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Fos in locus coeruleus neurons following audiogenic seizure in the genetically epilepsy-prone rat: comparison to electroshock and pentylenetetrazol seizure models
Neuroscience Letters 233 (1997) 21–24
Fos in locus coeruleus neurons following audiogenic seizure in the genetically epilepsy-prone rat: comparison to electroshock and pentylenetetrazol seizure models Jeffrey B. Eells a, Rich W. Clough b ,*, Ronald A. Browning a, Phillip C. Jobe c a Department of Physiology, Southern Illinois University School of Medicine at Carbondale, Carbondale, IL 62901, USA Department of Anatomy, Southern Illinois University School of Medicine-Carbondale, Carbondale, IL 629001-6523, USA c Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA b
Received 23 July 1997; received in revised form 8 August 1997; accepted 8 August 1997
Abstract Seizures in genetically epilepsy-prone rats (GEPRs) may result from hypoactivity of locus coeruleus (LC) neurons during seizures. This study examined Fos-like-immunoreactivity (FLI) in the LC following audiogenic seizures in two strains of GEPRs (GEPR-9s and -3s), and following pentylenetetrazol (PTZ) or maximal electroshock seizures (MES) in normal rats. After tonic seizure, GEPR-9s showed an identical LC-FLI response to that of normal rats following tonic seizures induced by either PTZ or MES. GEPR-3s, having clonic seizures, had less FLI in the LC. Therefore, stimulus-transcription coupling in the GEPR LC is apparently normo-typic in its FLI response to seizure and thus is not likely the root cause of NE abnormalities in this seizure model. 1997 Elsevier Science Ireland Ltd. Keywords: Epilepsy; Norepinephrine; Immediate early gene expression; c-Fos
Genetically epilepsy-prone rats (GEPRs) exhibit audiogenic seizures (AGS) in response to acoustic stimulation (AS). Two strains of GEPRs have been developed: GEPR3s display AS-induced clonic-seizures whereas GEPR-9s display severe tonic-seizures culminating in tonic hindlimb extension. Deficiencies in the norepinephrine (NE) neurotransmitter system serve as determinants of seizure severity in GEPR-9s and GEPR-3s [9]. Depletion of brain NE also exacerbates seizures in maximal electroshock (MES) [3], excitant amino acid [5], cobalt [17] and pentylenetetrazol (PTZ)-induced seizures [8], and promotes the development of kindling seizures [12]. Also, as is true in GEPRs, augmentation of NE activity or stimulation of the locus coeruleus (LC) has seizure-suppressant effects in non-genetic models of epilepsy such as kindling [1], penicillin [14], metrazol [15] and MES [3]-induced seizures. GEPRs have an innate deficiency in NE and, as a result, exhibit reflex epilepsy that can be alleviated with NE augmentation [6].
* Corresponding author. Tel.: +1 618 4531578; fax: +1 618 4531527; e-mail: [email protected]
Whether the innate problem is within perikarya of NE neurons or in their terminals in target nuclei is not known. We have shown that seizures in GEPR-9s are paralleled by the rapid accumulation of the proto-oncoprotein Fos in several distinct regions of the brain including the LC [7]. However, since there exists a widespread deficiency in NE in GEPR brains and the LC innervates the majority of the neuraxis with NE, neurons in the LC of GEPRs may show a reduced activation pattern of Fos expression following AGS when compared to phenotypically similar seizures induced in control animals by other means (i.e. MES and PTZ). Reduced Fos activation in the LC following AGS in GEPRs would be indicative of reduced genomic activity in these neurons in general and may help explain the overall reduced integrity of the NE system in these animals. Alternatively, if LC perikarya show a comparatively similar pattern of Fos expression across seizure models, this would support an alternate hypothesis that the NE deficiencies described in target regions of LC neurons may be attributable to deficiencies in NE terminals per se. Seizure-naive (snGEPR-9s; never having experienced an AGS) and seizure-experienced female GEPR-9s and GEPR-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940 (97 )0 0611- 3
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3s were obtained from the colony at the University of Illinois College of Medicine-Peoria (Peoria, IL, USA). Female Sprague–Dawley rats (controls) were obtained from Harlan Labs (Indianapolis, IN, USA). Rats were housed (3/cage) in a vivarium with a 12:12 h light/dark cycle with free access to food and water. On the day of the experiment, individual snGEPR-9s (n = 4), GEPR-9s (n = 4), GEPR-3s (n = 4) or AS control rats (n = 4) were placed in a cylindrical Plexiglas chamber of 40 cm diameter and exposed for 60 s (or until onset of seizure) to AS of approximately 110 dB intensity. Severity-score 9 seizures were characterized by a brief (approximately 0–2 s) wild-running phase culminating in a generalized tonic-convulsion with complete hindlimb extension. Severity-score 3 seizures were characterized by a brief running episode culminating in a generalized clonic activity of all limbs. Additional control groups consisted of normal Sprague–Dawley rats (n = 3) and GEPR-9s (n = 3) not exposed to AS. Two additional brainstem-evoked seizure models were prepared: MES seizures were produced in Sprague–Dawley rats (n = 3) by transcorneal stimulation (150 mA, 60 Hz, 0.2 s), and high-dose PTZ-induced seizures were induced in Sprague–Dawley rats (n = 2) by administration of PTZ (50 mg/kg, i.p., in saline). Seizure severity following MES or PTZ was evaluated as described by Browning et al. [4], using the following rating scale: A, clonus only, without loss of posture; B, clonus with loss of posture; C, forelimb extension (tonic) with hindlimb flexion; D, forelimb extension (tonic) and partial hindlimb extension; E, full tonic hindlimb extension. Two and onehalf hours following AGS, MES, or PTZ-seizure, each rat was deeply anesthetized (pentobarbital sodium, 150 mg/kg,
Fig. 1. Immunohistochemistry of Fos in the locus coeruleus (LC). (A) Shows the typically minimal FLI in an acoustically-stimulated control rat 2.5 h following acoustic stimulation. (B) Shows the typically robust FLI in the LC of a GEPR-9, 2.5 h following tonic-seizure. A strikingly similar pattern was observed in all other tonic-seizure groups (see Fig. 2). scp, superior cerebellar peduncle; arrows, neurons of the mesencephalic trigeminal nucleus; brackets delineate the LC and immunoreactivity is evident over the nuclei of LC neurons (punctata); *, fourth ventricle.
