memory (Hampson and Deadwyler, 1998; Heyser et al., 1993; Lichtman et al., 1995). The involvement of cannabinoids in memory, learning and motor behavior has triggered the hypothesis of the ability of these drugs to induce plastic changes in the CNS (Kim and Thayer, 2001). Early findings by Lawston et al. (2000) showed that a CB1 agonist is able to produce morphological alterations in the hippocampal dendrites. However, morphological evidences of neuronal plasticity and synaptic modifications by cannabinoids are absent in the literature. The goal of the present work was to analyze the morphological changes in the neuronal cytoskeleton as well as the alterations in the area covered by neuronal processes induced by chronic treatment with the synthetic cannabinoid agonist WIN 55,212. Neurofilaments of 160 kDa (Nf-160) and 200 kDa (Nf-200), microtubule-associated protein-2 (MAP-2) expression was analyzed in anatomical areas with high density of CB1 receptors such as the stratum radiatum of the CA1 hippocampal area, corpus striatum, cerebellum and frontal cortex. Since cannabinoids are also involved in the regulation of neurotransmitter release and that effect is also proposed for neuroprotection, the ultrastructure of the synaptic connections as well as the expression of synaptophysin (Syn), a surface protein present in the synaptic vesicles, was analyzed to study the morphological changes that could be induced by the long-term treatment with WIN. The astroglial cells were studied to detect changes such as hypertrophy that could indicate CNS damage induced by the treatment (Tagliaferro et al., 1997; Ramos et al., 2002, 2004).

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Fig. 1 – Nf-160 immunostaining. Photomicrographs show the Nf-160 immunoreactivity in the CA1 hippocampal area (A: control; B: Win-treated); in the corpus striatum (C: control; D: Win-treated); in the cerebellum (E: control; F: Win-treated); and in the frontal cortex (G: control; H: Win-treated). Primary magnification: 400×.

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2.

Results

2.1.

160 and 200 kDa neurofilaments (Nf-160; Nf-200)

Neurofilaments are the major cytoskeletal components in neurons. They are intrinsic determinants of axonal caliber (Nixon and Sihag, 1991), and their dynamic remodeling is essential for axonal growth and guidance. By regulating axonal caliber, neurofilaments can affect both axonal transport and neuronal function (Hoffman et al., 1987; Ramos et al., 2000). In the stratum radiatum of the hippocampal CA1 area from control animals, the presence of thin and long neuronal processes immunoreactive either for Nf-160 (Fig. 1A) or Nf200 was observed (Fig. 3A). In WIN-treated animals, Nf-160 expression was increased and cellular projections were thicker and presented an irregular morphology (Fig. 1B). Nf-200 expression was also increased when compared to the control animals (Fig. 3B). Digital image analysis demonstrated a statistically significant increase in the tissue area covered by either Nf-160 (Fig. 2)- or Nf-200-immunoreactive neuronal processes between control and WIN-treated animals (Fig. 4). In the striatum, Nf-160-immunostained neuronal processes were observed in the striatal matrix and patches (Fig. 1C). In WIN-treated animals, Nf-160 expression was increased in the two striatal areas (Fig. 1D). In the striatal patches, the Nf-160 morphology appeared fragmented and irregular, while in the matrix they were observed as a fine network of neuronal processes (Fig. 1D). Nf-200-immunostained neuronal processes showed the same pattern of distribution of Nf-160 (Fig. 3C). In the WIN-treated animals, Nf-200 expression was increased in both the striatal matrix and patches (Fig. 3D). These observations were confirmed by digital image analysis that showed significant differences in the relative area covered

