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Infusion of 3
a
,5
a
-THP to the pontine reticular formation attenuates
1
PTZ-induced seizures
a ,
*
b cCheryl A. Frye
, Joaquin Manjarrez , Ignacio Camacho-Arroyo
a
Department of Psychology, Biological Sciences & The Neurobiology Research Center, The University at Albany-SUNY, 1400 Washington Avenue,
Albany, NY 12222, USA
b
Instituto Nacional de Neurologia y Neurocirugia MVS, Mexico, Mexico
c
Departamento de Biologia, Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Coyoacan 04510, D.F., Mexico,
Mexico
Accepted 31 August 2000
Abstract
Whether progesterone (P ) and its metabolite, 54 a-pregnan-3a-ol-20-one (3a,5a-THP) have anti-seizure effects through actions in the pontine reticular formation (PRF) was investigated. Concentrations of P and 34 a,5a-THP in the PRF were greater in proestrous and hormone-primed rats, that are typically more resistant to seizure-induction, than diestrous and males rats. Ovx, Long-Evans rats with unilateral microinjections into the PRF of 3a,5a-THP (5mg / 0.2ml), but not P (114 mg / 0.2ml) or vehicle (b-cyclodextrin), had a greater latency and lower incidence of tonic-clonic seizures induced by pentylenetetrazol (PTZ; 70 mg / kg, IP) administration. Infusions that missed the PRF were not effective. These data suggest 3a,5a-THP has anti-seizure effects in part through actions in the PRF. 2000 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Epilepsy: anti-convulsant drugs
Keywords: Progesterone; Allopreganollone; Tetrahydroprogesterone; Non-genomic; Neurosteroid; Pentylenetetrazol
The brain regions that are involved in progesterone’s ences in seizure susceptibility. Second, to compare P ’s4
(P ) and its 54 a-reduced metabolite, 5a-pregnan-3a-ol-20- and 3a,5a-THP’s ability to prevent seizures when infused one’s (3a,5a-THP) anti-seizure effects are unknown; into the PRF.
however, the pontine reticular formation (PRF) may be a Sexually mature female (n5126; Experiment 1; n540, site of action. The PRF is activated during seizures [4] and Experiment 2) or male (n542; Experiment 1) Long-Evans infusions of the g-aminobutyric acid (GABA)A receptor rats (Taconic Farms) were housed in polypropylene cages (GBR) antagonist bicuculline to the PRF has anti-convul- (25323320 cm). The rats were maintained on a 12 / 12 h sant effects [21]. reversed light cycle (lights off 8:00 am) with access to Progesterone has functionally relevant actions in the Purina Rat Chow and tap water in their home cages. The PRF. For example, infusions of P decrease the latency to4 estrous cycle of some females was determined by daily REM sleep in male and female rats [3]. Neurosteroids, lavage [7] for 2–3 cycles before inclusion in the experi-such as 3a,5a-THP, also have robust sleep-enhancing ment. Other rats had their ovaries removed (ovx) under effects [13]. The purpose of these experiments was Rhompun (12 mg / kg IP) and Ketaset (80 mg / kg IP) twofold. First, to examine progestin levels in the PRF in anesthesia and a week later were hormone-primed with hormonal and reproductive states associated with differ- estradiol benzoate (10mg SC in sesame oil at hour 0) and P (5004 mg SC in sesame oil at hour 44; tissues were collected 4 h later).
1
Published on the World Wide Web on 13 September 2000.
In Experiment 1, P and 34 a,5a-THP were measured by *Corresponding author. Tel.: 11-518-442-4836; fax: 1
1-518-442-radioimmunoassay [8] in PRF. Tissue from PRF of 6 rats 4247.
E-mail address: cafrye@cnsunix.albany.edu (C.A. Frye). of the same condition were obtained at 1000 and later
pooled for progestin measurement. There were 7 diestrous, 7 proestrous, 7 intact male, and 7 ovx hormone-primed pools. NB: Although the ovx rats were surgerized the cycling and male rats did not undergo sham surgery.