Fig. 2. Mean ( ± SEM) numerical volume density of locus coeruleus (LC) neurons showing Fos-like-immunoreactivity (FLI). Acoustically stimulated control rats did not experience seizures but did show a moderate amount of FLI in the LC. GEPR-3 experienced moderate clonic-seizures and a significantly greater number of FLI neurons within the LC than the AS controls (P , 0.05). All the remaining groups experienced more severe seizures characterized by tonic components and exhibited robust FLI within the LC. Tonic-seizure groups (GEPR-9s, MES and PTZ treated rats) did not differ between one another with regard to induction of FLI in the LC but each was higher than the GEPR-3 group (P , 0.01, except PTZ, see text). Numbers within the columns represent the number of animals per group. Different letters denote significant differences (ANOVA).
i.p.) and transcardially perfused with cold phosphate-buffered-saline (PBS; 0.1 M, pH 7.4, 40 ml) followed by icecold 4% paraformaldehyde (150 ml) in PBS. Brains were removed, post-fixed in the same fixative for 90 min, placed in 25% sucrose for 48 h (4°C), frozen-sectioned and processed for Fos immunohistochemistry as previously described [7]. The primary antiserum (5 mg/ml) was rabbit polyclonal anti-Fos raised against amino acid residues 4–17 (AB-2, Oncogene Sci.). Immunohistochemical controls consisted of sections processed with either omission of the primary or secondary antibodies, or sections processed with the negative antibody control provided by Oncogene Sci. (staining was absent in these controls). The microscope slides were coded by an unbiased third party in order that they be analyzed blind to the treatment groups. The numerical area density (NA = number of FLI nuclei/mm2 area) of FLI profiles was determined using a stereological method in which the image of each LC section was magnified and projected by a drawing tube attachment onto a grid of known and magnification-corrected dimensions. The mean number of stained nuclei within several randomly-selected representative areas of the LC was tabulated to obtain the NA. From the NA data, the numerical volume density (NV = number of FLI nuclei/mm3) was calculated for the LC as previously detailed [7]. The number of sections/LC/rat were from 6 to 14 and the LC were quantified bilaterally. It
J.B. Eells et al. / Neuroscience Letters 233 (1997) 21–24
should be noted that the calculated NV is not comparable to the absolute number of neurons in the LC and, thus, this number is not comparable to previous published reports of total neuron number in this nucleus. Rather, the NV is the number of stained neurons per a standardized unit volume of 1 mm3. The standardized numbers are unbiased and comparable between groups irrespective of possible differences in shape or size of the LC. After all quantifications were complete, data from each rat was decoded, grouped into appropriate groups and subjected to statistical analysis. Due to the observation that no FLI profiles were found in any brain area of GEPRs or control rats not subjected to the AS, and, only two rats comprised the PTZ-treated group, these animals were not included in the comparisons of FLI numerical densities. Therefore, statistical comparisons were made by one-way analysis of variance (ANOVA) with a post-hoc Sheffe test between AS-control rats, AS-GEPR9s, AS-snGEPR-9s, AS-GEPR-3s, and MES rats. Each GEPR-9 had a score-9 tonic-seizure whereas each GEPR-3 had a score-3 clonic-seizure in response to AS. None of the control Sprague–Dawley rats exposed to the AS experienced a seizure; however, they did show an acoustic-startle response to the AS followed immediately by normal exploratory and grooming behavior. MES-treated animals experienced tonic-seizures of class E (n = 2) or class C (n = 1) whereas PTZ-treated rats showed eventual tonic-seizures of class E after progression from class A. The final-severity phenotype of the MES, high-dose PTZ and GEPR-9 seizures were similar in that they were severe tonic-seizures. Fig. 1 shows representative tissue sections through the LC of an AS-control rat (Fig. 1A) and a snGEPR-9 (Fig. 1B). The FLI staining patterns of the LC of snGEPR-9s, GEPR-9s, MES-treated, and PTZ-treated animals were very similar and thus are not differentially shown. Numerical densities of FLI neurons in all groups are shown in Fig. 2. Untreated control rats and GEPRs not subjected to the AS showed virtually no FLI in the LC (or other brain areas). In contrast, an appreciable number of FLI neurons were found within the LC of AS-control rats as well as snGEPR-9s, GEPR-9s and GEPR-3s following AS; however, the number of FLI neurons in all GEPR groups were significantly higher than that found in the AS-control rats. Moreover, the number of FLI neurons in the GEPR-9 groups were significantly higher than that of the GEPR-3s. Also shown in Fig. 2, MES treatment induced a significant increase in the number of FLI profiles in the LC over that of the AS-control animals and GEPR-3s but similar to that of the GEPR-9 groups. Tonic seizures resulting from highdose PTZ-administration appeared to induce similar FLI in the LC as the other tonic seizure groups however this was not included in the statistical comparisons. Even so, the large and significant increase in FLI neuron number in the LC following generalized tonic-seizures appears equally manifest across three distinct generalized tonic-seizure models (audiogenic, chemical, and electroshock). Alternatively, generalized but comparatively moderate clonic-sei-