Fig. 2 – Relative area of Nf-160-immunoreactive intermediate filaments in the CA1 hippocampal area (CA1) (control: 0.038 ± 0.002; WIN-treated: 0.058 ± 0.003), in the striatal matrix (StrtM) (control: 0.035 ± 0.006; WIN-treated: 0.074 ± 0.008) and striatal patches (StrtP) (control: 0.187 ± 0.006; WIN-treated: 0.211 ± 0.013), in cerebellum (Cereb) (control: 0.038 ± 0.005; WIN-treated: 0.104 ± 0.016) and frontal cortex (FCx) (control: 0.123 ± 0.028; WIN-treated: 0.252 ± 0.041). Note the increased relative area in all the areas except for the StrtP. *P b 0.05; **P b 0.01 and ***P b 0.0001 after two-tailed Student's t test. Bars represent mean ± SD.

either by Nf-160 (Fig. 2) and Nf-200-immunoreactive neuronal processes between control and WIN-treated groups (Fig. 4). In the cerebellum, the expression of both Nf-160 (Figs. 1E and F) and Nf-200 (Figs. 3E and F) was increased in the three layers of the cerebellar cortex of WIN-treated animals. The increase was especially evident in the basket around the base of the Purkinje cells (see Fig. 1F). In the molecular and granular layers, the neuronal processes immunoreactive either for Nf160 or Nf-200 were more numerous and thicker than in the control animals. Digital image analysis clearly showed the increased expression of Nf-160 (Fig. 2) and Nf-200 (Fig. 4) in the WIN-treated animals. In the frontal cortex, Nf-160 expression showed a significant increase in all cortical layers of WIN-treated animals (Figs. 1G and H). The relative area covered by the Nf-160-immunostained neuronal processes was significantly increased in the WIN-treated animals (Fig. 2). The pattern of Nf-200 expression in the frontal cortex also showed differences between control and WIN-treated animals (Figs. 3G and H). The increase in the relative area covered by Nf-160- and Nf-200-immunostained neuronal processes was statistically significant (Figs. 2 and 4). The two markers used for the neurofilament processes showed an increased expression in the areas studied following a chronic treatment with WIN.

2.2.

Microtubule-associated protein-2 (MAP-2)

MAP-2 is a microtubular-associated protein found in dendrites, and it is commonly used as a specific marker to visualize these neuronal processes to analyze the dendritic tree and its extension. The stratum radiatum of the CA1 hippocampal area of control animals showed thin and regular MAP-2-immunostained dendrites (Fig. 5A). WIN-treated animals showed changes in the morphology of MAP-2-immunostained dendrites, being thicker, irregular and waved (Fig. 5B). An increase in the tissue area covered by these MAP-2-immunostained dendrites was also observed in WIN-treated animals. This observation was confirmed by the results obtained from the digital image analysis (Fig. 6). In the striatum, the immunostaining for MAP-2 was limited to few and thin dendrites present in the matrix in both control and WIN-treated animals. It was not possible to measure the relative area covered by these MAP-2-immunostained neuronal processes. In the molecular layer of the cerebellum from WIN-treated rats, the MAP-2-immunostained dendritic trees coming from the Purkinje cells were more evident, longer and thicker than in control rats (Figs. 5C and D). In the granular layer, there was also an increase in the MAP-2 immunostaining, indicating the presence of thin and irregular neuronal processes (Fig. 5D). Digital image analysis showed significant differences between control and WIN-treated animals in the relative area covered by these MAP-2-immunostained neuronal processes (Fig. 6). MAP-2 immunostaining in the frontal cortex showed the same expression pattern in control (Fig. 5E) and WIN-treated animals, but the relative area covered by the MAP-2-immunostained dendrites was higher in the WIN-treated animals (Fig. 5F). Digital image analysis confirmed these observations (Fig. 6).

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Fig. 3 – Nf-200 immunostaining. Photomicrographs show the Nf-200 immunoreactivity in the CA1 hippocampal area (A: control; B: Win-treated); in the corpus striatum (C: control; D: Win-treated); in the cerebellum (E: control; F: Win-treated); and in the frontal cortex (G: control; H: Win-treated). Primary magnification: 400×.