For Experiment 2, 40 rats were ovx and a week later were stereotaxically implanted with unilateral, 30 gauge, guide cannula aimed over the PRF (coordinates from bregma: AP:29.7, ML:60.8, DV29.0). Five days later rats were randomly assigned to be infused with 0.2 ml
b-cyclodextrin vehicle [9] (n513), P (114 mg; n513), or 3a,5a-THP (5 mg; n514), 30 min prior to pentylenetet-razol (PTZ; 70 mg / kg, IP) induction of seizures. Immedi-ately after PTZ, ictal behaviors were recorded for 1 h [5]. Ictal behavior, recorded while rats were in a plexiglas arena (26330350 cm), was based on the latency and incidence of myoclonus, characterized by facial twitching
Fig. 1. Mean 3a,5a-THP levels (ng / g6SEM) in tissue pools (n57 and forelimb clonis, and tonic clonic seizures, identified by
observations per group) from the rat pontine reticular formation. * rearing and a complete loss of balance. Rats from each
Indicates significant difference between tissues from ovariectomized group (n52–3 per group) were also implanted with estradiol (10mg) and P (500mg)-primed rats and diestrous and males
4
electrodes in the PRF and cortex so that we could monitor rats (P,0.05).[Indicates a significant difference between tissues from
proestrous and males rats (P,0.05). changes in seizures and electrical activity following steroid
infusions and PTZ.
Following testing all rats were infused with indigo blue
and then intracardially perfused with 0.9% saline and 10% infusions to the PRF had seizures, while seven of ten of the formalin. Fixed frozen brains were sliced at 40mM so that rats with P or vehicle to the PRF had tonic clonic seizures.4
infusion and electrode placement could be determined. Of Notably, the ten rats that had infusion sites in the dorsal the 40 rats that had infusions, 30 (n510 per group) were medial tegmental nucleus, that is those with ‘missed’ PRF localized to the PRF. The 10 rats that had infusions infusions, all had tonic clonic seizures.
localized to the dorsal medial tegmental nucleus were not 3a,5a-THP infusions to the PRF also tended to enhance included in the analyses because these infusions had no the latency to myoclonic seizures (F2, 2752.66, P50.08; impact on ictal activity. All rats with ‘missed’ infusions 75.169.7 s), versus that seen in vehicle (60.367.2 s) or P4
had tonic clonic seizures within 75 s of infusion irre- infused (48.466.8 s) rats; however, there were no differ-spective of whether vehicle (n53), P (n4 53), or 3a,5a- ences in incidence of myoclonus across groups.
THP (n54) was infused. Statistical analyses consisted of Electrographic recordings from the PRF and motor multiple one-way analysis of variances (ANOVAs) to cortex were examined from rats in each PRF infusion examine effects of hormone condition on progestin levels condition. Progesterone did not modify the electrographic or effects of PRF infusion condition on ictal activity. recording prior to PTZ administration but 3a,5a-THP Where appropriate, ANOVA’s that revealed significant reduced EEG. The latter markedly reduced ictal events differences at the P,0.05 level were followed by Dun- induced by PTZ. Fig. 3 shows a representative recording can’s post-hoc tests. during the most intense ictal activity after PTZ administra-Progesterone (F3,2459.734, P50.002) and 3a,5a-THP tion of rats previously infused with 3a,5a-THP, P , or4
(F3,2455.867, P50.003) concentrations in the PRF varied vehicle to the PRF. 3a,5a-THP infusions produced fewer significantly across groups. Although concentrations of P4 spikes in the PRF, compared to that observed in rats were very low in the PRF the P levels were highest in the4 infused with vehicle or P ; however, P infusions also4 4
hormone-primed (1.3810.20 ng / g) and proestrus (1.271 reduced epileptiform activity compared to vehicle. In the 0.24 ng / g).diestrus (0.4710.10 ng / g)5males (0.381 motor cortex both 3a,5a-THP and P reduced ictal activity4
0.06 ng / g). This same pattern was seen for 3a,5a-THP; (see Fig. 3).
