- Email: [email protected]
Temporal relationship between the expression of Fos, Jun and Krox-24 in the guinea pig vestibular nuclei during the development of vestibular compensation for unilateral vestibular deafferentation
BRAIN RESEARCH ELSEVIER
Brain Research 735 (1996) 173-176
Short communication
Temporal relationship between the expression of Fos, Jun and Krox-24 in the guinea pig vestibular nuclei during the development of vestibular compensation for unilateral vestibular deafferentation Cynthia L. Darlington a, *, Patricia Lawlor b, Paul F. Smith c, Michael Dragunow b a Department of Psychology and the Neuroscience Research Center, University ofOtago, Dunedin, New Zealand b Department of Pharmacology and Clinical Pharmacology, University of Auckland School of Medicine, Auckland, New Zealand c Department of Pharmacology, School of Medical Sciences, University of Otago Medical School, Dunedin, New Zealand
Accepted 16 July 1996
Abstract
Immediately following unilateral vestibular deafferentation (UVD), expression of the c-fos protein increased in both vestibular nucleus complexes (VNCs). By 10 h post-UVD, c-fos proteins were more evident in the bilateral medial vestibular nuclei (MVN), but their expression had decreased in the bilateral superior vestibular nuclei (SVN) and lateral vestibular nuclei (LVN) compared to immediately post-UVD. At 50 h post-UVD, c-fos protein expression was still evident in the bilateral VNC, but had decreased relative to immediately post-UVD. Immediately post-UVD, c-jun protein expression increased in the bilateral VNC, but was more noticeable at 10 h post-UVD. By 50 h post-UVD, c-jun protein expression had decreased markedly throughout the bilateral VNC. The krox-24 protein could not be seen anywhere in the immediate post-UVD condition. However, it was expressed at a low level in the bilateral MVN at 10 h post-UVD; by 50 h post-UVD, this expression had decreased. No evidence of apoptosis was observed following UVD. Keywords: Immediate early gene; c-fos; c-jun; Krox-24; Vestibular nucleus; Vestibular compensation; Unilateral labyrinthectomy
Some immediate early gene proteins (IEGPs) are transcription factor proteins which regulate gene expression within cells. Certain forms of cellular activation lead to immediate early gene transcription, and following translation, the IEGPs re-enter the nucleus and activate 'late-response genes', leading to further gene transcription [7]. The biochemical cascade involving IEGPs has been demonstrated to be an important part of the way that cells produce phenotypic changes in response to environmental events [7]. Unilateral peripheral vestibular deafferentation (UVD) results in the loss of vestibular input to the vestibular nucleus complex (VNC) ipsilateral to the lesion, causing a large decrease in resting activity in these neurons and consequently, abnormal vestibular reflex activity. However, over time, resting activity recovers in the ipsilateral VNC, producing a partial recovery in vestibular reflexes in a process known as 'vestibular compensation' [12,14]. Recent studies using rat and cat have reported that UVD
* Corresponding author. Fax: (64) (3) 479-8335.
results in the expression of the c-fos IEGP in the bilateral VNC soon after UVD [1,8,10,11]. Although there is disagreement regarding the basal expression of c-fos in the VNC [1,5,10,11], it is generally agreed that c-fos induction following UVD subsides by approximately 3 days post-op. [9,10]. However, studies in other areas of the CNS have shown that c-fos induction is only one part of a complex biochemical cascade involving the induction of many other immediate early genes, such as c-jun and krox-24 (also known as zif268) [7]. To date, only one study of the VNC has analysed c-jun expression, and, in this case, no c-jun IEGP induction was found up to 4 weeks post-UVD [11]; to date, krox-24 expression in the VNC has not been studied following UVD. Since previous studies have described only c-fos IEGP expression in the VNC following UVD, the objective of the present study was to examine, under the same experimental conditions, the temporal relationship between the expression of the c-fos, c-jun and krox-24 IEGPs in the VNC, at various times following UVD which correlate with the development of vestibular compensation and recovery of neuronal activity in that species. Previous studies