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features of astroglial reaction is the increased large size of cellular projections, it is possible to evaluate the astroglial reaction by studying changes in the area of GFAP expression (Tagliaferro et al., 1997). We did not observe any change in the astrocytic cell area, shape or organization pattern in none of the four evaluated areas. There were no statistical differences between control and WIN-treated groups in the stratum radiatum of the CA1 hippocampal area, in the striatum, in the frontal cortex or in the cerebellum (see Figs. 10 and 11).

2.5.

Fig. 4 – Relative area of Nf-200-immunoreactive intermediate filaments in the CA1 hippocampal area (CA1) (control: 0.053 ± 0.014; WIN-treated: 0.083 ± 0.016), in the striatal matrix (StrtM) (control: 0.07 ± 0.009; WIN-treated: 0.10 ± 0.012) and striatal patches (StrtP) (control: 0.146 ± 0.028; WIN-treated: 0.198 ± 0.026), in cerebellum (Cereb) (control: 0.057 ± 0.006; WINtreated: 0.01 ± 0.004) and frontal cortex (FCx) (control: 0.224 ± 0.037; WIN-treated: 0.352 ± 0.029). Note the increased relative area in all the studied areas. *P b 0.05; and **P b 0.01 after twotailed Student's t test. Bars represent mean ± SD.

2.3.

Synaptophysin (Syn)

Syn is a protein present in the synaptic vesicles at the presynaptic terminal. In the CA1 hippocampal area of control animals, the expression of Syn was observed as a diffuse and granulated immunostaining in all the layers except the pyramidal cell layer that presented unlabeled pyramidal neurons as negative images surrounded by Syn immunostaining (Fig. 7A). In the WIN-treated animals, the pattern of expression of this protein was similar to that from the control animals, but there was a tendency to an increase in the Syn immunostaining (Fig. 7B) that was not significant in the ROD values obtained by digital image analysis (Fig. 8). In the cerebellum of control animals, a diffuse immunostaining was observed in the molecular layer, but, in the granular layer, the immunostaining for Syn was observed as irregular and small structures, probably representing the glomerulum (Fig. 7C). In the WIN-treated animals, the pattern of expression was similar to the control animals, but the intensity of the immunostaining was increased (Fig. 7D). In the granular layer, the Syn-immunostained structures appeared also bigger. The intensity of Syn immunostaining was evaluated by digital image analysis, and there were statistically significant differences in the ROD values between control and WIN-treated animals in both the molecular and granular layer (Fig. 8). Digital image analysis also showed significant differences in the relative tissue area occupied by these Synimmunostained structures (Fig. 9).

2.4.

Glial fibrillary acidic protein (GFAP)

GFAP is the main intermediate filament in astrocytes, and it defines the astrocytic morphology. Since one of the main

Electron microscopy

The electron microscopy study showed ultrastructural differences between control and WIN-treated animals in two areas, stratum radiatum of the CA1 hippocampal area and in the granular layer of the cerebellum. In the hippocampus of control animals, the synapses presented the classical ultrastructural components such as synaptic vesicles and mitochondria at the presynaptic terminal (Fig. 12A). Accordingly with their morphology, they were probably gabaergic (symmetrical). The postsynaptic densities were thin but well defined. In the treated animals, postsynaptic densities were thicker and darker, and the presynaptic densities were also thicker (Fig. 12B). Image analysis confirmed this result (Fig. 12E). In the cerebellum of control animals, synaptic contacts also showed the typical aspect with clear vesicles and diffuse postsynaptic densities (Fig. 12C). Accordingly with their morphology, they were asymmetrical (probably glutamatergic). In the WIN-treated animals, the thickness of the postsynaptic densities was increased (Fig. 12D). An increase in the number of synaptic vesicles was observed in WIN-treated animals.

3.