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zures, as exhibited by GEPR-3s, are associated with fewer FLI LC neurons than tonic-seizure models. It is well documented that GEPRs have an innate and global deficiency in NE and that this deficiency is a partial determinant of seizure severity in these animals [9]. Indeed, NE has been found to be seizure-modulatory in a wide variety of seizure models and is usually reported as an endogenous anticonvulsant [1,3,5,8,12,14,15,17]. The deficiencies in the NE system in GEPRs [9] have been characterized in terminal regions that are void of noradrenergic perikarya. It is unknown whether these deficiencies result from a generalized lack of NE perikaryal activity coincident with seizure. Thus, to assess perikaryal activity, we examined the elaboration of Fos in association with AGS in these animals and compared their responses to phenotypically-similar seizures induced by other means. In this study, the AS-control animals showed an impressive induction of FLI in the LC (approximately 6900 LC neurons/mm3) that may be indicative of a mild stress related to acoustic startle. GEPR-3s experienced clonic-seizures subsequent to AS and displayed at least three times as many FLI neurons in the LC (approximately 19 400/mm3) than did the AS-control rats. It was tempting to speculate that seizures in GEPRs may reflect an aberrant (i.e. seizure-prone) acoustic startle response; however, although there is some overlap in neural circuitry, the brainstem pathways which mediate acoustic startle [10] and audiogenic seizures [2] have distinct features. Most impressive was the degree of FLI following generalized tonic-seizures. In GEPR-9s and snGEPR-9s subjected to AS, as well as in MES- and PTZ-treated rats, approximately six times as many in FLI neurons were found in the LC (approximately 35 000/mm3) compared to the AS-controls and nearly twice as many as that found in the GEPR-3s. The observations of increased FLI neuron number in the more severe types of tonic-seizures (GEPR-9s, MES, and highdose PTZ-seizures) compared to comparatively moderate clonic-seizures (GEPR-3 seizures) suggests that seizure severity rather than inductive stimulus is positively correlated to FLI numerical density in the LC. Questions remain as to the interpretation of increased immediate-early gene expression after seizures. For example, it could be hypothesized that the LC is proconvulsant in each of these seizure models in light of the presumed increased activity (FLI) of the neurons therein. Alternatively, the LC may become activated in an attempt to terminate or recover from the seizure as may be expected based on the myriad of data that shows NE to be anticonvulsant, especially in the GEPR [9]. Although the profiles of NE release in the brain coincident with seizures are not presently known for the GEPR, extracellular accumulation NE in the thalamus of GEPRs following uptake inhibition, although appreciable, is significantly reduced compared to non-epileptic controls [18]. These findings suggest that synaptic release of NE from terminals of LC neurons, coincident with seizure, is deficient in the GEPR. Further support of the hypothesis of deficient NE terminals in GEPRs, as opposed to deficient NE neurons
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in general, are the findings of Lauterborn and Ribak [11] who showed diminished numbers of NE axons within the inferior colliculus of the GEPR-9. The present data show that LC neurons appear normo-typic in their cellular profile of FLI coincident with or subsequent to seizure and seem to support a notion that LC perikarya receive appropriate afferent signals sufficient to induce elaboration of Fos. Since no differences in LC-Fos induction could be found between various tonic-seizure models, it appears that stimulus-transcription coupling [13] of LC neurons is not altered in GEPRs. These findings support the hypothesis that LC neuron perikaryal responses to afferent signals are not altered in GEPRs, particularly in their FLI responsiveness to seizures, and that the innate problems with the NE neurotransmitter system reside within NE terminals in various target regions. Another possibility may be a deficiency in excitation-secretion coupling of GEPR LC neurons; however, this possibility has not been examined. Interestingly, only one other published report has examined the LC per se in the GEPR [16] and that study found a modest decrease in alpha-2 adrenergic receptor binding sites within this nucleus of GEPR-9s and GEPR-3s. It is theoretically possible that the described diminution in alpha-2 adrenergic receptors in the LC of GEPR-9s may contribute to increased expression of Fos subsequent to seizure; however, one would expect differences between the GEPR-9s and the animals of other tonic-seizure models that, presumably, have normal complements of intra-LC alpha-2 adrenergic receptor binding sites. In summary, the present data shows that perikarya of the LC appear to be equally responsive in the induction of FLI coincident with seizures between three distinct generalized tonic-seizure models. Therefore, these data suggest that the deficiencies in norepinephrine in the GEPR model of epilepsy are not likely due to diminished activity within the LC. The authors are grateful for the assistance of Hari Ailinani and Amanda Solomon who were precollege apprentices in the laboratory of R.W.C. during performance of this research. Supported by a SIU program project grant funded by Bayer Pharmaceuticals. [1] Applegate, C.D., Konkol, R.J. and Burchfiel, J.L., Kindling antagonism: a role for hindbrain norepinephrine in the development of site suppression following concurrent, alternate stimulation, Brain Res., 407 (1987) 212–217. [2] Browning, R.A., Neuroanatomical localization of structures responsible for seizures in the GEPR: lesion studies, Life Sci., 39 (1986) 857–867.
