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Keywords:

  • Seizures;
  • Flurothyl;
  • Pentylenetetrazole;
  • N-Methyl-d-aspartate;
  • Kainic acid

Summary

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Purpose:  Rapamycin (RAP) has certain antiepileptogenic features. However, it is unclear whether these effects can be explained by the anticonvulsant action of RAP, which has not been studied. To address this question, we tested potential anticonvulsant effects of RAP in immature and adult rats using different seizure models and treatment paradigms. In addition, we studied changes in the expression of neuropeptide Y (NPY) induced by RAP, which may serve as an indirect target of the RAP action.

Methods:  A complex approach was adopted to evaluate the anticonvulsant potential of RAP: We used flurothyl-, pentylenetetrazole (PTZ)–, N-methyl-d-aspartate (NMDA)–, and kainic acid (KA)–induced seizures to test the effects of RAP using different pretreatment protocols in immature and adult rats. We also evaluated expression of NPY within the primary motor cortex, hippocampal CA1, and dentate gyrus (DG) after different pretreatments with RAP in immature rats.

Key Findings:  We found the following: (1) RAP administered with short-term pretreatment paradigms has a weak anticonvulsant potential in the seizure models with compromised inhibition. (2) Lack of RAP efficacy correlates with decreased NPY expression in the cortex, CA1, and DG. Specifically in immature rats, a single dose of RAP (3 mg/kg) 4 or 24 h before seizure testing had anticonvulsant effects against PTZ-induced seizures. In the flurothyl seizure model only the 4-h pretreatment with RAP was anticonvulsant in the both age groups. Short-term pretreatments with RAP had no effects against NMDA- and KA-induced seizures tested in immature rats. Long-term pretreatments with RAP over 8 days did not show beneficial effect in all tested seizure models in developing rats. Moreover, the long-term pretreatment with RAP had a slight proconvulsant effect on KA-induced seizures. In immature rats, any lack of anticonvulsant effect (including proconvulsant effect of multiple doses of RAP) was associated with downregulation of NPY expression in the cortex and DG. In immature animals, after a single dose of RAP with 24 h delay, we found a decrease of NPY expression in DG, and CA1 as well.

Significance:  Our data show weak age-, treatment paradigm-, and model-specific anticonvulsant effects of RAP as well as loss of those effects after long-term RAP pretreatment associated with downregulation of NPY expression. These findings suggest that RAP is a poor anticonvulsant and may have beneficial effects only against epileptogenesis. In addition, our data present new insights into mechanisms of RAP action on seizures indicating a possible connection between mammalian target of rapamycin (mTOR) signaling and NPY system.

Recent attempts to link the serine/threonine kinase, mammalian target of rapamycin (mTOR) signaling to epileptogenesis with symptomatic origin seem promising. Syndromes (including those with epileptic complications) related to tissue pathologies such as tuberous sclerosis complex, cortical malformations, brain injuries, or cancers are directly or indirectly associated with impaired function in the mTOR signaling and respond to therapy with mTOR inhibitors (Rosner et al., 2008; Zeng et al., 2008, 2009; Huang et al., 2010; Krueger et al., 2010; Wong, 2010; Pitkanen & Lukasiuk, 2011; Raffo et al., 2011). Some studies show beneficial effects of mTOR inhibitor rapamycin (RAP) against epileptogenesis, as RAP treatment decreases status epilepticus (SE)–induced mossy fiber sprouting (Buckmaster et al., 2009; Zeng et al., 2009; Huang et al., 2010; McDaniel & Wong, 2011) with a subsequent decrease in the frequency of spontaneous seizures (Zeng et al., 2009; Huang et al., 2010). On the other hand, RAP-induced decrease of sprouting was not associated with changes in the occurrence of pilocarpine-induced spontaneous seizures in mice (Buckmaster & Lew, 2011). A recent study reported no impact of the long-lasting posttreatment with RAP on mossy-fiber sprouting or development of spontaneous seizures in the SE model of temporal lobe epilepsy induced by continuous stimulation of the amygdala in rats (Sliwa et al., 2011). Finally, we found that pretreatment with RAP does not affect development of NMDA-induced spasms in prenatally betamethasone-primed model of cryptogenic infantile spasms (Chachua et al., 2011).

Despite the fact that RAP has certain antiepileptogenic features in experimental genetic/acquired epilepsies (Wong, 2010; Pitkanen & Lukasiuk, 2011), there is still limited knowledge about whether the mTOR pathway is involved in direct modulation of seizures as a signaling pathway with anticonvulsant properties.