Discussion

It has been described that cannabinoids cause modifications in different hippocampal and striatum-related functions: learning and memory (Hampson and Deadwyler, 1998), decreased locomotor activity by an inhibition of GABA and dopamine uptake in the striatum (Ameri, 1999) and psychomotor function (Rodriguez de Fonseca et al., 1998). It is also now recognized that cannabinoids have interesting neuroprotective properties in several animal models of human pathological states and diseases (see for example Alger, 2004; De Lago et al., 2005; Jackson et al., 2005; Lastres-Becker et al., 2005; Khaspekov et al., 2004). The cannabinoid effects in the hippocampal formation also lead to the proposal of using cannabinoids to improve memory and cognitive deficits in Alzheimer's disease (Grotenhermen, 2005). The physiological basis for the effects on neuroprotection ranges from antiinflammatory effects, free radical scavenging to inhibition of glutamate release (and excitotoxicity) (see for review Van der Stelt and Di Marzo, 2005). The brain derived neurotrophic factor (BDNF) involvement in the cannabinoid effects (Marsicano et al., 2003; Khaspekov et al., 2004); the neurofilament damage in CB1-null mice (Jackson et al., 2005) and the cannabinoids blockage effects on synapses (Karanian et al., 2005) are evidences that point out the cannabinoids involvement in

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Fig. 5 – MAP-2 immunostaining. Photomicrographs show the MAP-2 immunoreactivity in the CA1 hippocampal area (A: control; B: Win-treated); in the cerebellum (C: control; D: Win-treated); and in the frontal cortex (E: control; F: Win-treated). Primary magnification: 400×.

neuronal plasticity and stabilization of connections in the CNS. The morphological correlate of these effects has never been shown in the literature. In the present work, we have demonstrated important changes in the neuronal cytoskeleton following a chronic treatment with the synthetic cannabinoid receptor agonist WIN. The treatment paradigm used in this study did not induce a detectable astroglial reaction in the studied areas since no alterations in the expression pattern of the main astroglial cytoskeletal protein were observed. We have also demonstrated ultrastructural changes at the synaptic contacts in the CA1 hippocampal area and in the cerebellum. This study was performed on four brain regions where the CB1 receptor is found

in high density (i.e., hippocampus, cerebellum, corpus striatum and frontal cortex), although the effects were also present in non-CB1-rich areas. After the twice-daily treatment for 14 days with the cannabinoid receptor agonist, we found an increase in the relative area covered by neuronal processes, probably indicating an increased expression of the cytoskeletal proteins or a reorganization of the cytoskeletal structure. MAP-2-immunostained dendrites from the stratum radiatum in the CA1 hippocampal area were thicker and presented an irregular structure, and the relative area covered by the dendrites was increased in the WIN-treated animals. In our experiments, the relative area covered by the dendrites was increased, probably reflecting an

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Fig. 6 – Relative area of MAP-2-immunoreactive filaments in the CA1 hippocampal area (CA1) (control: 0.124 ± 0.027; WIN-treated: 0.218 ± 0.036), in cerebellum (Cereb) (control: 0.060 ± 0.004; WIN-treated: 0.109 ± 0.025) and frontal cortex (FCx) (control: 0.317 ± 0.046; WIN-treated: 0.509 ± 0.072). Note the increased relative area in all the studied areas. **P b 0.01 after two-tailed Student's t test. Bars represent mean ± SD.

increased dendrite thickness and/or an increased branching characteristic of sprouting phenomena detected with MAP-2 immunostaining (Ramos et al., 2004). Interestingly, in other brain areas like the molecular layer of the cerebellum, where the MAP-2 immunostaining allowed the observation of the Purkinje cells dendritic trees, and in the frontal cortex, we

Fig. 8 – Relative optical density (ROD; in ROD units) of the Syn-immunostained structures in the stratum radiatum of the CA1 hippocampal area (CA1 Str rad) (control: 0.489 ± 0.083; WIN-treated: 0.544 ± 0.075) and in the granular layer of the cerebellar cortex (Cereb GL) (control: 0.612 ± 0.062; WIN-treated: 0.925 ± 0.088). Note the increased ROD only in the latter area. ***P b 0.0001 after two-tailed Student's t test. Bars represent mean ± SD.