anti-Fig. 2. Panel A represents latency (6SEM) to tonic clonic seizures following infusions to the pontine reticular formation of vehicle, P , or 34 a,5a-THP (n510 observations per group) and systemic pentylenetetrazol (70 mg / kg, IP). Panel B represents incidence (6SEM) of tonic clonic seizures following infusions to the pontine reticular formation of vehicle (open bars), P (stripped bars) or 34 a,5a-THP (black bars) and systemic pentylenetetrazol. * Indicates significant difference between 3a,5a-THP infusion compared to vehicle or P infusion (P4 ,0.05).
seizure effects in part through actions in the PRF. These potent than valium at modulating GBRs [20]. Anti-seizure findings are intriguing because of the importance of effects of some progestins like 3a,5a-THP, which can localizing the CNS site at which progestins may have directly activate the GABA-chloride channel [18], may be anti-seizure effects and also because of implications re- partially understood through its actions at GBRs.
garding the putative mechanism of 3a,5a-THP’s action. In order to elucidate the CNS substrates through which Traditionally P4 is understood to work via passive progestins have anti-seizure effects, it is important to diffusion into cells, binding with specific intracellular consider progestin metabolism because P and 34 a,5a-THP receptors in target cells to form a steroid receptor complex, typically have different mechanisms of action. Previous that in the nucleus binds to an acceptor site on the DNA, findings suggest that P has anti-seizure effects [12] but4
and enhances transcriptional and translational processes. that metabolites of P may mediate these effects. Systemic4
However, the actions of progestins at these intracellular administration of either P or 34 a,5a-THP prevent seizures; receptors do not fully account for their effects on seizure however, the latency for P ’s anti-convulsant effects are4
activity. There is a great disparity in affinity for intracellu- longer than 3a,5a-THP’s and more P than 34 a,5a-THP is lar progestin receptors between progestins, such as P and4 required to produce similar anti-seizure effects [10]. 3a,5a-THP [14]. That P has a high affinity for intracellu-4 Blocking P ’s metabolism to 34 a, 5a-THP attenuates its lar progestin receptors, and 3a,5a-THP is devoid of anti-seizure actions [11]. In people and rats, decreased activity at intracellular progestin receptors, and both can seizure susceptibility is better correlated with increases in have anti-seizure effects, suggest that P ’s and 34 a,5a- 3a,5a-THP than P4 [6,19]. In the present study P ’s4
THP’s effects on seizure threshold are not the sole metabolism to 3a,5a -THP in the PRF may have been consequence of actions at traditional intracellular progestin limited and precluded P ’s anti-seizure effects. The de-4
receptors. crease in ictal activity in the PRF and cortex following P4
Many anti-epileptic drugs used therapeutically have their infusions to the PRF suggest that P4 possesses some effects by increasing the activity of the primary inhibitory anticonvulsant effects. It is possible that the molecular neurochemical in the brain, GABA; progestins can also encapsulization vehicle or the 30 min latency may have enhance activity of this system through actions at GBRs. diminished P ’s conversion to 34 a,5a-THP and thereby Progestins effects on GBRs accounts for their rapid precluded P ’s anti-seizure effects. Another possibility is4
analgesic [9], anxiolytic [2], and anesthetic [1] actions, and there may not have been enough P4 to metabolize to may also underlie some of progestins’ anticonvulsant 3a,5a-THP.
Fig. 3. Representative electrographic recordings from the motor cortex and pontine reticular formation (PRF) from a rat in each condition during the most intense ictal activity. Note that commiserate with the lower ictal activity are fewer spikes in the PRF of the rat infused with 3a,5a-THP compared to the rats infused with vehicle or P .4
seizures [4,17]. Concentrations of 3a, 5a-THP were high References in the PRF of proestrous and hormone-primed rats that are
typically less susceptible to seizures. Infusions of 3a,5a- [1] D. Belelli, N.C. Lan, K.W. Gee, Anticonvulsant steroids and the GABA / benzodiazepine receptor-chloride ionophone complex, THP to the PRF markedly diminished the number of
Neurosci. Biobehav. Rev. 14 (1990) 315–322. animals with seizures, produced longer latencies and lower
[2] D. Bitran, M. Shiekh, M. McLeod, Anxiolytic effect of progesterone incidences of tonic clonic seizures than did vehicle or P4 is mediated by the neurosteroid allopregnanolone at brain GABA
A
infusions. Because of the important implications of these receptors, J. Neuroendocrinol. 7 (1995) 171–177.
findings, our laboratory will continue to examine the [3] I. Camacho-Arroyo, R. Hernandez-Gollas, J. Manjarrez, R. Al-varado, Progesterone microinjections into the pontine reticular mechanisms and role of progestins in the PRF to prevent
formation modify sleep in male and female rats, Neurosci. Lett. 269 ictal activity.
