Molecular Brain Research, 12 (1992) 141-147 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0169-328X/92/$03.50

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BRESM 70362

Induction of transcription factors in somatosensory cortex after tactile stimulation Kenneth J. Mack and Pat A. Mack Department of Neurology and Waisman Center on Mental Retardation, University of Wisconsin, Madison, WI 53705 (U.S.A.) (Accepted 6 August 1991)

Key words: NGFI-A; NGFI-B; c-fos; Gene induction; Barrel cortex

Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcription factors in cerebral cortex by a physiological stimulus had not previously been demonstrated. In this study, tactile stimuli induced multiple transcription factors in the somatosensory cortex. Adult male rats were lightly anesthetized with urethane. Tactile stimuli was delivered by a paint brush gently stroking an animals whiskers on one side of its face for a 15 rain period. Two h later, the animals were sacrificed. Cortex contralateral to the stimulation was compared with ipsilateral cortex using antibodies raised against immediate early response gene products NGFI-A, NGFI-B, and c-los. The different transcription factors showed shghtly different patterns of response to the tactile stimulus. However, the induction of immunohistochemical staining was most prominent in layer 4 with all antibodies under study. This increase in the number of cell bodies stained was less robust than that seen in the somatosensory cortex after a seizure, and showed more of a predominance in layer 4 cells. These data demonstrate that physiologic stimulation can induce immediate early response genes in cortical cells, and that multiple immediate early response genes react to a stimulus. INTRODUCTION

Initially it was shown that pharmacological treatments that stimulate postsynaptic neurons, such as high KC! or muscarinic agonists, could induce the immediate early response gene (IEG) c.fos in neuronal-like PC12 cells5' n. Later, Morgan et al. 12 used a seizure as a model of neuronal stimulation to show that the induction of c-los can also occur in neurons in vivo. Other investigators have shown that lEGs such as N G H - A ~'t3't~, N G H - B m, and c-]un 13 can also be induced after seizure stimulation in rodents. This led to question if these genes could be induced after more specific or physiologic stimuli. Trans-synaptic electrical stimulation can induce b o t h c-los 14 and Krox 24 (synonymous to NGFI-A) 6 at various levels in the central nervous system. Using paradigms involving painful stimuli to a limb, investigators have shown the induction of c-fos in the spinal cord 1'9 and thalamus t. Additionally, water deprivation can induce c-fos in hypothalamic nuclei s4. The induction of IEGs may play a proximal role in the expression of other target genes such as proenkephalin t6. This phenomen~n of IEG inducibility may have implications as a possible, but unproven, mechanism in plasticity3't5. All of the above paradigms showing physiologic stimulation of IEGs demonstrated inductions in subcortical

regions. This study attempted to address the question of whether physiological tactile stimulation can lead to induction of transcription factors in the cortex. The barrel cortex was chosen because of an easily defined peripheral receptive field, as well as its large representation in rodent somatosensory cortex 2°. Antibodies to a variety of inducible transcription factors, representing a prototypic leucine zipper (c-fos), zinc finger (NGFI-A), and steroid.hormone binding proteins (NGFI-B), were used to identify IEGs in the barrel cortex, and address the possible sensory induction of the IEGs. MATERIALS AND METHODS

Materials Adult (200-300 g) male albino Sprague-Dawley rats were obtained from breeding colonies at the Waisman Center. Metrazol was obtained from Sigma. Antibodies against c-fo~ were obtained from Cambridge Research Biochemicals (OA-11-823; Valley Stream, NY). Preimmune serum, biotinylated secondary antibodies, and ABC kits were obtained from Vector Labs (Burlingame, CA). Antibodies against NGFI-A (A310) 2 and NGFI-B (B1350, B1440)4 were a generous gift from Dr. Jeffrey Milbrandt of Washington University, St. Louis.