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0006- 8993(96)00889-X
174
C.L. Darlington et al. / Brain Research 735 (1996) 173-176
of IEGP expression in the VNC have been confined to rat and cat [1,5,8-11]; in the present study we used guinea pig since many vestibular compensation studies have been conducted in this species and the guinea pig vestibular system is very similar to that in humans [4,12-14]. In addition, we also looked for evidence of apoptosis in the VNC following UVD; to our knowledge, apoptosis has not been investigated previously under these circumstances. Data were obtained from 12 guinea pigs (300-600 g) randomly divided into the following groups: (1) sham UVD: the bony labyrinth was exposed but was not opened (n = 3); (2) immediate post-UVD (n = 3); (3) 10 h postUVD (n = 3); and (4) 50 h post-UVD (n = 3). These times were chosen on the basis of our previous studies showing that UVD symptoms are maximally expressed at 10 h post-UVD, whereas by 50 h, they are largely compensated [4,14]. All animals were anesthetised with 0.4 m l / k g , i.m. fentazin (0.4 m g / m l fentanyl citrate, 58.3 m g / m l xylazine HC1 and 3.2 m g / m l azaperone; Parnell, New Zealand [13]) and received a right surgical unilateral vestibular deafferentation (UVD) as described in detail previously [4]. Briefly, the right temporal bone was exposed using blunt dissection and, under microscopic control, drilled open to expose the bony labyrinth. Using a dental drill with a fine burr, the horizontal and anterior semicircular canal ampullae were opened and the sensory epithelia aspirated. The utricular duct was also exposed and aspirated; the saccule and the posterior canal ampulla were probed blindly and aspirated. At the designated time, animals were re-anesthetized with fentazin (Groups 3 and 4) and were transcardially perfused with saline and then with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brainstem was then removed and stored in 4% paraformaldehyde in PBS until the time of the immunohistochemistry. The brainstems were incubated in sucrose for cryoprotection and sectioned (30 /,tm) on a cryostat; in each of the three animals in each group, representative sections were selected from three different rostrocaudal areas of the VNC. The sections were washed 3 X 5 min in 0.01 M PBS containing 0.2% Triton-X, before being incubated in 1% hydrogen peroxide in 100% methanol for 5 rain (to remove endogenous peroxidase activity and enhance antibody penetration into the tissue). Sections were then again washed for 3 X 5 min with PBS before being incubated for 24 h with rabbit polyclonal antibodies to immediate early gene proteins at various dilutions (see below) in immunobuffer (0.01 M PBS, 1% normal goat serum, and 0.4 m g / m l methiolate). The antisera used for these studies recognise c-Fos (Oncogene Science, 1:500 dilution #PCO5), c-Jun (Oncogene Science, 1:500 dilution #PCO6) and Krox-24 (generously provided by Dr. R. Bravo, Princeton, USA, 1:50000 dilution). Following primary antibody incubations, sections were washed in PBS, 3 × 5 min, and then incubated for 3 h with biotinylated goat anti-rabbit serum (Sigma) in immunobuffer (1:500 dilution). After washing
again in PBS, 3 × 5 rain, sections were incubated with extravidin (Sigma, 1:500 dilution in immunobuffer) for 2 h, before finally being washed in PBS and placed in 3,3'-diaminobenzidine (DAB, Sigma) containing hydrogen peroxide to develop [6]. Specificity of staining for the c-fos and c-jun IEGPs was tested by incubating sections with c-fos and c-jun antisera preadsorbed with their respective peptides [2,3]. Sections were analysed for evidence of apoptosis using in situ labelling of DNA fragments based on the TUNEL method [3]. All incubations were performed at room temperature. Slides were examined using light microscopy and intensity and extent of neuronal nuclear staining was evaluated by two observers, one of whom was blind to the experimental conditions [1]. Following Cirelli et al. [1], the two observers agreed on a rating of the intensity and extent of staining of the VNC subnuclei, using a rating scale of 0-3, where 3 represents maximal intensity [1]. Although this rating scale did not distinguish