As a regulator of protein synthesis, mTOR pathway is involved in establishing protein-synthesis–dependent long-term potentiation (LTP) (Tang et al., 2002; Cammalleri et al., 2003; Tsokas et al., 2005) as well as in the metabotropic glutamate receptor–dependent long-term depression (LTD) (Huber et al., 2001, 2002; Hou & Klann, 2004; Sharma et al., 2010). RAP blocks both LTP and memory consolidation in mammals in a number of behavioral tasks (Tang et al., 2002; Tischmeyer et al., 2003; Dash et al., 2006; Stoica et al., 2011) without affecting basal neuronal transmission (Cammalleri et al., 2003; Ruegg et al., 2007). However, high-dose of RAP may induce a moderate increase in the field excitatory responses in the hippocampal CA1 region (Daoud et al., 2007), probably by activation of intracellular Ca2+ signaling mechanism (Terashima et al., 2000). These findings indicate that RAP may directly modulate seizures by affecting synaptic strength and efficacy. However, effects of RAP on acute seizures have not been yet studied.

Neuropeptide Y (NPY) is an endogenous peptide with anticonvulsant features against experimental seizures and a promising candidate for gene therapy of epilepsy (Baraban, 1998; Woldbye & Kokaia, 2004; Noe et al., 2007, 2009). Effects of NPY on neuronal transmission are complex, as it decreases presynaptic release of glutamate onto excitatory neurons as well as γ-aminobutyric acid (GABA) release onto GABAergic neurons in number of the brain areas (Chen & van den Pol, 1996; McQuiston & Colmers, 1996; van den Pol et al., 1996; Qian et al., 1997; Rhim et al., 1997; Sun et al., 2001a,b; Bacci et al., 2002). Recently, a concomitant increase in NPY mRNA and phosphorylated Akt (Ser473) has been reported after pilocarpine-induced SE in the rat hippocampi (Goto et al., 2010). Phospho-Akt is an essential upstream activator of mTORC1 through the inhibition of TSC1/TSC2. Akt per se is regulated by a negative feedback from mTORC1 downstream target, S6K1 (Laplante & Sabatini, 2009; Zoncu et al., 2011). In addition, there is strong crosstalk between mTOR signaling and NPY system in the hypothalamus (Cota et al., 2006). Therefore, NPY may serve as an indirect target of RAP action and contribute to its effects on seizures.

In the present study, we tested effects of RAP on flurothyl-, pentylenetetrazole (PTZ)–, N-methyl-d-aspartate (NMDA) – and kainic acid (KA) –induced seizures by using different pretreatment protocols in immature and adult rats. We also evaluated RAP-induced changes in NPY expression in the cortex and hippocampus as a possible target of RAP action on seizures.

Methods and Procedure

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Animals

Experiments have been approved by the Albert Einstein College of Medicine as well as New York Medical College Institutional Animal Care and Use Committees, and conform to the National Institutes of Health (NIH) Revised Guide for the Care and Use of Laboratory Animals. Sprague-Dawley male rats were used (Taconic Farms, Germantown, NY, U.S.A.). We tested immature male rats at postnatal day 15 (PN15; the day of birth counted as PN0) and young adult male rats between PN 55 and 60 (140–180 g of body weight). Animals were kept in the controlled environment of either the Albert Einstein College of Medicine or New York Medical College AAALAC-approved animal facilities with food and water ad libitum and 12 h light:12 h dark cycle with lights on at 07:00 h. Immature rats were housed in a cage with a foster dam (10 rats per dam); the adult animals were housed in groups of three per cage. All efforts were made to keep the number of animals to a minimum while keeping the results meaningful.

Rapamycin pretreatment design

Rapamycin (RAP; LC Laboratories, Woburn, MA, U.S.A.) was injected intraperitoneally (i.p.) in a dose of 3 mg/kg in PN15 rats and 3 or 6 mg/kg in the adult rats. The dose regimen was chosen according to the previous reports (Zeng et al., 2008, 2009). Because pretreatment with 3 mg/kg of RAP in PN15 rats significantly affected body weight, we did not use a higher dose of RAP for this age group. RAP was dissolved in 100% ethanol and administered as 1% ethanol solution in the final dilution for the 3 mg/kg dose or 2% ethanol for the 6 mg/kg dose. Controls received 1% or 2% ethanol vehicle, respectively. In P15 rats, RAP was injected using the following pretreatment paradigms: (1) A single injection 4 h before seizure testing; (2) a single injection 24 h before seizure testing; (3) three daily injections on PN12–14; (4) eight daily injections on PN7–14. A seizure test was always performed on P15. Adult rats were injected with a single dose of RAP either 4 or 24 h before seizure testing. We did not assess chronic pretreatment with RAP in adult rats because others reported no effect of chronic pretreatment on development of acute seizures (Zeng et al., 2009; Huang et al., 2010), and also we found generally unimpressive anticonvulsant effects of the acute dose and a complete lack of any anticonvulsant effects with 24-h pretreatment paradigm as well as with a higher dose of RAP in present study. According to the in vitro studies, RAP exerts its effect on synaptic function as quickly as within 1 h after application and initiates long-lasting changes in the function of neuronal network (Tang et al., 2002; Cammalleri et al., 2003; Tsokas et al., 2005). Based on these findings, we selected the 4-h pretreatment with RAP to study a possible anticonvulsant effect, which may involve mechanisms other than inhibition of the mTORC1 complex. The 24-h pretreatment was chosen to test the anticonvulsant effect of mTORC1 cascade blockade, and the long-lasting treatments were chosen to test the possible involvement of mTORC2 system blockade, in addition to mTORC1 (Sarbassov et al., 2006).