Stimulation Animals were anesthetized with 1.1 g/kg intraperitoneal injection of urethane, a level previously shown to result in mild anesthesia with m i n i ~ ! offects on electrophysiological activity in many brain regionss. After the animals stopped moving, typically within a 5 rain time period, the animals whiskers were stimulated unilaterally

Correspondence: K.J. Mack, Waisman Center, University of Wisconsin, 1500 Highland Ave., Madison, WI 53705, U.S.A.

142 by stroking with a artist's paint brush over a 15 rain time interval. Two h after the start of stimulation, the animals were given chloral hydrate to induce complete anesthesia. The animals were then perfused with b0 ml phosphate buffered saline (PBS), and 400 ml of 1.5% paraformaldehyde solution in phosphate buffer. Some animals were given an intraperitoneal injection of 50 mg/kg metrazol, resulting in a brief 2-3 rain generalized seizure. Control animals to this seizure group were given an injection of distilled water. Two h after the seizure, the animals were given chloral hydrate anesthesia and perfused as above.

Histology After perfusion, the tissue was immersed in the fixative solution for 1 h at 4 °C, and then overnight in 30% sucrose at 4 °C. The sections are then cut on a cryostat at -20 °C in ten micron thick sections, and stored at -70 °C until use. For NGFI-A and NGFI-B antibodies, the sections were pretreated with 10% normal goat serum to block non-specific binding for 30 min, 0.5% Triton X-100 to improve penetration for 30 min, and then exposed to primary antibody for 60 min. All reagents were diluted in PBS. The sections were then exposed to the biotinylated secondary anti-rabbit antibody and processed via the ABC ~ystem (Vectastain) according to the manufacturers protocols. Control sections incubated with preimmune serum showed only a diffuse brown background stain. Preabsorption of the antibodies with the appropriate bacterial fusion proteins blocked staining. 1"he anti-fos antibody obtained from Cambridge Research Biochemicals were noted by the manufacturer to recognize fos-related antigens. The manufacturers protocol for this antibody was followed. Essentially, a blocking step of 3% normal rabbit serum in 0.05% Tween 20 and PBS for 30 min was used to block non-specific binding. The primary antibody was used at a 1:1000 dilution in 0.05% Tween 20/PBS solution for 4 h. The sections were then exposed to a biotinylated anti-sheep secondary antibody and processed via the Vectastaia ABC system. If anti-NGFI-A or antiNGFI-B was used in this Tween-based protocol, a slight decrease in staining intensity was seen, although the qualitative results were unchanged. No staining was seen if the anti-los antibodies were used in the Triton-based protocol. Seizure and control injection animals were processed and stained simultaneously. In animals that received unilateral whisker stimulation, the contralateral (stimulated) cortex was compared to the ipsilateral (unstimulated) cortex on the same coronal sections. Tissue sections from 3 to 8 animals per group were used for cell counts. Cells were counted at 200x power using a grid that spanned 500/~m in width. Cells from layer 1 through 6 of the cortex wer-~ counted in the 10/~m thick sections. The cells were counted with the observer being blind to the status (control vs seizure, stimulated side vs unstimulated side) of the tissue section. However, the patterns of staining unique to each treatment often made the identity of the group from which the slide came obvious, particularly in regards to intensity of staining. Data obtained from a second blinded observer on a subset of tissue sections revealed similar resuits. RESULTS Previous immunohistochemical analysis showed prominent immediate early response gene (IEG) antigen 2 h after stimulation by a seizure 7'12 (and unpublished observations with NGFI-B). Based on this similar time course between sets of I E G antigens, 2 h after a sensory stimulation was chosen as an appropriate time to observe antigen induction after a stimulus. Urethane anesthesia was used because of its well-documented minimal effects on electrophysiology, generally thought not to inhibit