found an increased relative area covered by the MAP-2immunostained dendrites, but without the structural features observed in hippocampal dendrites. Since cannabimimetic drugs can induce changes in the pattern of synaptic connections, these kinds of structural changes in dendrites could be more likely, indicating a branching phenomena leading to the

Fig. 7 – Synaptophysin immunostaining. Photomicrographs show the Syn immunoreactivity in the CA1 hippocampal area (A: control; B: Win-treated) and in the cerebellum (C: control; D: Win-treated). Primary magnification: 400×.

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Fig. 9 – Relative area of the Syn-immunostained structures located in the granular layer of the cerebellar cortex. Note the increased value in WIN-treated animals (control: 0.0017 ± 0.0003; WIN-treated: 0.0025 ± 0.0003). ***P b 0.0001 after two-tailed Student's t test. Bars represent mean ± SD.

development of new neuronal connections and reflecting an induction of plasticity in the CNS. The CB1 receptor has been implicated in different forms of synaptic plasticity involving the coupling of postsynaptic activation with decreases in the probability of the presynaptic neurotransmitter release (Ronesi et al., 2004). These forms of plasticity include depolarization-induced suppression of inhibitory and excitatory transmission (Kreitzer and Regehr, 2001; Wilson and Nicoll, 2001) and several types of long-term depression at both excitatory and inhibitory synapses (for review, see Gederman and Lovinger, 2003). This may play an important role in the control of neuronal circuits, particularly in cerebellum and hippocampus (Iversen, 2003). By means of ultrastructural studies, we demonstrated increased thickness of the postsynatic densities in WIN-treated animals. The synapses observed in the control cerebellar glomerular layer are asymmetrical (probably glutamatergic), and the ones shown in the control hippocampus are symmetrical (probably gabaergic). Increased number of vesicles was also observed, supporting the hypothesis of a direct effect of cannabinoids (probably mediated by FAK and ERK1/2) on the synaptic stabilization (Karanian et al., 2005). In this scenario, the observed increase in Syn immunostaining in cerebellum and hippocampus could be associated with the increased number of synaptic vesicles observed by EM. Our observations demonstrated the morphological and cytoskeletal changes following a treatment with the cannabinoid receptor agonist WIN 55,212-2 and represent morphological evidences that cannabinoids are able to induce neuronal plasticity. This is an interesting fact considering the long-term effects of cannabinoids in neuroprotection and the reported evidences of neurotrophin involvement in the cannabinoid effects (Marsicano et al., 2003; Khaspekov et al., 2004), where the sprouting phenomenon is likely to occur. However, we can only speculate about the benefits or the detrimental effects of the observed increased dendritic arborization and synaptic ultrastructural modifications in the CNS, especially in long-term treatments or in pathological states of the CNS. Neurofilaments are one of the most important components of the axonal cytoskeleton, being mainly responsible for the

