- Email: [email protected]
Multiple promoters direct stimulus and temporal specific expression of brain-derived neurotrophic factor in the somatosensory cortex
Molecular Brain Research 62 Ž1998. 216–219
Short communication
Multiple promoters direct stimulus and temporal specific expression of brain-derived neurotrophic factor in the somatosensory cortex Steven Nanda, Kenneth J. Mack
)
Neuroscience Training Program, UniÕersity of Wisconsin— Madison, Madison, WI 53705, USA 707 Waisman Center, UniÕersity of Wisconsin— Madison, Madison, WI 53705, USA Accepted 25 August 1998
Abstract Neuronal activity rapidly induces expression of brain-derived neurotrophic factor ŽBDNF. in the adult rat cortex. The rat BDNF gene has four differentially regulated promoters, each of which produce an mRNA containing a unique 5X exon ŽI–IV. and a common 3X exon ŽV. that encodes the mature BDNF protein. The present study used an exon-specific RT-PCR analysis to determine the time course of the induction from both seizures and whisker stimulation. Our data show that specific promoters are utilized at different stages of the activity-dependent induction of the BDNF gene. Furthermore, the data show a differential utilization of the four promoters following a specific stimulus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Brain-derived neurotrophic factor; Immediate-early gene; Barrel cortex
Learning and memory are believed to involve changes in synaptic structure w3,10x and the efficiency of communication between neurons w4x. Brain-derived neurotrophic factor ŽBDNF. plays a key role in regulating synaptic function w7,13x. Neuronal activity regulates the expression of BDNF w11,15,16x, and BDNF has been shown to enhance synaptic transmission w1,2,5x. These studies suggest that BDNF may play a significant role in activity-dependent synaptic plasticity. Previous studies have shown an upregulation of BDNF in the hippocampus following kainate acid treatment and stimulation paradigms which elicit long-term potentiation w11,16x. These studies looked at the combined expression from all four promoters, and did not look at expression within the cortex. Additional work has shown an upregulation of BDNF in the cortex following sensory stimulation w12x, but the in situ hybridization technique that was used is not quantitative, and no attempt was made to determine promoter specific expression. The rat BDNF gene has four differentially regulated promoters, each of which produce an mRNA containing a unique 5X exon ŽI–IV. and a common 3X exon ŽV. that
) Corresponding author. 707 Waisman Center, University of Wisconsin —Madison, Madison, WI 53705, USA. Fax: q1-608-265-4103; E-mail: [email protected]
encodes the mature BDNF protein Žsee Fig. 1. w14x. The current study asks which of the specific BDNF promoters are involved in the activity dependent expression of this gene. This study examines the levels of BDNF mRNA in the rat barrel cortex following whisker sensory stimulation and following seizure. Levels of expression from each of BDNF’s four promoters were determined individually, through a quantitative RT-PCR analysis. An understanding of the regulation of the BDNF gene will give us greater insight into BDNF’s role in activity-dependent synaptic plasticity. Two methods of stimulation included a pentylenetetrazol-induced seizure Ž50 mgrkg of pentylenetetrazol. and direct sensory stimulation of the vibrissae Žwhiskers. w8x. The somatosensory cortex was collected from the rats at 0.5, 1, 2, 4, 8, and 24 h post-stimulation. In total, 80 animals were used; six at each time point for both stimulus paradigms, plus eight water injected controls. Total RNA was extracted using the SNAP Total RNA isolation kit ŽInvitrogen; Carlsbad, CA.. The RT-PCR reactions were performed as previously described w9x, with the following modifications. Unique 5X primers were designed within each of BDNF’s first four exons and were paired with a common 3X primer located within the fifth exon. ŽPlease refer to Fig. 1 for BDNF gene diagram. Exon 1: 5X dCAAGACACATTACCTTCC, exon 2: 5X dCTCCGGGTTGGTATACTG, exon 3: 5X dGACCGGTCTTCCCCA-
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 4 2 - 3
S. Nanda, K.J. Mack r Molecular Brain Research 62 (1998) 216–219
217
Fig. 1. Genomic structure of the rat BDNF gene and RT-PCR primer locations. Exons are indicated by boxes, and introns by lines. The hatched portion of exon V indicates the coding region of the BDNF gene. Rightward arrows indicate forward RT-PCR primers, and the leftward arrow indicates the common reverse primer. The BDNF gene is transcribed from four different promoters, which immediately precede each of the first four exons. Each transcript X X contains a unique 5 exon Žexons I–IV. and a common 3 exon Žexon V. which encodes the mature protein.