electrical activity in many cell populations at low dosage s . With this dose, the animals showed no spontaneous movement besides blinking and breathing. In this state, the animals appeared awake and alertable, as evidenced by the fact that they would b!ink in response to visual threat, and move their body away from a strong painful stimuli. There may be over 100 IEGs available to cells to use in response to stimulation ~s. The specific antigens chosen represent multiple families of transcription factors (leucine zippers, zinc fingers and steroid hormone binding proteins). Much of the work in the field of neuronal I E G induction addresses c-fos expression. The anti-fos antibody used in these studies is directed against a conserved region of c-fos, and therefore ~ecognizes several los-related antigens (personal communication, Cambridge Research Biochemicals). To assure that this antibody allows appropriate comparison to previous work, the expression of los related antigens was studied in animals receiving either a control injection or those that had a metrazol-induced seizure. In the control, nonseized, non-anesthetized animal, only light nuclear expression of the antigen was seen. Cortical cells exhibited only very faint staining in multiple cortical cell layers. Two h after a seizure, expression of los antigens was seen throughout multiple cortical cell layers, including but not predominantly layer 4. In contrast, aRer somatosensory stimulation, cells of the contralateral cortex showed an increased staining primarily in layer 4, as compared to the control cortex from the same animal which showed no obvious induction (Figs. 1 and 2). After tactile stimulation, the induction of c-fos and related antigens was observed primariiy in somatosensory cortex (Fig. 3). Some fainter induction of immunohistochemical staining was observed to a lesser degree in motor and primary olfactory cortex on the side contralateral to the peripheral staining. This induction is visually most apparent with c-fos, because of the low number and very faint staining observed in the control sections. N G F I - A (also called Krox 24, egr-1), a neuronal zinc finger protein, is another I E G inducible after seizures 7' 13,17. Differing from c-fos, N G F I - A was more readily detectable at basal levels in the central nervous system by Northern blot ~3 and immunohistochemistry 7. In this study, post-seizure animals demonstrated a ,,~,,~,~!;~,~ induction of this antigen throughout the cortical layers. The number of I E G positive cells detected was greatest using the antibody to N G F I - A (Table I). In the whisker stimulated animals, this antigen apDeared to be induced at multiple layers (including layers 2, 3, and 6), with layer 4 induction being the most prominent (Figs. 1 and 2). This latter observation may be due in part to the higher level of detectable expression in other cell layers

143 (relative to NGFI-B and c-fos), rather than true differences in expression patterns between transcription factors. NGFI-B, a member of the steroid hormone binding

family, was also expressed in the control central nervous system. Using the current antibodies, staining for NGFI-A was more prominent than for NGFI-B. After a seizure, increa~d NGFI-B staining was detected throug~5o

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Fig. 1. Induction of antigens. Sections illustrate antibody staining in layer 4 of somatosensory cortex, on either the ipsilateral (unstimulated) or contralateral (stimulated) cortex. Comparison panels are taken from the same 10/~m slide section. A: anti-NGFI-A staining. B: antiNGFI-B staining. C: anti-c-los staining.

144 cells after a seizure was seen using all 3 antibodies, c-fos immunoreactivity showed the largest percentage increase after a seizure. In contrast, NGFI-A immunoreactive cells were increased the most in absolute number, although the percentage increase was lower because of the higher num. her of cells stained in the control ~tate. The intensity of staining should be a reflection of the amount of antigen present within cells. Control animals, or control ipsilateral cortex, typically showed lighter stained cells than their stimulated counterparts. This was most oh-

out the cortex, with some layer 4 cells being prominem. In whisker stimulated animals, an induction (Figs. 1 and 2) over the non-stimulated side in layer 4 is seen, although some increased cell staining in the superficial layers is also observed. The results presented here represent data using two different polyclonal antibodies (B1350;B1440), with similar qualitative and quantitative results. Cell counts of immunohistochemically stained cells were obtained in both seizure and sensory stimulation paradigms. induction in the number of positively staining

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Fig. 2. Comparison of stimulated versus non-stimulated cortex. Coronal sections illustrating representative tissue sections of either non-stimulated or stimulated barrel cortex. A: Nissl-stained section through barrel cortex. Along side it are photomicrographs demonstrating NGFI-A immunohistochemical staining from either ipsilateral (control) or contralateral (stimulated) cortex to the side of peripheral stimulation. B: composite drawings of unstimulated and stimulated cortex stained with antibodies to the transcription factors. The drawing of NGFI-A is of the same cells as shown in the photomicrograph of A. Note that differences are most apparent in layer 4 cells.

