Dendritic Targeting of mRNAs for Plasticity Genes in Experimental Models of Temporal Lobe Epilepsy

Epilepsia, 43(Suppl. 5):153–158, 2002 Blackwell Publishing, Inc. © International League Against Epilepsy

Dendritic Targeting of mRNAs for Plasticity Genes in Experimental Models of Temporal Lobe Epilepsy *M. Simonato, *G. Bregola, †M. Armellin, †P. Del Piccolo, *D. Rodi, *S. Zucchini, and †E. Tongiorgi *Department of Clinical and Experimental Medicine, Section of Pharmacology and Neuroscience Center, University of Ferrara, Ferrara; and †BRAIN Centre for Neuroscience, Department of Biology, University of Trieste, Trieste, Italy

Summary: Purpose: To analyze whether the subcellular localization of the messenger RNAs (mRNAs) coding for the neurotrophin brain-derived neurotrophic factor (BDNF), its receptor TrkB, and the ␣ and ␤ subunits of calcium-calmodulin– dependent kinase II (CaMKII) are modified after pilocarpine and kindled seizures. Methods: Epilepsy models: pilocarpine and kindling. Analysis of mRNA levels in the dendrites: high-resolution, nonradioactive in situ hybridization. Results: Nonstimulated rats: BDNF, TrkB, and CaMKII-␤ mRNAs localized in the soma and in the proximal dendrites of hippocampal pyramidal cells, and in the soma only of dentate gyrus (DG) granule cells; CaMKII-␣ mRNA localized throughout the dendritic length in neurons of all hippocampal subfields. Pilocarpine seizures: increased staining levels of CaMKII-␣ mRNA throughout the whole dendritic length in all hippocampal subfields; induction of CaMKII-␤, BDNF, and TrkB

mRNAs dendritic targeting in CA1, CA3, and DG neurons. Class 2 kindled seizures: increase in dendritic staining intensity for CaMKII-␣ in CA1, CA3, and DG neurons; induction of dendritic localization of CaMKII-␤, BDNF, and TrkB mRNAs in CA3 neurons. Fully kindled seizures: no change in the subcellular distribution of BDNF, TrkB and CaMKII-␤ mRNAs; reduction of CaMKII-␣ mRNA dendritic staining, as compared with unstimulated kindled animals. Conclusions: Data provide evidence that BDNF, TrkB, and CaMKII-␣ and -␤ mRNAs are accumulated in the dendrites of specific hippocampal neurons during pilocarpine seizures and kindling development. The dendritic targeting of these genes may be causally involved in epileptogenesis and thus may represent a new therapeutic target for some forms of partial epilepsy. Key Words: Pilocarpine—Kindling—Brain-derived neurotrophic factor—Calcium-calmodulin–dependent kinase II—mRNA dendritic targeting.

Epileptogenesis is associated with extensive reorganization of synaptic connections and complex changes in gene expression (1,2). Research in animal models of epilepsy has identified several plasticity genes whose messenger RNA (mRNA) and protein expression levels change in response to seizures. Among these genes, calcium-calmodulin kinase II (CaMKII) and brainderived neurotrophic factor (BDNF), with its receptor TrkB, are involved in the control of hippocampal plasticity (3–5) and are thought to play an important role in epileptogenesis (6,7). Targeting of mRNA to dendrites with subsequent local protein synthesis at activated synapses is considered a key mechanism for a fine regulation of synaptic plasticity (8–10). The mRNAs for BDNF and TrkB (11) and for the ␣ subunit of CaMKII (12) can be targeted to the

dendrites, where they can be locally translated into proteins in response to electrical activity (11,13,14). Much is known about the regulation of expression of these mRNAs: several lines of evidence indicate that, in both animal models and patients, a chronic state of epilepsy is associated with increased levels of BDNF and TrkB, and lowered levels of CaMKII-␣ mRNA (15–18). At present, however, the dendritic localization of these mRNAs during epileptogenesis is still unknown. The aim of this study was to evaluate the subcellular localization of the mRNAs coding for BDNF, its receptor TrkB, and the ␣- and ␤-subunits of CaMKII in the pilocarpine and kindling models of epilepsy. METHODS Preparation of the animals Male Sprague–Dawley rats were used for all experiments. Procedures involving animals and their care were carried out in accordance with European Community and national laws and policies. All efforts were made to mini-

Address correspondence and reprint requests to Dr. M. Simonato at Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy. E-mail: [email protected]