beween number of labelled nuclei and density of nuclear staining, in general, the density of staining was similar across experimental conditions for the same antibody dilution factor. Great care was taken to distinguish labelling of neuronal nuclei from glial cell labelling. In the sham condition, the c-fos protein was expressed at a low level in the bilateral rostral VNC, including the superior vestibular nucleus (SVN), the lateral vestibular nucleus (LVN), and also in abducens nucleus. No or very little basal expression could be seen in the medial vestibular nucleus (MVN). Immediately post-UVD, expression of the c-fos protein increased in both VNC, in the rostral SVN, LVN and more caudally in the MVN. By 10 h post-UVD, c-fos proteins were more evident in the bilateral MVN, but their expression had decreased in the bilateral SVN and LVN compared to immediately postUVD. At 50 h post-UVD, c-fos protein expression was still evident in the bilateral VNC, but had decreased relative to immediately post-UVD. In all cases, staining for the c-fos, c-jun and krox-24 IEGPs was approximately symmetrical between the ipsilateral and contralateral VNCs; therefore, Fig. 1 illustrates staining in the bilateral VNCs over time post-UVD (Fig. 1). In the sham condition, very few c-jun proteins could be seen in the bilateral rostral VNC (mainly LVN and SVN). Immediately post-UVD, c-jun protein expression increased in the bilateral VNC, but was more noticeable at 10 h post-UVD, where intense expression could be seen in the rostral and caudal VNC on both sides: the distribution of c-jun protein included the SVN, LVN, MVN, descending vestibular nucleus (DVN), and also the abducens nucleus. By 50 h post-UVD, c-jun protein expression had decreased markedly throughout the bilateral rostro-caudal VNC relative to 10 h post-UVD (Fig. 1). The krox-24 protein could not be seen anywhere in the VNC in the sham and immediate post-UVD conditions. However, it was expressed at a low level in the bilateral caudal MVN at 10 h post-UVD; by 50 h post-UVD, this
C.L. Darlington et al./Brain Research 735 (1996) 173-176 A 3 •
[] []
rostral mid caudal
~2-
I.I.
control immediate Time
10 h
50 h
post- UVD
B3 ¸
=m2
175
low level of basal c-fos IEGP expression in the VNC. These discrepancies may be due to species differences, to differences in the environmental conditions of the animals prior to the experiment or may be the result of the sham operation that was performed in the control conditions. In contrast to the results of Kitihara et al. [11], we did find c-jun IEGP expression in the VNC following UVD, although its temporal and spatial pattern was markedly different to that for c-fos IEGPs. In general, many studies have reported that c-jun expression occurs later than c-fos expression [7]. At present, the significance of the transient and low level of krox-24 IEGP expression in the caudal VNC following UVD is unclear. With respect to c-fos, a recent study suggests that c-fos induction may be related to some form of injury or 'error' signal in the VNC, since when compensation was reversed using an injection of an Nmethyl-D-aspartate (NMDA) receptor/channel antagonist at 3 days post-UVD, c-fos IEGPs were again induced in the VNC [10]. Future studies of IEGP induction in the VNC during vestibular compensation will need to explore its relationship to the neurophysiological basis of the compensation process, possibly using c-fos antisense oligonucleotides [ 1].
0 control immediate Time
C 3
10 h
50 h
post-UVD
_.~ 2
Acknowledgements This research was supported by a Project Grant from the New Zealand Neurological Foundation (to PS) and a Programme Grant from the Health Research Council of New Zealand (to MD). We thank the Department of Psychology and the Division of Sciences, University of Otago, for travel support.
7~ //1 // /p
- -
References o
control
immediate Time
10 h
50 h
post- UVD
Fig. 1. Intensity and extent of Fos (A), Jun (B) and Krox-24 (C) labelling in the rostral, caudal and mid bilateral VNC in control animals and at various times post-UVD. The y-axis represents an ordinal scale of labelling, where '3' indicates maximum intensity. Note that the histograms illustrate staining in the bilateral VNC because staining was approximately symmetrical in the two VNCs for all IEGPs.