[3] Browning, R.A., The role of neurotransmitters in electroshock seizure models. In P.C. Jobe and H.E. Laird II (Eds.), Neurotransmitters and Epilepsy, Humana Press, Totowa, NJ, 1987, pp. 277–320. [4] Browning, R.A., Turner, F.J., Simonton, R.L. and Bundman, M.C., Effect of midbrain and pontine tegmental lesions on the maxi0mal electroshock seizure pattern in rats, Epilepsia, 22 (1981) 583– 594. [5] Browning, R.A., Wang, C. and Faingold, C.L., Effect of norepinephrine depletion on audiogenic-like seizures elicited by microinfusion of an excitant amino acid into the inferior colliculus of normal rats, Exp. Neurol., 112 (1991) 200–205. [6] Clough, R.W., Browning, R.A., Maring, M.L., Statnick, M.A., Wang, C. and Jobe, P.C., Effects of intraventricular locus coeruleus transplants on seizure severity in genetically epilepsy-prone rats following depletion of brain norepinephrine, J. Neural Trans. Plast., 5 (1994) 65–79. [7] Clough, R.W., Eells, J.B., Browning, R.A. and Jobe, P.C., Seizures and proto-oncogene expression of Fos in the brain of adult genetically epilepsy-prone rats (GEPRs), Exp. Neurol., 146 (1997) 341– 353. [8] Corcoran, M.E., Fibiger, H.C., McCaughran, J.A. and Wada, J.A., Potentiation of amygdaloid kindling and metazol-induced seizures by 6-hydroxydopamine in rats, Exp. Neurol., 45 (1974) 118–133. [9] Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E. and Jobe, P.C., Noradrenergic abnormalities in the central nervous system of seizure-naive genetically epilepsy-prone rats, Epilepsia, 32 (1991) 168–173. [10] Davis, M., Gendelman, D.S., Tischler, M. and Gendelman, P.M., A primary acoustic startle circuit: lesion and stimulation studies, J. Neurosci., 2 (1982) 791–805. [11] Lauterborn, J.C. and Ribak, C.E., Differences in dopamine-betahydroxylase immunoreactivity between the brains of genetically epilepsy-prone and Sprague–Dawley rats, Epilepsy Res., 4 (1989) 161– 176. [12] McIntyre, D.C. and Edson, N., Facilitation of amygdala kindling after norepinephrine depletion with 6-hydroxydopamine in rats, Exp. Neurol., 74 (1981) 748–757. [13] Morgan, J.I. and Curran, T., Stimulus-transcription coupling in neurons: role of cellular immediate-early genes, Trends Neurosci., 12 (1989) 459–462. [14] Neuman, R.S., Suppression of penicillin-induced focal epileptiform activity by locus ceruleus stimulation: mediation by an alpha 1adrenoceptor, Epilepsia, 27 (1986) 359–366. [15] Papanicolaou, J., Summers, R.J., Vajda, F.J.E. and Louis, W.J., Anticonvulsant effects of clonidine mediated through central alpha2 adrenoceptors, Eur. J. Pharmacol., 77 (1882) 163–166. [16] Razani-Boroujerdi, S., Tso-Olivas, D.Y., Hoffman, T.J., Weiss, G.K. and Savage, D.D., Decrease in locus coeruleus [3H]idazoxan binding site density in genetically epilepsy-prone (GEPR) rats, Brain Res., 600 (1993) 181–186. [17] Trottier, S., Lindvall, O., Chauvel, P. and Bjorklund, A., Facilitation of focal cobalt-induced epilepsy after lesions of the noradrenergic locus coeruleus system, Brain Res., 454 (1988) 308–314. [18] Yan, Q.-S., Jobe, P.C. and Dailey, J.W., Thalamic deficiency in norepinephrine release detected via intracerebral microdialysis: a synaptic determinant of seizure predisposition in the genetically epilepsy-prone rat, Epilepsy Res., 14 (1993) 229–236.