TABLE I

Positively stained cell number NGFI-A

NGFI-B

c-FOS

Control Seizure % Difference

86.3 111.4 29%

9.1 13.8 52%

9.7 18.0 85%

Control Stimulated % Difference

99.7 115.5 16%

8.2 10.2 23%

9.3 13.1 41%

Values expressed are the mean number of cells positively stained by immunohistochemistry within a 500-/~m-wide column of somatosensory cerebral cortex. Typically, values are the means of at least 20 observations from at least 3 animals in each group. The standard error of the mean ranged from 5 to 15%. Use of the Student's t-test shows that the differences between seizure and their controls, and between control and sensory stimulation are significant at the 0.05 level.

Fig. 3. Camera iucida drawing of fos-immunoreactive cells. Drawing shows location of cell bodies darkly stained with anti-fos antibody. Note that most of the cells stained are in layer 4 of the somatosensory cortex contralateral to the peripheral stir,,.ulation. CONTRA, contralateral to peripheral stimulation; IPSI, ipsilateral to peripheral stimulation; MC, motor cortex; SC, somatosensory cortex; PO, primary olfactory cortex.

146 servable with c-fos and NGFI-B, but also was noted with NGFI-A. Although this study can address the number of detectable cells by each antibody, the exact amount of antigen that was increased could not be directly addressed within such a defined cell population. DISCUSSION Immediate early response genes (lEGs) are rapidly inducible transcription factors that have the ability to affect the rate of transcription of other target genes. Recently it has been shown that a variety of these IEGs were inducible within neurons in the central nervous system (see Sheng and Greenberg t5 for review), and may affect specific target genes such as proenkepahlin ~. Previously published paradigms of IEG induction looked at models of cortical stimulation, in the form of seizures, or transynaptic electrical activity. This study addresses the question whether a 'physiological' peripheral stimulus can affect gene induction. The animals used in these studies are arousable, yet show limited movement that may have affected background levels of these transcription factors. Although a 15 min stimulation with a paint brush is not a natural stimulus for a rat, one could conceive of rodent behaviors (such as foraging) where the rat's continual body movement would result in prolonged stimulation of its whiskers, and presumably barrel somatosensory cortex. The three transcription factors under study have previously been shown to be inducible after seizure stimuli at the mRNA and/or protein level 7'12'tJ'lT,~H. In this study, differences were notable in the number of cells stained as well as the intensity of staining for the IEG antigens. The data here confirms the induction of these factors after a seizure, but additionally demonstrates that this induction occurs in different cell numbers for each transcription factor. Peripheral stimulation of the rat's whiskers also reREFERENCES 1 Bullitt, E., Induction of c-fos-like protein within the lumbar

spinal cord and thalamus of the rat following peripheral stimulation, Brain Res., 493 (1989) 391-397. 2 Day, M.L., Fahrner, T.J,, Aykent. S. and Milbrandt, J,, The zinc finger protein NGFI-A exists in both nuclear and cytoplasmic forms in NGF-stimulated PC,2 celts, J. Biol. Chem., 265 (1990) 15253-15260. 3 Dragunow, M., Currie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate.early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 4 Fahrner, T.J., Carroll, S.L. and Milbrandt, J., The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally, Mol. Cell. Biol., 10 (1990) 6454-6459. 5 Greenberg, M.E., Ziff, E.B. and Greene, L.A., Stimulation of