expression had decreased (Fig. 1). Using the TUNEL test, no evidence of apoptosis was seen in any section. The results of the present experiment are consistent with those from previous studies in indicating that UVD results in the increased expression of the c-fos IEGP in the bilateral VNC shortly following UVD [1,8,10,11]. Similarly, we found that by 50 h post-UVD, the level of c-fos IEGP induction had markedly decreased [9,10]. However, unlike some previous studies using rat [10,11], but in agreement with others using rat and cat [1,5], we did find a
[1] Cirelli, C., Pompeiano, M., D'Ascanio, P., Arrighi, P. and Pompeiano, O., c-fos expression in the rat brain after unilateral labyrinthectomy and its relation to the uncompensated and compensated stages, Neuroscience, 70 (1996) 515-546. [2] Dragunow M., Beilharz, E., Sirimanne, E,, Lawlor, P., Williams, C., Bravo, R. and Gluckman, P., IEGP expression in neurons undergoing delayed death, but not necrosis, following hypoxic-ishemic injury to the young rat brain, Mol. Brain Res., 25 (1994) 19-33. [3] Dragunow, M., Preston, K., Dodd, J., Young, D., Lawlor, P. and Christie, D., Clusterin accumulates in dying neurons following status epilepticus, Mol. Brain Res., 32 (1995) 279-290. [4] Gilchrist, D.P.D., Darlington, C.L. and Smith, P.F., A dose-response analysis of the beneficial effects of the ACTH-(4-9) analogue, Org 2766, on behavioural recovery following unilateral labyrinthectomy in guinea pig, Br. J. Pharmacol., 111 (1994) 358-363. [5] Herdegen, T., Kovary, K., Buhl, A., Bravo, R., Zimmermann, M. and Gass, P., Basal expression of the inducible transcription factors c-Jun, JunB, JunD, c-Fos, FosB and Krox-24 in the adult rat brain, J. Comp. Neurol., 354 (1995) 39-56. [6] Hughes, P., Dragunow, M., Beilharz, E., Lawlor, P. and Gluckman, P., MK801 induces immediate-early gene proteins and BDNF mRNA in rat cerebrocortical neurons, NeuroReport, 4 (1993) 183-186.
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C.L. Darlington et a l . / Brain Research 735 (1996) 173-176
[7] Hughes, P. and Dragunow, M., Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system, Pharmacol. Rev., 47 (1995)133-175. [8] Kaufman, G.D., Anderson, J.H. and Beitz, A.J., Brainstem Fos expression following acute unilateral labyrinthectomy in the rat, NeuroReport, 3 (1992) 829-832. [9] Kaufman, G.D., Anderson, J.H. and Beitz, A.J., Otolith-brainstem connectivity: Evidence for differential neural activation by vestibular hair cells based on quantification of Fos expression in unilateral labyrinthectomized rats, J. Neurophysiol., 70 (1993) 117-127. [10] Kitihara, T., Takeda, N., Saika, T., Kubo, T. and Kiyama, H., Effects of MK801 of Fos expression in the rat brainstem after unilateral labyrinthectomy, Brain Res., 700 (1995) 182-190. [11] Kitihara, T., Saika, T., Takeda, N., Kiyama, H. and Kubo, T.,
Changes in Fos and Jun expression in the rat brainstem in the process of vestibular compensation, Acta Otolaryngol. (Stockh.), Suppl. 520 (1995) 401-404. [12] Ris, L., de Waele, C., Serafin, M., Vidal, P.P. and Godaux, E., Neuronal activity in the ipsilateral vestibular nucleus following unilateral labyrinthectomy in the alert guinea pig, J. Neurophysiol., 74 (1995) 2087-2099. [13] Sansom, A.J., Smith, P.F. and Darlington, C.L., Fentazin anesthesia for labyrinthine surgery in guinea pigs, J. Vest. Res., 6 (1996) 49-52. [14] Smith, P.F. and Curthoys, I.S., Neuronal activity in the ipsilateral medial vestibular nucleus of the guinea pig following unilateral labyrinthectomy, Brain Res., 444 (1988) 308-319.