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mize animal suffering and to reduce the number of animals used. For pilocarpine experiments, animals were administered pilocarpine (340 mg/kg i.p.) and killed at different times thereafter (3, 6, and 24 h). For kindling, a twisted bipolar electrode was implanted in the right amygdala (coordinates: 4.8 mm lateral and 0.8 mm posterior to bregma, 8.3 mm deep from dura) under halothane anesthesia. Animals were allowed 7 days to recover. Beginning the following day, rats were stimulated once daily with a single 1-s train of bipolar pulses [1 ms, 60 Hz, intensity 25% above afterdischarge (AD) threshold]. Behavior [staging according to (19)] and the duration of the AD measured in the right amygdala were recorded after each stimulation. A group of animals was killed 3 h after the first class 2 seizure. Kindling criteria were three consecutive class 4 or 5 seizures. One week after reaching these criteria, kindled rats were killed either without any further stimulus or after a stimulation evoking a class 4–5 seizure. Sham-stimulated rats were aged-matched littermates, which had undergone surgery and daily handling but had not been stimulated. In situ hybridization Rats were transcardially perfused with 4% paraformaldehyde under ketamine anesthesia, and their brains were removed and kept in 4% paraformaldehyde/20% sucrose at 4°C for ⱖ3 days before sectioning. All probes were prepared from cDNA inserts cloned in a pBluescript plasmid. After linearization of the plasmid, digoxigenin-labeled riboprobes were synthesized with a SP6-T7 DIG-RNA labeling kit (La Roche, Mannheim, Germany), according to the manufacturer’s instructions. In situ hybridization was performed as previously described (20). In brief, free-floating, 40-␮m coronal sections at the level of the dorsal hippocampus were postfixed in paraformaldehyde, 4%, washed in phosphatebuffered saline (PBS), treated with proteinase K, and washed again in PBS. They were then prehybridized at 55°C for 60–90 min, with a mixture containing 50% deionized formamide, 20 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 300 mM NaCl, 100 mM dithiothreitol (DTT), 1× Denhart’s solution, 0.5 mg/ml ssDNA, 0.5 mg/ml polyadenylic acid (all reagents from Sigma-Aldrich, Milan, Italy). In situ hybridization was performed overnight at 55°C in the prehybridization mix, to which 10% dextran sulphate and the riboprobe (50– 100 ng/ml) were added. After hybridization, highstringency washes were performed in 0.1× SSC/0.1% Tween-20 at 60°C. Sections hybridized with digoxigeninlabeled riboprobes were incubated overnight at 4°C with anti-DIG Fab fragments coupled to alkaline phosphatase (La Roche), diluted 1:500 in 10% fetal calf serum. Sections were then washed 4 times in PBS, and reacted with 4-nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylEpilepsia, Vol. 43, Suppl. 5, 2002

phosphate (both reagents from La Roche) in 100 mM Tris-HCl, 50 mM MgCl2, 100 mM NaCl, and 1 mM levamisol. To obtain reproducible and comparable results and to avoid saturation of the reaction, alkaline phosphatase development was always performed for 4 h at room temperature. Finally, sections were mounted on gelatin-coated slides, dried for 30 min at 55°C, rinsed in methanol for 30 s, in methanol/xylene (1:1) for 30 s, in xylene for 3 min, and then coverslips were mounted with DPX-mountant for histology. RESULTS Control animals In control rats, intense labeling for CaMKII-␣ mRNA was found on the soma of hippocampal pyramidal cells and of dentate gyrus (DG) granule cells; furthermore, moderate labeling also was found throughout the dendritic length in neurons of all hippocampal subfields (Figs. 1A and 2A). In contrast, CaMKII-␤ (Figs. 1C and 2D), BDNF (Figs. 1E and 2G), and TrkB mRNAs (Figs. 1G and 2J) were localized in the soma and in the proximal dendrites of hippocampal pyramidal cells, and in the soma only of DG granule cells. Pilocarpine seizures Rats began experiencing generalized seizures and entered into status epilepticus 16 ± 3 min after pilocarpine administration (340 mg/kg, i.p.). Three hours after pilocarpine administration, a robust increase in the dendritic levels of CaMKII-␣ mRNA was observed in all hippocampal subfields (Fig. 1B). This effect gradually declined in the following hours. Resetting to pretreatment levels was observed within 24 h in CA1 and DG, but not in CA3, dendrites (data not shown). Furthermore, pilocarpine induced CaMKII-␤ (Fig. 1D), BDNF (Fig. 1F), and TrkB (Fig. 1H) mRNA targeting to the distal part of the dendrites (ⱖ100–200 ␮m from the cell soma) of CA1 and CA3 neurons and to the proximal third of the dendrites of DG neurons. These events were not unspecific, in that growth-associated protein 43 (GAP43) mRNA, whose subcellular distribution under normal conditions overlaps those of CaMKII␤, BDNF, and TrkB mRNAs, did not undergo dendritic targeting after pilocarpine seizures (data not shown). Kindling A group of rats was killed 3 h after the first class 2 kindled seizure. This seizure class was reached after 4 ± 1 stimulations (cumulative AD duration, 164 ± 27 s, last AD duration, 55 ± 12 s). With respect to controls (Fig. 2A, D, G, and J for CaMKII-␣ CaMKII-␤, BDNF, and TrkB, respectively), class 2 seizures induced an increase in dendritic localization of CaMKII-␣ mRNA in CA1, CA3, and DG neurons (Fig. 2B), as well as dendritic localization of CaMKII-␤ (Fig. 2E), BDNF (Fig. 2H),