suited in an observable induction of the transcription factors. Most of the prominent staining was in layer 4, a layer where thalamic and other subcortical input would be expected. With NGFI-A, some inducible immunohistochemical staining was also observed in other layers (ll and III; VI) of the cortex as well. The intensity of the stained cells was increased, suggesting more antigen in each cell. Additionally, the number of cells was also increased in each group at about 50% of the level of induction seen in a seizure (Table I). Quantifying the exact increases in IEG induction is a difficult question to approach. A 50% induction in the amount of cells stained may not mean that there is 50% more antigen. A previous study looking at the amount of NGFI-A antigen induction after a seizure 7 had noted increases observed by Western blot technique that parallel the increases seen in immunohistochemical techniques. However, even with the Western blot method, it is still difficult to quantify the exact amount of antigen increase within CNS tissue. Immunohistochemistry can offer multiple advantages when asking questions of gene induction, including providing information on cell location, regional specificity, and cell number. One important question that these results raise are if the increases in IEG antigen expression have any significant physiological effect. Presumably, one would expect these IEOs to affect other neuronal target genes. One potential target gene, glutamic acid decarboxylase, has already been shown by immunohistochemistry to be increased in the mouse barrel cortex after prolonged intermitte~lt stimulation of the animals whiskers ~9. The induction of other possible transcription factors, their potential targe~ genes, and the precise physiological significance of this phenomenon remain to be determined.

Acknowledgements. This work was supported in part by grants from the PMA Foendation and the AMA.

neuronal acetylcholine receptors induce rapid gene transcrip. tion, Science, 234 (1986) 80-83. 6 Herdegen, T., Walker, T., Leah, J.D., Bravo, R. and Zimmerman, M., The Krox-24 protein, a new transcription regulating factor: expression in the rat central nervous system following afferent somatosensory stimulation, Neurosci. Lett., 120 (1990) 21-24. 7 Mack, K.J., Day, M., Milbrandt, J. and Oottlieb, D.I., Localization of the NGFI-A protein in rat brain, Mol. Brain Res., 8 (1990) 177-180. 8 Maggi, C.A. and Meli, A,, Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part 1: General considerations, Experientia, 42 (1986) 109--210. 9 Menetrey, D., Gannon, A., Levine, J.D. and Basbaum, A.I., Expression of c.fos protein in interneurons and projection neurons of the rat spinal cord in response to noxious somatic, articular, and visceral stimulation, J. Comp. Neurol., 285 (1989)

147 177-195. 10 Milbrandt, J., A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor, Science, 238 (1987) 797-799. 11 Morgan, J.l. and Curran, T., Role of ion influx in the control of c-fos expression, Nature, 322 (1986) 552-555. 12 Morgan, J.l., Cohen, D.R., Hempstead, J.L. and Currsln, T., Mapping patterns of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 13 Saffen, D.W., Cole, A.J., Worley, P.E, Christy, B.A., Ryder, K. and Baraban, J.M., Convulsant-induced increase in transcription factor message RNAs in rat brain, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7795-7799. 14 Sagar, S.M., Sharp, ER. and Curran, T., Expression of c-fos protein in brain: metabolic mapping at the cellular level, Science, 240 (1988) 1328-1331. 15 Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 16 Sonnenberg, J.L., Rauscher, EJ., Morgan, J.I. and Curran, T.,

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Regulation of proenkephalin by los and jun, Science, 246 (1989) 1622-1625. Sukhatme, V.P., Cao, X., Chang, L.C., Tsai-Morris, C.-H., Stamenkovich, D., Ferreira, EC.E, Cohen, D.R., Edwards, S.A., Shows, T.B., Curran, T., LeBeau, M.M. and Adamson, E.D., A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization, Cell, 53 (1988) 37-43. Watson, M.A. and Milbrandt, J., The NGFI-B gene, a transcriptionally inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction, Mol. Cell. Biol., 9 (1989) 4213-4219. Weiker E., Soriano, E., Dorlf, J. and VanderLoos, H., Plasticity in the barrel cortex of the adult mouse: transient increase of OAD-immunoreactivity following sensory stimulation, Exp. Brain Res., 78 (1989) 659-664. Woolsey, T.A. and VanDerLoos, H., The structural organization of layer IV in the somatosensory region (S I) of mouse cerebral cortex, Brain Res., 17 (1970) 205-242.

Induction of transcription factors in somatosensory cortex after tactile stimulation.

Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcript...
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