Brain Research 735 (1996) 173-176
Short communication
Temporal relationship between the expression of Fos, Jun and Krox-24 in the guinea pig vestibular nuclei during the development of vestibular compensation for unilateral vestibular deafferentation Cynthia L. Darlington a, *, Patricia Lawlor b, Paul F. Smith c, Michael Dragunow b a Department of Psychology and the Neuroscience Research Center, University ofOtago, Dunedin, New Zealand b Department of Pharmacology and Clinical Pharmacology, University of Auckland School of Medicine, Auckland, New Zealand c Department of Pharmacology, School of Medical Sciences, University of Otago Medical School, Dunedin, New Zealand
Accepted 16 July 1996
Abstract
Immediately following unilateral vestibular deafferentation (UVD), expression of the c-fos protein increased in both vestibular nucleus complexes (VNCs). By 10 h post-UVD, c-fos proteins were more evident in the bilateral medial vestibular nuclei (MVN), but their expression had decreased in the bilateral superior vestibular nuclei (SVN) and lateral vestibular nuclei (LVN) compared to immediately post-UVD. At 50 h post-UVD, c-fos protein expression was still evident in the bilateral VNC, but had decreased relative to immediately post-UVD. Immediately post-UVD, c-jun protein expression increased in the bilateral VNC, but was more noticeable at 10 h post-UVD. By 50 h post-UVD, c-jun protein expression had decreased markedly throughout the bilateral VNC. The krox-24 protein could not be seen anywhere in the immediate post-UVD condition. However, it was expressed at a low level in the bilateral MVN at 10 h post-UVD; by 50 h post-UVD, this expression had decreased. No evidence of apoptosis was observed following UVD. Keywords: Immediate early gene; c-fos; c-jun; Krox-24; Vestibular nucleus; Vestibular compensation; Unilateral labyrinthectomy
Some immediate early gene proteins (IEGPs) are transcription factor proteins which regulate gene expression within cells. Certain forms of cellular activation lead to immediate early gene transcription, and following translation, the IEGPs re-enter the nucleus and activate 'late-response genes', leading to further gene transcription [7]. The biochemical cascade involving IEGPs has been demonstrated to be an important part of the way that cells produce phenotypic changes in response to environmental events [7]. Unilateral peripheral vestibular deafferentation (UVD) results in the loss of vestibular input to the vestibular nucleus complex (VNC) ipsilateral to the lesion, causing a large decrease in resting activity in these neurons and consequently, abnormal vestibular reflex activity. However, over time, resting activity recovers in the ipsilateral VNC, producing a partial recovery in vestibular reflexes in a process known as 'vestibular compensation' [12,14]. Recent studies using rat and cat have reported that UVD
* Corresponding author. Fax: (64) (3) 479-8335.
results in the expression of the c-fos IEGP in the bilateral VNC soon after UVD [1,8,10,11]. Although there is disagreement regarding the basal expression of c-fos in the VNC [1,5,10,11], it is generally agreed that c-fos induction following UVD subsides by approximately 3 days post-op. [9,10]. However, studies in other areas of the CNS have shown that c-fos induction is only one part of a complex biochemical cascade involving the induction of many other immediate early genes, such as c-jun and krox-24 (also known as zif268) [7]. To date, only one study of the VNC has analysed c-jun expression, and, in this case, no c-jun IEGP induction was found up to 4 weeks post-UVD [11]; to date, krox-24 expression in the VNC has not been studied following UVD. Since previous studies have described only c-fos IEGP expression in the VNC following UVD, the objective of the present study was to examine, under the same experimental conditions, the temporal relationship between the expression of the c-fos, c-jun and krox-24 IEGPs in the VNC, at various times following UVD which correlate with the development of vestibular compensation and recovery of neuronal activity in that species. Previous studies
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0006- 8993(96)00889-X
174
C.L. Darlington et al. / Brain Research 735 (1996) 173-176
of IEGP expression in the VNC have been confined to rat and cat [1,5,8-11]; in the present study we used guinea pig since many vestibular compensation studies have been conducted in this species and the guinea pig vestibular system is very similar to that in humans [4,12-14]. In addition, we also looked for evidence of apoptosis in the VNC following UVD; to our knowledge, apoptosis has not been investigated previously under these circumstances. Data were obtained from 12 guinea pigs (300-600 g) randomly divided into the following groups: (1) sham UVD: the bony labyrinth was exposed but was not opened (n = 3); (2) immediate post-UVD (n = 3); (3) 10 h postUVD (n = 3); and (4) 50 h post-UVD (n = 3). These times were chosen on the basis of our previous studies showing that UVD symptoms are maximally expressed at 10 h post-UVD, whereas by 50 h, they are largely compensated [4,14]. All animals were anesthetised with 0.4 m l / k g , i.m. fentazin (0.4 m g / m l fentanyl citrate, 58.3 