NPY immunohistochemistry

Brains were collected after the RAP pretreatment on PN15 without seizure testing. We tested the effects of RAP on NPY expression using only the following three pretreatment paradigms: A single 3 mg/kg dose of RAP administered (1) 4 or (2) 24 h before tissue collection or, (3) eight times daily injections of 3 mg/kg RAP. Animals were transcardially perfused with saline followed by the ice-cold 4% paraformaldehyde (PFA) and cryoprotected in 30% sucrose in phosphate-buffered saline (PBS; 0.01 M, pH – 7.3–7.4). Frozen sections of 40-μm thickness were cut sagittally by Leica cryostat and collected in PBS. At least three sections with the primary motor cortex (M1) ∼40–80 μm apart were taken per animal, starting at 1.9 mm laterally from the midline. M1 is a brain region involved in motor expression of seizures. Anti-NPY immunohistochemistry (IHC) experiments were done on the free-floating sections using the avidin–biotin peroxidase technique routinely used in our laboratory (Velíšková & Velíšek, 2007). Briefly, sections were processed in 1% H2O2 for 20 min and blocked with a solution of 0.6% bovine serum albumin (BSA), 10% normal goat serum (NGS), and 0.4% Triton 100 in PBS for 90 min. Sections were then incubated in anti-NPY rabbit antibody (1:2,000; Sigma-Aldrich, St. Louis, MO) overnight at +4°C. After we incubated in biotinylated anti-rabbit secondary antibody (1:500; Vector Laboratories, Burlingame, CA, U.S.A.) for 60 min at room temperature, NPY-immunopositive cells were visualized by 3′-3′-diaminobenzidine using the avidin–biotin horseradish peroxidase method (Vectastain ABC Elite kit; Vector Laboratories). Sections were mounted, dried, and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA, U.S.A.).

Cell counting

Sections were scanned using a Nikon upright microscope (E400) (Morrell Instruments, Melville, NY, U.S.A.), and the M1 area was captured by the Nikon digital camera under 10× magnification. M1 area pictures were constructed from individual images using Adobe PhotoShop CS3 (San Jose, CA, U.S.A.). In addition to M1, we also evaluated hippocampal CA1 and dentate gyrus (DG) from the same sections as areas of interest for additional possible RAP site of action (Ruegg et al., 2007). At least three sections per animal were used for evaluation. An investigator blinded to experimental condition performed the cell counting using the approach described in Ravizza et al. (2002). Briefly, the NPY immunopositive cells were counted in subfields of M1 and CA1, and the entire dentate gyrus (DG) for each section. The M1 subfield was outlined by a rectangle box (width/height = 345 μm/1207.5 μm) per section placed across all cortical layers in the middle of M1 area identified by the rat brain atlas (Paxinos & Watson, 2007) (Fig. 5B). In the CA1 region, three equal-size square boxes (width/height = 185/185 μm) were placed across strata oriens, pyramidale, and radiatum 60–125 μm apart from each other (Fig. S1D). Numbers of the NPY immunopositive cells from the three sections per each region of interest per animal were averaged. Because we were interested in changes of NPY expression as a function of RAP exposure and not in the total number of NPY immunopositive cells, mean number of NPY immunopositive cells from animals in the vehicle-injected group was considered as 100% and individual number of the NPY immunopositive cells per animal in control and treatment groups was expressed in percentage relative to the mean of controls.

Seizure models

If possible, seizure testing was done with operator blinded to experimental conditions. However, in chronic treatments with rapamycin, the significant weight decrease revealed the condition.

Flurothyl-induced seizures

Flurothyl is a volatile convulsant liquid, which after inhalation of vapors induces seizures starting with myoclonic jerks (twitches) followed by clonic seizures and culminating in tonic–clonic seizures (Velíšková et al., 1994). Flurothyl was infused in an airtight chamber (25/25/45 cm) at a constant rate of 40 μl/min. Flurothyl immediately evaporated and the animal breathed its vapors. Flurothyl administration continued until onset of tonic–clonic seizures. Afterward, the animal was removed from the chamber and the chamber was flushed with the vacuum and aerated before starting the next session. We measured the latency to onset of clonic and tonic–clonic seizures. Flurothyl threshold for seizures is expressed as the amount of flurothyl in microliters necessary to induce the first clonic and first tonic–clonic seizure (Velíšková & Moshé, 2001) regardless of body weight, since there is no correlation between the body weight and the effects of flurothyl (Lánský et al., 1997; Schwechter et al., 2003).

Pentylenetetrazole (PTZ)–induced seizures

PTZ induces myoclonic twitches, clonic seizures, and eventually tonic–clonic seizures. PTZ (dissolved in saline) was injected subcutaneously (sc) in a concentration of 100 mg/kg in PN15 and 120 mg/kg in adult rats (Velíšek et al., 1992). We determined latency to onset of the first clonic and the first tonic–clonic seizure.