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axonal caliber. But, neurofilaments are also present in dendrites. Their dynamic remodeling is necessary for the axonal growth and maintenance. In the matrix of the corpus striatum, we showed increased number of fibers, statistically demonstrated by an increase in the relative area covered by Nf160- or NF-200-immunoreactive cellular processes. Most of these processes have the structural characteristic of axons and appear as a fine network of very thin fibers, but some of them may also be dendrites. The increase in the neurofilament immunostaining was even more evident in the cerebellum, where the three cortical layers showed increased Nf-160immunostained and Nf-200-immunostained neuronal processes. This increase in the expression of cytoskeletal proteins might represent sprouting phenomena in the local neuronal population since this network was more evident in the WINtreated animals than in control ones. Again, we could speculate that the observed sprouting, as well as the dendritic branching, might be reflecting plastic changes tending to the establishment of transitory or permanent new connections induced by the WIN treatment. Astroglial cells are involved in the reuptake and release of neuroactive substances (Kimelberg and Katz, 1985; Shain et al., 1986), in the storage of glycogen (Tsacopoulos and Magistretti, 1996) and in the maintenance of CNS homeostasis (Walz, 1989); they also provide trophic factors that induce the development and survival of other cellular populations and are capable of reacting when different kinds of pre- and postnatal injuries affect CNS (Azmitia et al., 1990; Evrard et al., 2003; Garcia et al., 2001; Ramos et al., 2002, 2004; Tagliaferro et al., 2002). The astroglial reaction is characterized by an increase in the cellular size, the presence of thicker and longer cellular processes as well as the alterations in the expression pattern of some specific proteins, especially GFAP. In the last years, it has become evident that the detection of astroglial reaction is a very sensitive parameter to evaluate neuronal damage but also relatively minor alterations in brain environment (Evrard et al., 2003; Tagliaferro et al., 1997). Interestingly, after the cannabinoid agonist treatment, we did not find any alteration in astroglial morphology or in the intensity of GFAP immunostaining, indicating that astroglial reaction was not associated with the chronic administration of the WIN compound. This is an important result that is in accordance with the reported absence of neuronal death after WIN treatment (Lawston et al., 2000). Astroglial reaction is commonly observed after glutamate-induced neurotoxicity. We may consider that a decreased glutamate release could be associated to the absence of an astroglial reaction in our studies. Evidence in support of a direct inhibitory, but neuroprotective, effect of cannabinoids on the NMDA receptor, via Ca2+-dependent mechanisms, has been previously reported (Nadler et al., 1993; Striem et al., 1997). In summary, in this work, we present evidences of structural and ultrastructural changes in the CNS that might be representing morphological evidence of synaptic plasticity phenomena in which the administration of an exogenous cannabinoid is involved. These morphological changes were not accompanied by astroglial reaction, which could indicate CNS damage. At present, the functional significance or the benefits or detrimental effects of the observed alterations in the neuronal cytoskeleton and synapses are not known. We demonstrated a plastic phenomenon involving sprouting and modifications in synapses;

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Fig. 10 – GFAP immunostaining. Photomicrographs show the GFAP-immunoreactive astrocytes in the CA1 hippocampal area (A: control; B: Win-treated); in the corpus striatum (C: control; D: Win-treated); in the cerebellum (E: control; F: Win-treated); and in the frontal cortex (G: control; H: Win-treated). Primary magnification: 400×.

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Fig. 11 – Cell area (in μm2) of GFAP-immunoreactive astrocytes in the CA1 hippocampal area (CA1) (control: 110.6 ± 12.15; WIN-treated: 108.8 ± 11.5), in corpus striatum (Strt) (control: 82.39 ± 12.07; WIN-treated: 91.87 ± 22.82), in cerebellum (Cereb) (control: 64.34 ± 13.67; WIN-treated: 74.2 ± 13.53) and frontal cortex (FCx) (control: 90.11 ± 9.32; WIN-treated: 93.21 ± 10.3). Note the similar values in all the four areas after two-tailed Student's t test. Bars represent mean ± SD. however, we can only speculate how these effects could contribute to the observed long-term effects of cannabinoids in neuroprotection in several models of CNS injury. Our results are in accordance with the proposed effects of cannabinoids on synapses (Karanian et al., 2005), neurofilaments (Jackson et al., 2005) and compatible with the physiological effects of neurotrophin release observed after CB1 receptor stimulation (Khaspekov et al., 2004; Marsicano et al., 2003). Under the light of the recent finding of CB2 receptors in the CNS (Van Sickle et al., 2005; Gong et al., 2005, in press; Onaivi et al., 2006) and the known nonCB1 effects of the WIN, it remains to be elucidated how much of the observed effects depend on CB1, CB2 or if it depends on the interaction between both types of receptors.

4.