(1999) 9–12.
[4] Z. Elazar, A. Berchanski, Excitatory amino acids modulate epi-leptogenesis in the brain stem, Neuroreport 11 (2000) 1777–1780.
Acknowledgements [5] C.A. Frye, The neurosteroid 3a, 5a-THP has antiseizure and
possible neuroprotective effects in an animal model of epilepsy, Brain Res. 696 (1995) 113–120.
Supported by grants to CAF from the National Science
exogenous progesterone increases kainic acid and perforant pathway [16] M.D. Majewska, N.L. Harrison, R.D. Schwartz, J.L. Barker, S.M. induced seizures, Pharmacol. Biochem. Behav. 62 (1999) 315–322. Paul, Steroid hormone metabolites are barbiturate-like modulators of [8] C.A. Frye, L.E. Bayon, Mating stimuli influence endogenous the GABA receptor, Science 232 (1986) 1004–1007.
variations in the neurosteroids 3a, 5a-THP and 3a-Diol, J. Neuroen- [17] P. N’Gouemo, C.L. Faingold, Phenytoin administration reveals a docrinol. 11 (1999) 839–847. differential role of pontine reticular formation and periaqueductal [9] C.A. Frye, J.E. Duncan, Progesterone metabolites, effective at the gray neurons in generation of the convulsive behaviors of
au-GABAA receptor complex, attenuate pain sensitivity in rats, Brain diogenic seizures, Brain Res. 859 (2000) 311–317.
Res. 643 (1994) 194–203. [18] V.K. Patchev, M. Shoaib, F. Holsboer, O.F. Almeida, The neuro-[10] C.A. Frye, T.J. Scalise, Anti-seizure effects of progesterone and 3a, steroid tetrahydroprogesterone counteracts corticotropin-releasing 5a-THP on kainic acid and perforant pathway models of epilepsy, hormone induce anxiety and alerts the release and gene expression Psychoneuroendocrinology 25 (2000) 407–420. of corticotropin-releasing hormone in the rat hypothalamus, Neuro-[11] C.A. Frye, T.J. Scalise, L.E. Bayon, Finasteride blocks the reduction sci. 62 (1994) 265–271.
in ictal activity produced by exogenous estrous cyclicity, J Neuroen- [19] D. Rosciszewska, B. Buntner, I. Guz, L. Zawisza, Ovarian hor-docrinol. 10 (1998) 291–296. mones, anticonvulsant drugs, and seizures during the menstrual [12] A.G. Herzog, Progesterone therapy in women with complex partial cycle in women with epilepsy, J. Neurol. Neurosurg. Psych. 49
and secondary generalized seizures, Neurology 45 (1995) 1660– (1986) 47–51.
1662. [20] R.D. Schwartz, The GABA receptor-gated ion channel: biochemi-A
[13] F. Holsboer, K. Wiedemann, R. Rupprecht, A. Steiger, Effects of cal and pharmacological studies of structure and function, Biochem. corticosteroids and neurosteroids on sleep EEG, Clin. Neurophar- Pharmacol. 37 (1988) 3369–3375.
macol. 15 (1992) 588A–589A. [21] S. Shelab, J. Guadagno, K. Ferguson, P. Redgrave, Regional [14] S. Iswari, A.E. Colas, H.J. Karavolas, Binding of 5a-dihydroproges- distribution of the anticonvulsant and behavioural effects of bicucul-terone and other progestins to female rat anterior pituitary nuclear line injected into the pontine reticular formation of rats, Eur. J. extracts, Steroids 47 (1988) 189–203. Neurosci. 9 (1997) 1875–1884.