NMDA-induced seizures

NMDA-induced seizures have a strong age-specific phenotype. With continuing postnatal development, the rat loses its sensitivity to NMDA, and very high doses of NMDA are required to elicit seizures. In addition, adult rats show less-consistent phenotype of NMDA-induced seizures with high mortality rate (Mareš & Velíšek, 1992). Excessive concentrations of excitatory amino acids (including NMDA) may lead to neuronal excitotoxicity (Sattler & Tymianski, 2001). Therefore, we decided to test only PN15 rat pups for NMDA-induced seizures. On PN15, NMDA-induced seizures start with initial tail shaking followed by repetitive flexion spasms (Mareš & Velíšek, 1992). We used 15 mg/kg of NMDA dissolved in saline injected i.p. as per previous studies (Mareš & Velíšek, 1992; Velíšek et al., 2007) and determined latency to onset of the flexion spasms as well as the number of flexion spasms during 70-min observation period.

Kainic acid (KA)–induced seizures

We tested only PN15 rats for KA-induced seizures, because the effects of RAP in adult rats have been reported previously (Zeng et al., 2009). At PN15, intraperitoneal administration of KA in a concentration of 5 mg/kg induces the following five stages of the motor seizures: (1) scratching, (2) unilateral forelimb clonus, followed by (3) bilateral forelimb clonus, later on (4) clonus of all limbs, and finally (5) tonic–clonic seizures (Velíšková et al., 1988). Latency to each stage of seizures was measured and compared between vehicle-injected and RAP-treated groups.

Statistical analysis

First, we tested normality of data by comparison to Gaussian distribution using the Kolmogorov-Smirnov test. Because the data were normally distributed with similar variance, the data were analyzed by Student’s t-test (two group comparisons). Multiple groups were compared using analysis of variance (ANOVA) with post hoc Fisher Protected Least Significant Difference (PLSD) test. Level of significance was preset to p < 0.05 and curtailed for multiple comparisons. The values are shown as mean ± standard error of the mean (SEM).

Results

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Effects of RAP pretreatment on body weight gain in PN15 rats

RAP pretreatment decreases the nutrient consumption as it inhibits the mTOR pathway (Cota et al., 2006; Sarbassov et al., 2006; Mori et al., 2009; Catania et al., 2011). At testing day (PN15), the RAP-treated animals were smaller than the vehicle-treated rats (Fig. 1A). Body weight gain was already decreased after a single administration of RAP 24 h prior to seizure testing (t-test p = 0.005; Fig. 1B). Long-lasting pretreatment with RAP (three or eight daily injections) dramatically affected body weight gain (t-test p < 0.001 for both groups; Fig. 1C,D). These data confirm that the 3 mg/kg dose of RAP was biologically effective.

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Figure 1.   Body weight gain after rapamycin in PN15 rats (mean ± SEM). (A) PN15 male rat on the testing day after pretreatment with eight doses of 3 mg/kg RAP (smaller) along with its vehicle-injected control (larger). (B) Gained body weight after the RAP pretreatment applied 24 h prior to seizure testing. This pretreatment significantly decreased the body weight gain compared to vehicle-treated controls (*p < 0.01). (C) Three doses of RAP were far more effective to decrease the body weight gain than a single injection; the actual body weight of RAP-treated animals at PN15 was much lower than that of the vehicle-injected control (*p < 0.0001). (D) The effect of RAP on body weight dramatically increased with eight daily doses of RAP. Values for the gained or actual body weights (at PN15) of RAP- and vehicle-injected rat pups clearly show how powerful this side effect of RAP can be (*p < 0.0001).

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Effects of RAP pretreatment on body weight in adult rats

A single pretreatment with RAP at 24 h prior to seizure testing significantly decreased the body weight gain possibly because of impaired feeding behavior (F2,46 = 13.045, ANOVA p < 0.001). The 6 mg/kg dose was far more effective than the 3 mg/kg dose (post hoc Fisher PLSD test p < 0.001 and p = 0.003 vs. control group, respectively). The actual body weights before and after each RAP pretreatment are given in the Table S1.

Effects of RAP pretreatment on seizures in PN15 rats

Flurothyl-induced seizures

A single 3 mg/kg dose of RAP (n = 8) 4 h prior to the flurothyl-induced seizure testing resulted in a trend to increased threshold (depicted in microliter of flurothyl) for clonic seizures compared to vehicle-treated controls (n = 7; t-test p = 0.089; Fig. 2A), whereas a threshold for tonic–clonic seizures was significantly elevated after this RAP pretreatment regimen (t-test p = 0.001; Fig. 2A). A single 3 mg/kg RAP dose 24 h prior to flurothyl-induced seizure testing (n = 9) did not affect the threshold for clonic seizures compared to vehicle-injected rats (n = 9; t-test p = 0.712). On the other hand, there was an apparent trend to increased threshold for tonic–clonic seizures (t-test p = 0.053; Fig. 2B).