Fos in locus coeruleus neurons following audiogenic seizure in the genetically epilepsy-prone rat: comparison to electroshock and pentylenetetrazol seizure models Jeffrey B. Eells a, Rich W. Clough b ,*, Ronald A. Browning a, Phillip C. Jobe c a Department of Physiology, Southern Illinois University School of Medicine at Carbondale, Carbondale, IL 62901, USA Department of Anatomy, Southern Illinois University School of Medicine-Carbondale, Carbondale, IL 629001-6523, USA c Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA b
Received 23 July 1997; received in revised form 8 August 1997; accepted 8 August 1997
Abstract Seizures in genetically epilepsy-prone rats (GEPRs) may result from hypoactivity of locus coeruleus (LC) neurons during seizures. This study examined Fos-like-immunoreactivity (FLI) in the LC following audiogenic seizures in two strains of GEPRs (GEPR-9s and -3s), and following pentylenetetrazol (PTZ) or maximal electroshock seizures (MES) in normal rats. After tonic seizure, GEPR-9s showed an identical LC-FLI response to that of normal rats following tonic seizures induced by either PTZ or MES. GEPR-3s, having clonic seizures, had less FLI in the LC. Therefore, stimulus-transcription coupling in the GEPR LC is apparently normo-typic in its FLI response to seizure and thus is not likely the root cause of NE abnormalities in this seizure model. 1997 Elsevier Science Ireland Ltd. Keywords: Epilepsy; Norepinephrine; Immediate early gene expression; c-Fos
Genetically epilepsy-prone rats (GEPRs) exhibit audiogenic seizures (AGS) in response to acoustic stimulation (AS). Two strains of GEPRs have been developed: GEPR3s display AS-induced clonic-seizures whereas GEPR-9s display severe tonic-seizures culminating in tonic hindlimb extension. Deficiencies in the norepinephrine (NE) neurotransmitter system serve as determinants of seizure severity in GEPR-9s and GEPR-3s [9]. Depletion of brain NE also exacerbates seizures in maximal electroshock (MES) [3], excitant amino acid [5], cobalt [17] and pentylenetetrazol (PTZ)-induced seizures [8], and promotes the development of kindling seizures [12]. Also, as is true in GEPRs, augmentation of NE activity or stimulation of the locus coeruleus (LC) has seizure-suppressant effects in non-genetic models of epilepsy such as kindling [1], penicillin [14], metrazol [15] and MES [3]-induced seizures. GEPRs have an innate deficiency in NE and, as a result, exhibit reflex epilepsy that can be alleviated with NE augmentation [6].
* Corresponding author. Tel.: +1 618 4531578; fax: +1 618 4531527; e-mail: [email protected]
Whether the innate problem is within perikarya of NE neurons or in their terminals in target nuclei is not known. We have shown that seizures in GEPR-9s are paralleled by the rapid accumulation of the proto-oncoprotein Fos in several distinct regions of the brain including the LC [7]. However, since there exists a widespread deficiency in NE in GEPR brains and the LC innervates the majority of the neuraxis with NE, neurons in the LC of GEPRs may show a reduced activation pattern of Fos expression following AGS when compared to phenotypically similar seizures induced in control animals by other means (i.e. MES and PTZ). Reduced Fos activation in the LC following AGS in GEPRs would be indicative of reduced genomic activity in these neurons in general and may help explain the overall reduced integrity of the NE system in these animals. Alternatively, if LC perikarya show a comparatively similar pattern of Fos expression across seizure models, this would support an alternate hypothesis that the NE deficiencies described in target regions of LC neurons may be attributable to deficiencies in NE terminals per se. Seizure-naive (snGEPR-9s; never having experienced an AGS) and seizure-experienced female GEPR-9s and GEPR-
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940 (97 )0 0611- 3
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3s were obtained from the colony at the University of Illinois College of Medicine-Peoria (Peoria, IL, USA). Female Sprague–Dawley rats (controls) were obtained from Harlan Labs (Indianapolis, IN, USA). Rats were housed (3/cage) in a vivarium with a 12:12 h light/dark cycle with free access to food and water. On the day of the experiment, individual snGEPR-9s (n = 4), GEPR-9s (n = 4), GEPR-3s (n = 4) or AS control rats (n = 4) were placed in a cylindrical Plexiglas chamber of 40 cm diameter and exposed for 60 s (or until onset of seizure) to AS of approximately 110 dB intensity. Severity-score 9 seizures were characterized by a brief (approximately 0–2 s) wild-running phase culminating in a generalized tonic-convulsion with complete hindlimb extension. Severity-score 3 seizures were characterized by a brief running episode culminating in a generalized clonic activity of all limbs. Additional control groups consisted of normal Sprague–Dawley rats (n = 3) and GEPR-9s (n = 3) not exposed to AS. Two additional brainstem-evoked seizure models were prepared: MES seizures were produced in Sprague–Dawley rats (n = 3) by transcorneal stimulation (150 mA, 60 Hz, 0.2 s), and high-dose PTZ-induced seizures were induced in Sprague–Dawley rats (n = 2) by administration of PTZ (50 mg/kg, i.p., in saline). Seizure severity following MES or PTZ was evaluated as described by Browning et al. [4], using the following rating scale: A, clonus only, without loss of posture; B, clonus with loss of posture; C, forelimb extension (tonic) with hindlimb flexion; D, forelimb extension (tonic) and partial hindlimb extension; E, full tonic hindlimb extension. Two and onehalf hours following AGS, MES, or PTZ-seizure, each rat was deeply anesthetized (pentobarbital sodium, 150 mg/kg,
Fig. 1. Immunohistochemistry of Fos in the locus coeruleus (LC). (A) Shows the typically minimal FLI in an acoustically-stimulated control rat 2.5 h following acoustic stimulation. (B) Shows the typically robust FLI in the LC of a GEPR-9, 2.5 h following tonic-seizure. A strikingly similar pattern was observed in all other tonic-seizure groups (see Fig. 2). scp, superior cerebellar peduncle; arrows, neurons of the mesencephalic trigeminal nucleus; brackets delineate the LC and immunoreactivity is evident over the nuclei of LC neurons (punctata); *, fourth ventricle.