Experimental procedures

Mouse monoclonal antibodies anti-Nf-160, anti-Nf-200, antiMAP-2 and anti-Syn, secondary biotinylated antibodies and streptavidin complex used for immunohistochemical studies were all purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal anti-GFAP was purchased from Dako (Glostrup, Denmark). WIN 55,212-2 mesylate (WIN) was purchased from Research Biochemicals International (Natick, MA). All other chemical substances were of analytical grade.

4.1.

Animal treatment

Ten young male Wistar rats weighing 150–170 g (40–45 days old) were used. The model of cannabinoid agonist treatment was modified from a previous report (Lawston et al., 2000). Rats were divided into two groups. One group of five rats was treated with WIN dissolved in dimethylsulfoxyde (DMSO) (treated group), and other five rats were treated only with DMSO (control group). The solution containing WIN was injected twice a day (every 12 h) subcutaneously (in the back neck region) over 14 days. Individual WIN doses were of 3 mg/ kg (6 mg/kg/day); injection volumes ranged from 0.2 to 0.3 ml.

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We did not observe any animal mortality during the treatment. Control animals received the same volume of sterile DMSO and were kept in the same environment as the WINtreated animals (12/12 h light–dark cycle, controlled humidity and temperature, free access to standard laboratory rat food and water). Another group of animals was not injected and used to exclude DMSO effects in the analyzed parameters. This non-injected group did not present differences when the parameters analyzed were compared with DMSO-treated ones. The animal care for this experimental protocol was in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the principles presented in the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience.

4.2.

Fixation

In the 15th day, animals were deeply anesthetized with 300 mg/kg of chloral hydrate. They were perfused through the left ventricle, initially with a cold saline solution containing 0.05% (w/v) NaNO2 plus 50 IU of heparin and subsequently with a cold fixative solution containing 4% (w/v) paraformaldehyde and 0.25% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were removed and kept in the same cold fixative solution for 4 h. Then, brains were washed three times in cold 0.1 M phosphate buffer pH 7.4 containing 5% (w/v) sucrose and left in this washing solution for 18 h at 4 °C. Sagittal 40-μmthick brain sections were obtained using a vibratome. The sections were cryoprotected by immersing them in a solution containing 25% (w/v) sucrose in 0.1 M phosphate buffer pH 7.4 and stored at −20 °C until the immunohistochemical studies were performed.

4.3.

Immunohistochemistry

Free-floating sections of both control and WIN exposed animal groups were simultaneously processed by immunohistochemistry for each marker. In order to inhibit endogenous peroxidase activity, tissue sections were previously dehydrated, treated with 0.5% (v/v) H2O2 in methanol for 30 min at room temperature and rehydrated. Unspecific binding sites were blocked by incubating the brain sections 1 h with 3% (v/v) normal goat serum in phosphate-buffered saline (PBS). After two rinses in PBS, the sections were incubated for 48 h at 4 °C with primary antibodies to Nf-160 diluted 1:2000, Nf-200 1:2000, MAP-2 1:1000, Syn 1:800 or GFAP 1:3000. Following five rinses in PBS, sections were incubated 1 h at room temperature with biotinylated secondary antibodies diluted 1:200. After further rinsing in PBS, sections were incubated for 1 h with a streptavidin–peroxidase complex solution diluted 1:400. After rinsing again five times in PBS and two times in 0.1 M acetate buffer pH 6.0 (AcB), development of peroxidase activity was carried out with 0.035% (w/v) 3,3′diaminobenzidine plus 2.5% (w/v) nickel ammonium sulfate and 0.1% (v/v) H2O2 dissolved in AcB. Sections were rinsed in AcB three times, once in distilled water, and then mounted on gelatin-coated slides, dehydrated and coverslipped using Permount for light microscopic observation. All the antibodies, as well as streptavidin complex, were dissolved in PBS containing 1% (v/v) normal goat serum and 0.3% (v/v) Triton X-100, pH 7.4.