image

Figure 2.   Effects of RAP pretreatment (3 mg/kg) on flurothyl- and PTZ-induced clonic and tonic–clonic seizures in PN15 rats (mean ± SEM). Upper panel: The amount of flurothyl required to induce clonic and tonic–clonic seizures in vehicle- and RAP-treated PN15 pups are shown (i.e., flurothyl threshold). (A) A single i.p. injection of RAP (3 mg/kg) administered 4 h prior to flurothyl infusion (n = 8) significantly increased threshold for flurothyl-induced tonic–clonic seizures compared to matching controls (n = 7;*p < 0.01). (B) A single i.p. injection of RAP 24 h prior to testing (n = 9) did not have anticonvulsant effects compared to control animals (n = 9). (C) RAP administered daily for 3 days (n = 6) was ineffective against the flurothyl-induced seizures compared to matching controls (n = 8). (D) Eight doses of RAP (n = 7) administered daily were also ineffective against flurothyl-induced seizures compared to controls (n = 6). Lower panel: Latency to onset of PTZ-induced clonic and tonic–clonic seizures in vehicle- and RAP-treated PN15 pups is shown. (E) A single i.p. injection of RAP (n = 7) 4 h prior to the PTZ s.c. injection, significantly delayed the onset of clonic as well as tonic–clonic seizures compared to control group (n = 6; *p < 0.05). (F) A single dose of RAP (n = 10) administered 24 h prior to PTZ-induced seizure testing showed anticonvulsant effect as well, as it increased the latency to onset of tonic-clonic seizures compared to matching controls (n = 9; *p < 0.01). (G) Unlike the acute pretreatments, daily i.p. injections of RAP for 3 days (n = 9) were ineffective against PTZ-induced seizures compared to controls (n = 7). (H) Similarly, there was no effect of a daily i.p. administration of total eight doses of RAP (n = 7) compared to controls (n = 7).

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Three daily doses of 3 mg/kg RAP (n = 6) did not have an effect on either the flurothyl-induced clonic (t-test p = 0.842) or tonic–clonic (t-test p = 0.718) seizures compared to control animals (n = 8; Fig. 2C). Similarly daily injections of 3 mg/kg RAP (n = 7) for 8 days did not affect the threshold for flurothyl-induced clonic as well as tonic–clonic seizures compared to vehicle-injected animals (n = 6; t-test p = 0.204 for clonic and t-test p = 0.145 for tonic-clonic seizures; Fig. 2D).

PTZ-induced seizures

In PN15 pups, a single 3 mg/kg dose of RAP administered 4 h prior to PTZ-induced seizure testing (n = 7) significantly delayed development of clonic seizures compared to vehicle-injected controls (n = 6; t-test p = 0.045; Fig. 2E). This pretreatment with RAP also increased the latency to PTZ-induced tonic–clonic seizures compared to control animals (t-test p = 0.017; Fig. 2E). A single dose of 3 mg/kg RAP administered 24 h prior to PTZ-induced seizure testing (n = 10) induced a trend to a delayed development of clonic seizures compared to control animals (n = 9; t-test p = 0.074; Fig. 2F). This pretreatment significantly delayed the onset of the PTZ-induced tonic–clonic seizures compared to vehicle-injected controls (t-test p = 0.007; Fig. 2F).

Three times daily injections of 3 mg/kg RAP (n = 9) were not effective against either PTZ-induced clonic or tonic–clonic seizures compared to control group (n = 7; t-test p = 0.338 for clonic, t-test p = 0.553 for tonic–clonic; Fig. 2G). Similarly, eight daily injections of 3 mg/kg RAP (n = 7) did not affect the development of either PTZ-induced clonic or tonic–clonic seizures compared to vehicle-injected animals (n = 7; t-test p = 0.311 for clonic, t-test p = 0.819 for tonic–clonic; Fig. 2H).

NMDA-induced seizures

In vehicle-injected PN15 pups (n = 6), ip. injection of NMDA-induced flexion spasms in about 17 min. A single injection of 3 mg/kg of RAP 4 h prior to NMDA challenge (n = 9) did not affect either the latency to onset of flexion spasms (t-test p = 0.214) or the number of the flexion spasms (t-test p = 0.722) compared to control group (Fig. 3A). Similarly a single pretreatment with 3 mg/kg RAP 24 h prior to NMDA injection (n = 6) did not affect the latency to onset of flexion spasms compared to control group (n = 5; t-test p = 0.681). This pretreatment was not also effective against the number of spasms (t-test p = 0.741; Fig. 3B).