Fig. 2. Mean ( ± SEM) numerical volume density of locus coeruleus (LC) neurons showing Fos-like-immunoreactivity (FLI). Acoustically stimulated control rats did not experience seizures but did show a moderate amount of FLI in the LC. GEPR-3 experienced moderate clonic-seizures and a significantly greater number of FLI neurons within the LC than the AS controls (P , 0.05). All the remaining groups experienced more severe seizures characterized by tonic components and exhibited robust FLI within the LC. Tonic-seizure groups (GEPR-9s, MES and PTZ treated rats) did not differ between one another with regard to induction of FLI in the LC but each was higher than the GEPR-3 group (P , 0.01, except PTZ, see text). Numbers within the columns represent the number of animals per group. Different letters denote significant differences (ANOVA).
i.p.) and transcardially perfused with cold phosphate-buffered-saline (PBS; 0.1 M, pH 7.4, 40 ml) followed by icecold 4% paraformaldehyde (150 ml) in PBS. Brains were removed, post-fixed in the same fixative for 90 min, placed in 25% sucrose for 48 h (4°C), frozen-sectioned and processed for Fos immunohistochemistry as previously described [7]. The primary antiserum (5 mg/ml) was rabbit polyclonal anti-Fos raised against amino acid residues 4–17 (AB-2, Oncogene Sci.). Immunohistochemical controls consisted of sections processed with either omission of the primary or secondary antibodies, or sections processed with the negative antibody control provided by Oncogene Sci. (staining was absent in these controls). The microscope slides were coded by an unbiased third party in order that they be analyzed blind to the treatment groups. The numerical area density (NA = number of FLI nuclei/mm2 area) of FLI profiles was determined using a stereological method in which the image of each LC section was magnified and projected by a drawing tube attachment onto a grid of known and magnification-corrected dimensions. The mean number of stained nuclei within several randomly-selected representative areas of the LC was tabulated to obtain the NA. From the NA data, the numerical volume density (NV = number of FLI nuclei/mm3) was calculated for the LC as previously detailed [7]. The number of sections/LC/rat were from 6 to 14 and the LC were quantified bilaterally. It
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should be noted that the calculated NV is not comparable to the absolute number of neurons in the LC and, thus, this number is not comparable to previous published reports of total neuron number in this nucleus. Rather, the NV is the number of stained neurons per a standardized unit volume of 1 mm3. The standardized numbers are unbiased and comparable between groups irrespective of possible differences in shape or size of the LC. After all quantifications were complete, data from each rat was decoded, grouped into appropriate groups and subjected to statistical analysis. Due to the observation that no FLI profiles were found in any brain area of GEPRs or control rats not subjected to the AS, and, only two rats comprised the PTZ-treated group, these animals were not included in the comparisons of FLI numerical densities. Therefore, statistical comparisons were made by one-way analysis of variance (ANOVA) with a post-hoc Sheffe test between AS-control rats, AS-GEPR9s, AS-snGEPR-9s, AS-GEPR-3s, and MES rats. Each GEPR-9 had a score-9 tonic-seizure whereas each GEPR-3 had a score-3 clonic-seizure in response to AS. None of the control Sprague–Dawley rats exposed to the AS experienced a seizure; however, they did show an acoustic-startle response to the AS followed immediately by normal exploratory and grooming behavior. MES-treated animals experienced tonic-seizures of class E (n = 2) or class C (n = 1) whereas PTZ-treated rats showed eventual tonic-seizures of class E after progression from class A. The final-severity phenotype of the MES, high-dose PTZ and GEPR-9 seizures were similar in that they were severe tonic-seizures. Fig. 1 shows representative tissue sections through the LC of an AS-control rat (Fig. 1A) and a snGEPR-9 (Fig. 1B). The FLI staining patterns of the LC of snGEPR-9s, GEPR-9s, MES-treated, and PTZ-treated animals were very similar and thus are not differentially shown. Numerical densities of FLI neurons in all groups are shown in Fig. 2. Untreated control rats and GEPRs not subjected to the AS showed virtually no FLI in the LC (or other brain areas). In contrast, an appreciable number of FLI neurons were found within the LC of AS-control rats as well as snGEPR-9s, GEPR-9s and GEPR-3s following AS; however, the number of FLI neurons in all GEPR groups were significantly higher than that found in the AS-control rats. Moreover, the number of FLI neurons in the GEPR-9 groups were significantly higher than that of the GEPR-3s. Also shown in Fig. 2, MES treatment induced a significant increase in the number of FLI profiles in the LC over that of the AS-control animals and GEPR-3s but similar to that of the GEPR-9 groups. Tonic seizures resulting from highdose PTZ-administration appeared to induce similar FLI in the LC as the other tonic seizure groups however this was not included in the statistical comparisons. Even so, the large and significant increase in FLI neuron number in the LC following generalized tonic-seizures appears equally manifest across three distinct generalized tonic-seizure models (audiogenic, chemical, and electroshock). Alternatively, generalized but comparatively moderate clonic-sei-