DENDRITIC TARGETING OF BDNF AND CAMKII mRNAS

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FIG. 1. Nonradioactive in situ hybridization for calcium-calmodulin–dependent kinase II (CaMKII)-␣ (A, B), CaMKII-␤ (C, D), brainderived neurotrophic factor (BDNF) (E, F), and TrkB (G, H) messenger RNAs under control conditions (A, C, E, G) and 3 h after injection of pilocarpine (340 mg/kg, i.p.). Representative sections taken across the dorsal hippocampus.

and TrkB (Fig. 2K) mRNAs in CA3 pyramidal neurons. These effects were more pronounced ipsilateral to stimulation. Another group of rats was stimulated until reaching kindling criteria (three consecutive class 4 or 5 seizures), and then killed 1 week later, either without any further stimulation or 3 h after a new stimulus-evoked seizure. Kindling criteria were reached after 12 ± 1 stimulations (cumulative AD duration, 644 ± 47 s). Kindled rats (left

unstimulated for a week) maintained a pattern of CaMKII-␣ mRNA expression overlapping the one described for class 2 seizures (i.e., increase in staining intensity in the soma of CA3 neurons and in the dendrites of CA1, CA3, and DG neurons). In contrast, the anatomic pattern of expression of CaMKII-␤, BDNF, and TrkB mRNAs in kindled rats was identical to the one observed in control and sham-stimulated rats (data not shown). Epilepsia, Vol. 43, Suppl. 5, 2002

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FIG. 2. Nonradioactive in situ hybridization for calcium-calmodulin–dependent kinase II (CaMKII)-␣ (A–C), CaMKII-␤ (D–F), brainderived neurotrophic factor (BDNF) (G–I), and TrkB (J–L) messenger RNAs under control conditions (A, D, G, J), 3 h after a class 2 kindled seizure (B, E, H, K), and 3 h after a class 5 fully kindled seizure (C, F, I, L). Representative sections taken across the dorsal hippocampus.

A new stimulation in kindled animals evoked a class 5 seizure (AD duration, 79 ± 8 s). Despite dramatically increasing the staining for BDNF and TrkB mRNAs in pyramidal and DG granule cell somas, and moderately increasing that for CaMKII-␣ and -␤, fully kindled seizures did not affect the subcellular distribution of these mRNAs with respect to the corresponding controls (Fig. 2C, I, and L). DISCUSSION The increased dendritic localization of BDNF, TrkB, CaMKII-␣, and CaMKII-␤ mRNAs reported in this study suggests that, under conditions of epileptogenesis, there is potential for an increased local, dendritic translation into proteins of these mRNAs. This event should take place diffusely, in all hippocampal subfields, during the first hours of pilocarpine seizures, and more focally Epilepsia, Vol. 43, Suppl. 5, 2002

in other instances: for example, in the mossy fiber–CA3 synaptic area at the early stages (class 2) of kindling development for BDNF, TrkB, and CaMKII-␤ mRNAs. In fully kindled animals, however, we observed a return to control levels in the dendritic staining for BDNF, TrkB, and CaMKII-␤ mRNAs; furthermore, fully kindled seizures produced a reduction in dendritic CaMKII-␣ mRNA, especially in CA3 apical dendrites (stratum lucidum). These findings provide circumstantial evidence for a possible involvement of mRNA dendritic targeting in hippocampal plasticity during epileptogenesis. BDNF and TrkB Consistent with these observations, BDNF mRNA can be transported to the dendrites in vitro (11), and BDNF protein can be synthesized and secreted (11,21,22) in an