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Figure 3.   Effects of RAP pretreatments (3 mg/kg) on NMDA- and KA-induced seizures in PN15 rats (mean ± SEM). Upper panel: Latency to onset of flexion spasms and number of flexion spasms in response to NMDA i.p. injection in vehicle- and RAP-treated PN15 rat pups is shown. (A) NMDA-induced flexion spasms were refractory to a single i.p. injection of RAP (n = 9) administered 4 h prior to NMDA challenge compared to matching controls (n = 6). (B) RAP injected 24 h prior to NMDA i.p. injection (n = 6) was ineffective against NMDA-induced motor seizures compared to controls (n = 5). (C) Eight times daily i.p. administration of RAP (n = 7) had no effect against NMDA-induced flexion spasms compared to controls (n = 5); Mean ± SEM. Lower panel: Latency to onset of different stages of the KA-induced motor seizures in response to KA i.p. injection in vehicle-injected and RAP-treated PN15 rat pups is shown. The observed stages of the KA-induced motor seizures are: scr, scratching; uniFlC, unilateral forelimb clonus; biFlC, bilateral forelimb clonus; clonus, clonus of all four limbs. (D) None of the grades of KA motor seizures responded to a single dose of RAP administered 4 h prior to the KA-induced seizure testing (n = 6) compared to vehicle-injected controls (n = 6). (E) A single dose of RAP injected 24 h prior to the KA-induced seizure testing (n = 8) did not affect the development of KA-induced motor seizures compared to the controls (n = 9). (F) Eight times daily injection of RAP (n = 5) revealed a slight proconvulsant effect against the KA seizures as it decreased the latency to the onset of the unilateral forelimb clonus compared to controls (n = 3; *p < 0.05).

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Following eight daily injections of vehicle, NMDA-induced flexion spasms developed in about 20 min (n = 5). Eight daily doses of RAP (n = 7) insignificantly decreased the latency to onset of flexion spasms compared to vehicle-injected rats (t-test p = 0.217). This long-term pretreatment compared to control group did not affect the number of flexion spasms either (t-test p = 0.921; Fig. 3C). Based on these observations we chose not to use the treatment paradigm with three daily RAP injections.

KA-induced seizures

In PN15 rats, none of the pretreatments with RAP had an anticonvulsant effect in KA-induced seizures. A single injection of 3 mg/kg RAP administered either 4 (n = 6) or 24 (n = 8) hours before induction of seizures had no effect compared to control groups (n = 6 and n = 9, respectively; Fig. 3D,E). Eight times daily pretreatment with 3 mg/kg RAP (n = 5) showed even a moderate proconvulsant effect on development of KA-induced seizures, as there was a trend to decreased latency to onset of scratching (t-test p = 0.089; Fig. 3F), and a significant acceleration of the onset of unilateral forelimb clonus (t-test p = 0.033; Fig. 3F) compared to vehicle-injected group (n = 3). Based on these observations, we chose not to test the effects of three daily RAP injections against the KA-induced seizures.

Effects of RAP pretreatment on seizures in adult rats

Flurothyl-induced seizures

None of the doses (3 or 6 mg/kg) of RAP 4 h prior to seizure testing was effective against flurothyl-induced clonic seizures (F2,20 = 0.08, ANOVA p = 0.924). On the other hand, ANOVA revealed a significant difference for tonic–clonic seizures between the groups at 4 h after the RAP pretreatment (F2,20 = 3.609, ANOVA p = 0.046). The post hoc test showed that only the 3 mg/kg dose of RAP (n = 7) increased the threshold of flurothyl-induced tonic–clonic convulsions compared to controls (n = 10; Fisher PLSD p = 0.020), whereas the 6 mg/kg of RAP had no effect (n = 6; Fisher PLSD p = 0.865; Fig. 4A). In contrast, neither 3 nor 6 mg/kg dose of RAP administered 24 h prior to flurothyl testing (n = 7 and n = 6, respectively) affected development of flurothyl-induced clonic seizures (F2,20 = 0.34, ANOVA p = 0.723; Fig. 4B), whereas ANOVA revealed a trend to decreased threshold of flurothyl-induced tonic–clonic seizures compared to vehicle-injected animals (n = 10; F2,20 = 2.97, ANOVA p = 0.074; Fig. 4B). The post hoc test indicated that this trend occurred only after the 6 mg/kg dose of RAP (Fisher PLSD p = 0.073; Fig. 4B).

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Figure 4.   Effects of RAP pretreatments (3 and 6 mg/kg) on flurothyl- and PTZ-induced seizures in young adult rats (mean ± SEM). The amount of flurothyl required to induce clonic and tonic–clonic motor seizures (flurothyl threshold; the upper panel) and the latency to onset of PTZ-induced clonic and tonic–clonic seizures (the lower panel) in the vehicle-injected and RAP-treated young adults are shown. (A) A single 3 mg/kg dose of RAP administered 4 h prior to seizure testing (n = 7) had anticonvulsant effects against flurothyl-induced tonic–clonic seizures compared to matching controls (n = 10; *p < 0.05). Pretreatment with 6 mg/kg of RAP (n = 6) was not effective. (B) Flurothyl-induced motor seizures were not modified by pretreatment with either dose (3 or 6 mg/kg) of RAP applied 24 h prior to flurothyl exposure (n = 7 and n = 6, respectively) compared to controls (n = 10). (C) Similarly, neither dose (3 or 6 mg/kg) of RAP administered 4 h prior to PTZ-induced seizure testing was effective against motor seizures (n = 7 and n = 5, respectively) compared to the control group (n = 6). (D) A single 3 or 6 mg/kg dose of RAP administered 24 h prior to PTZ challenge (n = 5 and n = 6, respectively) was ineffective against PTZ-induced seizures compared to vehicle-injected controls (n = 8).