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zures, as exhibited by GEPR-3s, are associated with fewer FLI LC neurons than tonic-seizure models. It is well documented that GEPRs have an innate and global deficiency in NE and that this deficiency is a partial determinant of seizure severity in these animals [9]. Indeed, NE has been found to be seizure-modulatory in a wide variety of seizure models and is usually reported as an endogenous anticonvulsant [1,3,5,8,12,14,15,17]. The deficiencies in the NE system in GEPRs [9] have been characterized in terminal regions that are void of noradrenergic perikarya. It is unknown whether these deficiencies result from a generalized lack of NE perikaryal activity coincident with seizure. Thus, to assess perikaryal activity, we examined the elaboration of Fos in association with AGS in these animals and compared their responses to phenotypically-similar seizures induced by other means. In this study, the AS-control animals showed an impressive induction of FLI in the LC (approximately 6900 LC neurons/mm3) that may be indicative of a mild stress related to acoustic startle. GEPR-3s experienced clonic-seizures subsequent to AS and displayed at least three times as many FLI neurons in the LC (approximately 19 400/mm3) than did the AS-control rats. It was tempting to speculate that seizures in GEPRs may reflect an aberrant (i.e. seizure-prone) acoustic startle response; however, although there is some overlap in neural circuitry, the brainstem pathways which mediate acoustic startle [10] and audiogenic seizures [2] have distinct features. Most impressive was the degree of FLI following generalized tonic-seizures. In GEPR-9s and snGEPR-9s subjected to AS, as well as in MES- and PTZ-treated rats, approximately six times as many in FLI neurons were found in the LC (approximately 35 000/mm3) compared to the AS-controls and nearly twice as many as that found in the GEPR-3s. The observations of increased FLI neuron number in the more severe types of tonic-seizures (GEPR-9s, MES, and highdose PTZ-seizures) compared to comparatively moderate clonic-seizures (GEPR-3 seizures) suggests that seizure severity rather than inductive stimulus is positively correlated to FLI numerical density in the LC. Questions remain as to the interpretation of increased immediate-early gene expression after seizures. For example, it could be hypothesized that the LC is proconvulsant in each of these seizure models in light of the presumed increased activity (FLI) of the neurons therein. Alternatively, the LC may become activated in an attempt to terminate or recover from the seizure as may be expected based on the myriad of data that shows NE to be anticonvulsant, especially in the GEPR [9]. Although the profiles of NE release in the brain coincident with seizures are not presently known for the GEPR, extracellular accumulation NE in the thalamus of GEPRs following uptake inhibition, although appreciable, is significantly reduced compared to non-epileptic controls [18]. These findings suggest that synaptic release of NE from terminals of LC neurons, coincident with seizure, is deficient in the GEPR. Further support of the hypothesis of deficient NE terminals in GEPRs, as opposed to deficient NE neurons
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in general, are the findings of Lauterborn and Ribak [11] who showed diminished numbers of NE axons within the inferior colliculus of the GEPR-9. The present data show that LC neurons appear normo-typic in their cellular profile of FLI coincident with or subsequent to seizure and seem to support a notion that LC perikarya receive appropriate afferent signals sufficient to induce elaboration of Fos. Since no differences in LC-Fos induction could be found between various tonic-seizure models, it appears that stimulus-transcription coupling [13] of LC neurons is not altered in GEPRs. These findings support the hypothesis that LC neuron perikaryal responses to afferent signals are not altered in GEPRs, particularly in their FLI responsiveness to seizures, and that the innate problems with the NE neurotransmitter system reside within NE terminals in various target regions. Another possibility may be a deficiency in excitation-secretion coupling of GEPR LC neurons; however, this possibility has not been examined. Interestingly, only one other published report has examined the LC per se in the GEPR [16] and that study found a modest decrease in alpha-2 adrenergic receptor binding sites within this nucleus of GEPR-9s and GEPR-3s. It is theoretically possible that the described diminution in alpha-2 adrenergic receptors in the LC of GEPR-9s may contribute to increased expression of Fos subsequent to seizure; however, one would expect differences between the GEPR-9s and the animals of other tonic-seizure models that, presumably, have normal complements of intra-LC alpha-2 adrenergic receptor binding sites. In summary, the present data shows that perikarya of the LC appear to be equally responsive in the induction of FLI coincident with seizures between three distinct generalized tonic-seizure models. Therefore, these data suggest that the deficiencies in norepinephrine in the GEPR model of epilepsy are not likely due to diminished activity within the LC. The authors are grateful for the assistance of Hari Ailinani and Amanda Solomon who were precollege apprentices in the laboratory of R.W.C. during performance of this research. Supported by a SIU program project grant funded by Bayer Pharmaceuticals. [1] Applegate, C.D., Konkol, R.J. and Burchfiel, J.L., Kindling antagonism: a role for hindbrain norepinephrine in the development of site suppression following concurrent, alternate stimulation, Brain Res., 407 (1987) 212–217. [2] Browning, R.A., Neuroanatomical localization of structures responsible for seizures in the GEPR: lesion studies, Life Sci., 39 (1986) 857–867.