DENDRITIC TARGETING OF BDNF AND CAMKII mRNAS activity-dependent manner (i.e., under conditions of intense synaptic activation, like those of epileptogenesis). One possible functional implication of these events is that locally synthesized BDNF and TrkB are involved in synaptic potentiation. Consistent with this idea, (a) BDNF can enhance synaptic transmission in the hippocampus in a long-lasting (23) and local, dendritic protein synthesis–dependent (24) manner; (b) BDNF facilitates kindling development (25,26); and (c) one particularly potentiated synapse in kindled animals is the one between mossy fibers and CA3 pyramidal cell (27). In contrast to this idea, however, other authors reported that exogenously administered BDNF retards kindling development (28,29). One possible explanation for this discrepancy may be that the regulation of expression (30) and subcellular targeting of different BDNF mRNA splice variants may differ, and thus BDNF may serve different functions when produced in the dendrites during epileptogenesis (synaptic potentiation?) as compared with that when produced in the soma after generalized seizures (neuroprotection?). Another possibility is that intraventricular administration of exogenous BDNF may interfere with kindling development by enhancing inhibitory neurotransmitter release (31) or by increasing ␥-aminobutyric acid (GABA)-receptor expression or differentiation of inhibitory synapses (31,32). Another interesting observation is that, in keeping with previous reports (33), BDNF and TrkB mRNAs appear to be regulated in a coordinated manner, in that their expression pattern is similar in all of the experimental conditions we tested. One implication is that BDNF may exert autocrine effects. Activation of TrkB receptors in the CA3 stratum lucidum has been reported to occur 24 h after partial kindled seizures (34). This observation is in striking anatomic and chronologic coincidence with our data on the dendritic targeting of BDNF and TrkB mRNAs 3 h after class 2 kindled seizures. Because BDNF protein also can be synthesized in the soma of granule cells and transported along the mossy fibers after seizures (35), the interesting possibility may be postulated that postsynaptic (on CA3 pyramidal cell dendrites) TrkB receptors will be activated by BDNF secreted by both the pre- and the postsynaptic element. CaMKII The pattern of subcellular localization of CaMKII-␣ and ␤ mRNAs reported in this study also suggests that increased dendritic accumulation may lead to enhanced local dendritic translation into proteins at early stages of epileptogenesis (pilocarpine-induced status epilepticus and class 2 kindled seizures). Furthermore, the different pattern of dendritic targeting of the mRNAs for different subunits suggests that specific changes in enzyme activity will take place in different dendritic fields and phases

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of epileptogenesis. In fact, for the ␣-subunit mRNA, increased dendritic translation into protein should take place diffusely, in all hippocampal subfields, during pilocarpine seizures and during early phases of kindling development. In contrast, seizures in fully kindled animals are followed by a reduction in CaMKII-␣ mRNA levels in the dendrites. For the ␤-subunit mRNA, local translation may occur more focally: for example, selectively in the mossy fiber–CA3 synaptic area during kindling development. Notably, this is the first demonstration that CaMKII-␤ mRNA can be targeted to dendrites. This hypothesis is in line with previous reports of increased CaMKII activity in hippocampal homogenates (36) and autophosphorylation in neuronal cell bodies and dendrites (37) in kindled animals, but is in contrast with a reported decrease in CaMKII protein levels in septally kindled rats (38). However, specific focus on the dendritic component should be made to provide valid comparisons with the present data. The functional implications of these observations are difficult to define. Because CaMKII facilitates long-term potentiation (LTP) (39,40), it is tempting to speculate that its local protein synthesis plays a role in synaptic potentiation, as suggested earlier for the BDNF–TrkB system. However, CaMKII-␣ may actually retard epileptogenesis (6,41). Nonetheless, our data stress the notion that the dendritic, as opposed to the somatic, synthesis of proteins have different mechanisms of regulation, and likely, different functional implications. CONCLUSIONS These data provide evidence that BDNF, TrkB, and CaMKII-␣ and -␤ mRNAs are accumulated in the dendrites of specific hippocampal neurons during pilocarpine seizures and kindling development. Activation of both CaMKII and TrkB is required for the downstream transcription of genes involved in synaptic plasticity (24,42), including BDNF itself (43); furthermore, BDNF can regulate the dendritic targeting of its own and of TrkB mRNAs (44). It can be suggested that the dendritic targeting of plasticity genes may be causally involved in synaptic potentiation associated with epileptogenesis and thus may represent a new therapeutic target for some forms of partial epilepsy. Acknowledgment: This study was supported by a grant from Telethon Italia (project E.0954).

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