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PTZ-induced seizures

In adult rats, both 3 mg/kg (n = 7) or 6 mg/kg dose (n = 5) of RAP administered 4 h prior to the PTZ seizure testing were ineffective against clonic seizures compared to vehicle-injected animals (n = 6; F2,15 = 1.839, ANOVA p = 0.193; Fig. 4C). Similarly, neither dose of RAP (3 or 6 mg/kg) affected development of PTZ-induced tonic–clonic seizures compared to control rats (F2,15 = 0.963, ANOVA p = 0.403; Fig. 4C). Administration of 3 mg/kg (n = 5) or 6 mg/kg (n = 6) dose of RAP 24 h prior to PTZ-induced seizure testing did not affect the onset of clonic (F2,16 = 0.643, ANOVA p = 0.539; Fig. 4D) or tonic–clonic seizures (F2,16 = 0.321, ANOVA p = 0.732; Fig. 4D) compared to vehicle-injected rats (n = 8).

Effects of RAP on NPY expression in PN15 rats

Because we found that the immature rat is more responsive to the RAP pretreatment than the adult rat in terms of the seizure susceptibility (Table S2), we tested the effects of RAP on NPY expression only in PN15 rats.

A single pretreatment with 3 mg/kg RAP delivered either 4 or 24 h prior to brain harvesting (without seizure induction) did not influence the NPY expression in the M1 area. The RAP-treated animals showed a similar number of NPY immunopositive cells in M1 (n = 2 for 4 and n = 4 for 24 h delay groups; t-test p = 0.664 and p = 0.373, respectively) as vehicle-injected controls (n = 2 for 4 and n = 4 for 24 h delay groups; Fig. 5A). Because the statistical analysis did not reveal any trend of difference between the RAP and vehicle groups with 4 h delay, we did not increase the sample numbers for these groups.

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Figure 5.   Effect of RAP pretreatments (3 mg/kg) on the NPY expression in the M1 area of PN15 rats. (A) The number of the NPY immunopositive cells (in % of controls) in M1 after different RAP pretreatments regimens (mean ± SEM) – 4 h prior (n = 2), 24 h prior (n = 4) and eight times daily injections (n = 4) together with matching vehicle-injected controls (n = 2 and n = 4 and n = 3, respectively). Eight times daily injection of RAP significantly decreased number of the NPY-immunopositive cells in the M1 area compared to controls (*p < 0.05). (B) A sagittal section (2.4 mm lateral from the midline) stained with cresyl-violet depicting the area of the primary motor cortex (M1), where the counts for NPY were performed (boxed area). Scale – 1 mm. (C,D) Photomicrographs of NPY-immunopositive cells in the M1 subfield in a PN15 control rat and a PN15 rat injected daily with 3 mg/kg of RAP over 8 days. Scale – 100 μm. Some of the NPY-immunopositive cells are marked with arrows. Note the large number of cells in controls, whereas there are only few cells after long-term RAP treatment.

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On the other hand, eight daily injections of 3 mg/kg RAP (n = 4) significantly decreased the number of NPY-immunopositive cells in the M1 area compared to the control group (n = 3; t-test p = 0.038; Fig. 5A,C,D). This decrease was not associated with any neuronal loss as evaluated using Nissl (cresyl violet)–stained sections (data not shown).

A single pretreatment with 3 mg/kg RAP delivered 24 h prior (n = 4) to brain collection (without seizure induction) significantly decreased the number of the NPY-immunopositive cells in hippocampal CA1 as well as in DG compared to the control group (n = 4; t-test p = 0.012 and 0.036 respectively; Fig. S1B,C). Similarly, there was a significant reduction in number of NPY-immunopositive cells in the DG region after eight daily injections of 3 mg/kg RAP (n = 4) compared to the control group (n = 5; t-test p = 0.029; Fig. 1C). The same eight daily injections of 3 mg/kg RAP (n = 4) did not affect NPY expression in the CA1 region compared to the control group (n = 5; Fig. S1B) receiving eight daily injections of vehicle. Of interest, there was a trend to a decrease of number of NPY-immunopositive cells in CA1 after eight daily injections of vehicle itself compared to the other control group receiving a single injection of vehicle (t-test p = 0.078; Fig. S1D).