[3] Browning, R.A., The role of neurotransmitters in electroshock seizure models. In P.C. Jobe and H.E. Laird II (Eds.), Neurotransmitters and Epilepsy, Humana Press, Totowa, NJ, 1987, pp. 277–320. [4] Browning, R.A., Turner, F.J., Simonton, R.L. and Bundman, M.C., Effect of midbrain and pontine tegmental lesions on the maxi0mal electroshock seizure pattern in rats, Epilepsia, 22 (1981) 583– 594. [5] Browning, R.A., Wang, C. and Faingold, C.L., Effect of norepinephrine depletion on audiogenic-like seizures elicited by microinfusion of an excitant amino acid into the inferior colliculus of normal rats, Exp. Neurol., 112 (1991) 200–205. [6] Clough, R.W., Browning, R.A., Maring, M.L., Statnick, M.A., Wang, C. and Jobe, P.C., Effects of intraventricular locus coeruleus transplants on seizure severity in genetically epilepsy-prone rats following depletion of brain norepinephrine, J. Neural Trans. Plast., 5 (1994) 65–79. [7] Clough, R.W., Eells, J.B., Browning, R.A. and Jobe, P.C., Seizures and proto-oncogene expression of Fos in the brain of adult genetically epilepsy-prone rats (GEPRs), Exp. Neurol., 146 (1997) 341– 353. [8] Corcoran, M.E., Fibiger, H.C., McCaughran, J.A. and Wada, J.A., Potentiation of amygdaloid kindling and metazol-induced seizures by 6-hydroxydopamine in rats, Exp. Neurol., 45 (1974) 118–133. [9] Dailey, J.W., Mishra, P.K., Ko, K.H., Penny, J.E. and Jobe, P.C., Noradrenergic abnormalities in the central nervous system of seizure-naive genetically epilepsy-prone rats, Epilepsia, 32 (1991) 168–173. [10] Davis, M., Gendelman, D.S., Tischler, M. and Gendelman, P.M., A primary acoustic startle circuit: lesion and stimulation studies, J. Neurosci., 2 (1982) 791–805. [11] Lauterborn, J.C. and Ribak, C.E., Differences in dopamine-betahydroxylase immunoreactivity between the brains of genetically epilepsy-prone and Sprague–Dawley rats, Epilepsy Res., 4 (1989) 161– 176. [12] McIntyre, D.C. and Edson, N., Facilitation of amygdala kindling after norepinephrine depletion with 6-hydroxydopamine in rats, Exp. Neurol., 74 (1981) 748–757. [13] Morgan, J.I. and Curran, T., Stimulus-transcription coupling in neurons: role of cellular immediate-early genes, Trends Neurosci., 12 (1989) 459–462. [14] Neuman, R.S., Suppression of penicillin-induced focal epileptiform activity by locus ceruleus stimulation: mediation by an alpha 1adrenoceptor, Epilepsia, 27 (1986) 359–366. [15] Papanicolaou, J., Summers, R.J., Vajda, F.J.E. and Louis, W.J., Anticonvulsant effects of clonidine mediated through central alpha2 adrenoceptors, Eur. J. Pharmacol., 77 (1882) 163–166. [16] Razani-Boroujerdi, S., Tso-Olivas, D.Y., Hoffman, T.J., Weiss, G.K. and Savage, D.D., Decrease in locus coeruleus [3H]idazoxan binding site density in genetically epilepsy-prone (GEPR) rats, Brain Res., 600 (1993) 181–186. [17] Trottier, S., Lindvall, O., Chauvel, P. and Bjorklund, A., Facilitation of focal cobalt-induced epilepsy after lesions of the noradrenergic locus coeruleus system, Brain Res., 454 (1988) 308–314. [18] Yan, Q.-S., Jobe, P.C. and Dailey, J.W., Thalamic deficiency in norepinephrine release detected via intracerebral microdialysis: a synaptic determinant of seizure predisposition in the genetically epilepsy-prone rat, Epilepsy Res., 14 (1993) 229–236.