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Fig. 12 – Electron microscopy photomicrographs of synapses in the hippocampus (A: control; B: Win-treated) and cerebellum (C: control; D: Win-treated). Primary magnifications: 30,000× (A and B); 40,000× (C and D). (E) Morphometrical evaluation of postsynaptical density thickness performed on digital 50,000× (primary magnification) photographs. The maximal longitude between the postsynaptic membrane and the postsynaptic complex was evaluated by tracing a line between them and evaluating the number of pixels. *P b 0.05 after two-tailed Student's t test. Bars represent mean ± SD.

4.4.

Morphometric analysis

In order to ensure objectivity, all measurements were performed on coded slides, blind conditions, by two observers for each experiment, carrying out the measures of control and treated sections simultaneously. The studied tissue sections were previously selected according to anatomical landmarks corresponding to the plates 83–85 of the Paxinos and Watson (1998) rat brain atlas. Each studied field in each tissue section was randomly selected within the limits of each anatomical area of interest to be morphometrically analyzed. Different parameters (mean gray, cell area and relative areas) were analyzed in an Axiophot Zeiss light microscope equipped with a video camera on-line with a Zeiss–Kontron VIDAS image analyzer. The images

were digitized into an array of 512 × 512 pixels corresponding to a tissue area of 140 × 140 μm (40× primary magnification). The resolution of each pixel was of 256 gray levels. Immunostained cytoskeleton area was obtained after a normalization of the digitized images and the selection of an interactive threshold. For the evaluation of cytoskeletal proteins, the total area of the immunolabeled cytoskeleton was related to the total area of the evaluated field, thus rendering a relative area parameter. In GFAP-immunoreactive astrocytes, the cell area was measured by interactively determining each cell's limits. Relative optical density (ROD) of Syn-immunostained structures was obtained after a transformation of mean gray values into ROD by using the formula: ROD = log (256 / mean gray). A background parameter was obtained from each section out of the labeled structures and

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subtracted to each cell ROD before statistically processing values. EM photographs were digitally processed to measure the postsynaptic density thickness in an Image Pro image analyzer. The postsynaptic density thickness was evaluated as the length of a perpendicular line traced from the postsynaptic membrane to the most convex part of the synaptic complex in 50,000× primary magnification EM photographs.

4.5.

Statistical analysis

Six to nine separate immunohistochemical experiments were run for each primary antibody. Individual experiments were composed of at least two to three tissue sections from each animal from each group. Ten to fourteen fields were analyzed for each brain region. Interexperimental differences were not statistically significant. The values represent the mean ± SD of experiments performed for each marker and treatment. The statistical analysis was performed by applying a two-tailed Student's t test to the results of the quantitative analysis and by assuming that data were normally distributed. The equality of variance for control and treated values was analyzed by an F test. For the same group, interanimal and interexperimental differences were not significant. Statistical significance was set to P b 0.05.

4.6.

Electron microscopy

Vibratome sections from the hippocampus and the cerebellum were selected according to a rat brain atlas (Paxinos and Watson, 1998) and transferred into phosphate buffer. Sections were postfixed in 1% osmium tetroxide in the same phosphate buffer for 30 min. Then, they were contrasted with uranyl acetate 5%, and after that sections were dehydrated in graded ethanol and embedded in Durcupan (Fluka AG, Chemische Fabrik, Buchs SG, Switzerland). Ultrathin sections were stained with lead citrate (standard method according to Reynolds, 1963) and then observed and photographed with a Zeiss 109 electron microscope.

Acknowledgments We thank Ms. Emérita Jorge Vilela de Bianchieri for her expert technical assistance. This work was supported by grants UBACYT M-031 (to AB) and M-072 (to AB). Image analysis, photomicroscopy and electron microscopy were carried out at the Laboratorio Nacional de Investigación y Servicios en Microscopía Electrónica (LANAIS-MIE) from the Universidad de Buenos Aires (UBA)-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). AB and AJR are researchers from CONICET (Argentina); SE and JL are fellows of the University of Buenos Aires (UBA).

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