Discussion

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Our study evaluated the anticonvulsant potential of mTOR pathway inhibitor, RAP, using the models of acute seizures in immature and adult rats. Table S2 summarizes our data showing an overall better response of immature rats than of adult rats to the RAP pretreatment. In immature rats, the flurothyl seizure threshold was increased following the 4-h pretreatment with RAP, whereas PTZ seizure onset was delayed after the 4- as well as 24-h pretreatments with RAP. Only the 4-h RAP pretreatment paradigm was effective against flurothyl seizures in adult rats. Long-term pretreatment paradigms with RAP in immature rats were ineffective or RAP had a slight proconvulsant effect. Of interest, the loss of efficacy or the proconvulsant effect of RAP correlated with a decrease in NPY expression in the primary motor cortex as well as in the hippocampal DG of immature rats.

Present data show that RAP has a weak model-specific anticonvulsant potential. We used two different approaches to induce the acute seizures: (1) blocking inhibition (flurothyl and PTZ models) and (2) facilitating excitation (NMDA and KA models). RAP was somewhat effective after a single-dose administration only in those models with impaired inhibition. This finding is consistent with the study showing decreased burst firing as well as reduced overall network excitability after RAP treatment of the bicuculline-bathed rat hippocampal cultures; RAP did not affect network excitability under control conditions (Ruegg et al., 2007). This phenomenon might be worthy of further investigation.

In immature rats, RAP exerted its anticonvulsant effect only after single-dose pretreatments (4 or 24 h prior to seizure testing), whereas a chronic administration of the same dose of RAP over 3 or 8 days did not provide any anticonvulsant effect. Therefore, these data suggest involvement of different mechanisms in the RAP action after a single acute and repeated long-term administration.

Our study shows that RAP, depending on pretreatment paradigm, differentially targets NPY system in terms of region-specific decrease in the expression of NPY, a powerful endogenous anticonvulsant neuropeptide (Woldbye & Kokaia, 2004). NPY expression in the M1 area was decreased after eight daily pretreatments with RAP correlating with a loss of anticonvulsant efficacy of RAP. A single dose of RAP administered 24 h prior to testing selectively decreased NPY expression in hippocampal CA1 and DG regions, which may explain inefficacy of RAP in KA seizures with an origin in the limbic structures.

There is no known direct connection between the mTOR pathway and NPY system; however, interaction between mTOR signaling and the NPY system in the hypothalamus has been reported in association with regulation of feeding behavior (Cota et al., 2006). After pilocarpine-induced SE in rats, concomitant increases in the hippocampal expressions of phosphorylated Akt (Ser473), mTOR pathway activator, and NPY mRNA have been reported (Goto et al., 2010). Because blockade of Akt phosphorylation decreases the expression of NPY mRNA in human neuroblastoma cells (Kurihara et al., 2000), Akt may serve as a bridge between mTOR pathway and NPY system.

We found a dramatic decline in body weight as well as in the brain weight (data not shown) after RAP pretreatment, especially following repeated administration. The TORC1 complex is involved in energy metabolism and represents the “fuel-sensing” machinery in cells, which is regulated by hormones and nutrient levels (branched-chain amino acids) (Catania et al., 2011). Insulin and leptin- as well as leucine-induced decrease in food intake are mediated by activation of the mTORC1 complex, which is cancelled/blunted by RAP (Cota et al., 2006; Plum et al., 2006). In the Tsc1 knockout mice but not in wild-type mice, long-lasting treatment with RAP effectively decreases brain weight and the ratio of brain/body weight (Zeng et al., 2008; Mori et al., 2009). Our studies clearly demonstrate the negative effects of inactivated mTOR pathway on body/brain weights, which may involve central as well as peripheral mechanisms of inactivated mTOR signaling (Catania et al., 2011) and can lead to malnutrition, which could further affect seizure susceptibility in our experiments. However, no correlation between the malnutrition-related decreased body weight and the threshold for flurothyl-induced seizures has been found previously (Nunes et al., 2000).

In conclusion, the presented data suggest that RAP has only a weak and seizure type-specific anticonvulsant potential, which could be related to RAP-mediated negative regulation of the NPY system. Reports that RAP administration impairs ovarian function, slows follicle growth and leads to oocyte loss (Thomson et al., 2010; McLaughlin et al., 2011), plus our findings of limited anticonvulsant potency of RAP in adults, may argue against the use of RAP as an anticonvulsant agent, particularly in female patients.

Acknowledgment

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

We appreciate excellent technical assistances of Edward Bergin and Thomas Azeizat. The study was supported in part by the March of Dimes grant #6-FY08-591 and by NIH grants NS-059504, NS-056093, and NS-072966.

Disclosure

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

None of the authors has any conflict of interest to disclose. We, the authors, confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Methods and Procedure
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure
  8. References
  9. Supporting Information

Figure S1. Effects of RAP pretreatment (3 mg/kg) on the NPY expression in hippocampal CA1 and DG of PN15 rats.

Table S1. Body weights of adult rats before and after RAP pretreatment administered 24 h prior to seizure testing.

Table S2. Summarized effects of RAP pretreatments against seizures.

FilenameFormatSizeDescription
EPI_3674_sm_FigureS1.tif656KSupporting info item
EPI_3674_sm_TableS1.doc35KSupporting info item
EPI_3674_sm_TableS2.doc63KSupporting info item

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