m g / m l xylazine HC1 and 3.2 m g / m l azaperone; Parnell, New Zealand [13]) and received a right surgical unilateral vestibular deafferentation (UVD) as described in detail previously [4]. Briefly, the right temporal bone was exposed using blunt dissection and, under microscopic control, drilled open to expose the bony labyrinth. Using a dental drill with a fine burr, the horizontal and anterior semicircular canal ampullae were opened and the sensory epithelia aspirated. The utricular duct was also exposed and aspirated; the saccule and the posterior canal ampulla were probed blindly and aspirated. At the designated time, animals were re-anesthetized with fentazin (Groups 3 and 4) and were transcardially perfused with saline and then with 4% paraformaldehyde in phosphate-buffered saline (PBS). The brainstem was then removed and stored in 4% paraformaldehyde in PBS until the time of the immunohistochemistry. The brainstems were incubated in sucrose for cryoprotection and sectioned (30 /,tm) on a cryostat; in each of the three animals in each group, representative sections were selected from three different rostrocaudal areas of the VNC. The sections were washed 3 X 5 min in 0.01 M PBS containing 0.2% Triton-X, before being incubated in 1% hydrogen peroxide in 100% methanol for 5 rain (to remove endogenous peroxidase activity and enhance antibody penetration into the tissue). Sections were then again washed for 3 X 5 min with PBS before being incubated for 24 h with rabbit polyclonal antibodies to immediate early gene proteins at various dilutions (see below) in immunobuffer (0.01 M PBS, 1% normal goat serum, and 0.4 m g / m l methiolate). The antisera used for these studies recognise c-Fos (Oncogene Science, 1:500 dilution #PCO5), c-Jun (Oncogene Science, 1:500 dilution #PCO6) and Krox-24 (generously provided by Dr. R. Bravo, Princeton, USA, 1:50000 dilution). Following primary antibody incubations, sections were washed in PBS, 3 × 5 min, and then incubated for 3 h with biotinylated goat anti-rabbit serum (Sigma) in immunobuffer (1:500 dilution). After washing
again in PBS, 3 × 5 rain, sections were incubated with extravidin (Sigma, 1:500 dilution in immunobuffer) for 2 h, before finally being washed in PBS and placed in 3,3'-diaminobenzidine (DAB, Sigma) containing hydrogen peroxide to develop [6]. Specificity of staining for the c-fos and c-jun IEGPs was tested by incubating sections with c-fos and c-jun antisera preadsorbed with their respective peptides [2,3]. Sections were analysed for evidence of apoptosis using in situ labelling of DNA fragments based on the TUNEL method [3]. All incubations were performed at room temperature. Slides were examined using light microscopy and intensity and extent of neuronal nuclear staining was evaluated by two observers, one of whom was blind to the experimental conditions [1]. Following Cirelli et al. [1], the two observers agreed on a rating of the intensity and extent of staining of the VNC subnuclei, using a rating scale of 0-3, where 3 represents maximal intensity [1]. Although this rating scale did not distinguish beween number of labelled nuclei and density of nuclear staining, in general, the density of staining was similar across experimental conditions for the same antibody dilution factor. Great care was taken to distinguish labelling of neuronal nuclei from glial cell labelling. In the sham condition, the c-fos protein was expressed at a low level in the bilateral rostral VNC, including the superior vestibular nucleus (SVN), the lateral vestibular nucleus (LVN), and also in abducens nucleus. No or very little basal expression could be seen in the medial vestibular nucleus (MVN). Immediately post-UVD, expression of the c-fos protein increased in both VNC, in the rostral SVN, LVN and more caudally in the MVN. By 10 h post-UVD, c-fos proteins were more evident in the bilateral MVN, but their expression had decreased in the bilateral SVN and LVN compared to immediately postUVD. At 50 h post-UVD, c-fos protein expression was still evident in the bilateral VNC, but had decreased relative to immediately post-UVD. In all cases, staining for the c-fos, c-jun and krox-24 IEGPs was approximately symmetrical between the ipsilateral and contralateral VNCs; therefore, Fig. 1 illustrates staining in the bilateral VNCs over time post-UVD (Fig. 1). In the sham condition, very few c-jun proteins could be seen in the bilateral rostral VNC (mainly LVN and SVN). Immediately post-UVD, c-jun protein expression increased in the bilateral VNC, but was more noticeable at 10 h post-UVD, where intense expression could be seen in the rostral and caudal VNC on both sides: the distribution of c-jun protein included the SVN, LVN, MVN, descending vestibular nucleus (DVN), and also the abducens nucleus. By 50 h post-UVD, c-jun protein expression had decreased markedly throughout the bilateral rostro-caudal VNC relative to 10 h post-UVD (Fig. 1). The krox-24 protein could not be seen anywhere in the VNC in the sham and immediate post-UVD conditions. However, it was expressed at a low level in the bilateral caudal MVN at 10 h post-UVD; by 50 h post-UVD, this
C.L. Darlington et al./Brain Research 735 (1996) 173-176 A 3 •
[] []
rostral mid caudal
~2-
I.I.
control immediate Time
10 h
50 h
post- UVD
B3 ¸
=m2
175
low level of basal c-fos IEGP expression in the VNC. These discrepancies may be due to species differences, to differences in the environmental conditions of the animals prior to the experiment or may be the result of the sham operation that was performed in the control conditions. In contrast to the results of Kitihara et al. [11], we did find c-jun IEGP expression in the VNC following UVD, although its temporal and spatial pattern was markedly different to that for c-fos IEGPs. In general, many studies have reported that c-jun expression occurs later than c-fos expression [7]. At present, the significance of the transient and low level of krox-24 IEGP expression in the caudal VNC following UVD is unclear. With respect to c-fos, a recent study suggests that c-fos induction may be related to some form of injury or 'error' signal in the VNC, since when compensation was reversed using an injection of an Nmethyl-D-aspartate (NMDA) receptor/channel antagonist at 3 days post-UVD, c-fos IEGPs were again induced in the VNC [10]. Future studies of IEGP induction in the VNC during vestibular compensation will need to explore its relationship to the neurophysiological basis of the compensation process, possibly using c-fos antisense oligonucleotides [ 1].
0 control immediate Time
C 3
10 h
50 h
post-UVD
_.~ 2
Acknowledgements This research was supported by a Project Grant from the New Zealand Neurological Foundation (to PS) and a Programme Grant from the Health Research Council of New Zealand (to MD). We thank the Department of Psychology and the Division of Sciences, University of Otago, for travel support.
7~ //1 // /p
- -
References o
control
immediate Time
10 h
50 h
post- UVD
Fig. 1. Intensity and extent of Fos (A), Jun (B) and Krox-24 (C) labelling in the rostral, caudal and mid bilateral VNC in control animals and at various times post-UVD. The y-axis represents an ordinal scale of labelling, where '3' indicates maximum intensity. Note that the histograms illustrate staining in the bilateral VNC because staining was approximately symmetrical in the two VNCs for all IEGPs.
expression had decreased (Fig. 1). Using the TUNEL test, no evidence of apoptosis was seen in any section. The results of the present experiment are consistent with those from previous studies in indicating that UVD results in the increased expression of the c-fos IEGP in the bilateral VNC shortly following UVD [1,8,10,11]. Similarly, we found that by 50 h post-UVD, the level of c-fos IEGP induction had markedly decreased [9,10]. However, unlike some previous studies using rat [10,11], but in agreement with others using rat and cat [1,5], we did find a
[1] Cirelli, C., Pompeiano, M., D'Ascanio, P., Arrighi, P. and Pompeiano, O., c-fos expression in the rat brain after unilateral labyrinthectomy and its relation to the uncompensated and compensated stages, Neuroscience, 70 (1996) 515-546. [2] Dragunow M., Beilharz, E., Sirimanne, E,, Lawlor, P., Williams, C., Bravo, R. and Gluckman, P., IEGP expression in neurons undergoing delayed death, but not necrosis, following hypoxic-ishemic injury to the young rat brain, Mol. Brain Res., 25 (1994) 19-33. [3] Dragunow, M., Preston, K., Dodd, J., Young, D., Lawlor, P. and Christie, D., Clusterin accumulates in dying neurons following status epilepticus, Mol. Brain Res., 32 (1995) 279-290. [4] Gilchrist, D.P.D., Darlington, C.L. and Smith, P.F., A dose-response analysis of the beneficial effects of the ACTH-(4-9) analogue, Org 2766, on behavioural recovery following unilateral labyrinthectomy in guinea pig, Br. J. Pharmacol., 111 (1994) 358-363. [5] Herdegen, T., Kovary, K., Buhl, A., Bravo, R., Zimmermann, M. and Gass, P., Basal expression of the inducible transcription factors c-Jun, JunB, JunD, c-Fos, FosB and Krox-24 in the adult rat brain, J. Comp. Neurol., 354 (1995) 39-56. [6] Hughes, P., Dragunow, M., Beilharz, E., Lawlor, P. and Gluckman, P., MK801 induces immediate-early gene proteins and BDNF mRNA in rat cerebrocortical neurons, NeuroReport, 4 (1993) 183-186.
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