GAG, exon 4: 5X dGACCAGGAGCGTGACAAC, exon 5: 5X dGGCCCATTCACGCTCTCC.. PCR primers were designed to amplify b-actin as well. A multiplex PCR reaction was performed so that levels of BDNF expression were normalized to the level of b-actin expression in order to eliminate tube to tube variations. The radioactive signals were quantified using a phosphorimager from Molecular Dynamics ŽSunnyvale, CA. and analyzed with their Imagequant software. The data was presented as a percentage of the control level of expression. Rats given injections of water served as controls for the seizure animals, and the somatosensory cortex ipsilat-
eral to the whisker stimulation served as controls in the sensory stimulation experiments. The RT-PCR results show a differential utilization of the four promoters for both the type of stimulation and the pattern of activation. Expression of transcripts containing exon I start at levels similar to control, rise above levels of control at 4 h post-stimulus, peaking at 8 h post-stimulus, and remain elevated out to 24 h post-stimulus Žsee Fig. 2 for whisker stimulation data and Fig. 3 for seizure data.. Expression of transcripts containing exon II are not significantly different from control levels at any time point. Expression of transcripts containing exons III and IV both
Fig. 2. Expression of BDNF mRNA in the contralateral somatosensory cortex following unilateral whisker stimulation. Expression of mRNAs specific to each promoter were normalized to expression of b-actin, and the results were presented as a percentage of control Ži.e., the ipsilateral or unstimulated somatosensory cortex.. q Indicates values that were significant at P - 0.01 at this time point, relative to control.
218
S. Nanda, K.J. Mack r Molecular Brain Research 62 (1998) 216–219
Fig. 3. Expression of BDNF mRNA in the somatosensory cortex following seizure. Expression of mRNAs specific to each promoter after induction by a pentylenetetrazol-induced seizure Ž50 mgrkg of pentylenetetrazol. were normalized to expression of b-actin, and the results were presented as a percentage of control ŽH 2 O injected rats.. q Indicates values that were significant at P - 0.01 at this time point, relative to control.
rise immediately, peak at 1 h post-stimulus, then return to levels of control by 8 h post-stimulus. The data indicates quantitative differences between the two forms of stimulation. Peak induction of the first promoter transcripts is greater after whisker stimulation than after seizure Ž P - 0.01.. In contrast, transcripts from the third and fourth promoters show a higher peak expression after seizures than whisker stimulation Ž P - 0.01.. These data indicate that the third and fourth promoters are responsible for an early phase of activity dependent regulation of the BDNF gene, while the first promoter appears to involve a later phase of expression. Additionally, the peak level of induction for promoter 1 expression is greater following whisker stimulation, and the peak level of expression for promoter 3 is greater following seizure Ž P - 0.01.. In short, our results show a differential utilization of the four promoters following specific stimuli. The present studies were undertaken to determine whether neuronal activity differentially regulated the expression of cortical BDNF mRNA through the four separate promoters. Timmusk et al. w14x have found that BDNF’s promoters collectively directed the expression of the BDNF gene. While that study determined that a regional distribution of the transcripts from the four promot-
ers existed, our goal was to quantify Žrelative to basal levels. this expression within the somatosensory cortex, a model of experience dependent plasticity used in our laboratory w8x. To this end, we have determined the time course and magnitude of BDNF expression relative to basal levels. Our data illustrate that the four promoters are differentially activated by type of stimulation and express a different temporal pattern of activation. Previous work has determined that activity induces the expression of BDNF exons III and IV containing transcripts as immediate-early gene ŽIEG. type responses w6x. This work showed that the time course of expression for BDNF exons III and IV transcripts in cultured hippocampal slices was not altered by anisomycin treatment, whereas BDNF exons I and II transcript expression was decreased. It is comforting that our results confirm that expression of exons III and IV transcripts have the time course of IEGs, but by additionally examining expression out to 24 h post-stimulus, we were able to notice a late phase of expression mediated through the first promoter. By determining the time course and magnitude of BDNF expression, we have gained additional understanding of the regulation of the BDNF gene. One may postulate that the early-phase of induction, mediated through the third and
S. Nanda, K.J. Mack r Molecular Brain Research 62 (1998) 216–219
fourth promoter, may be responsible for the initiation of BDNF expression. The late-phase of induction, mediated through the first promoter, may be responsible for the long-term maintenance of changes observed with BDNF expression. Furthermore, the quantitative differences between the two stimuli add yet another degree of complexity to BDNF regulation. We speculate that the four promoters of the BDNF gene allow for a highly orchestrated temporal response to a specific stimulus.
w6x
w7x w8x
w9x
Acknowledgements This research was supported by NIH grant NS33913 to KJM.
References w1x Y. Akaneya, T. Tsumoto, S. Kinoshita, H. Hatanaka, Brain-derived neurotrophic factor enhances long-term potentiation in rat visual cortex, J. Neurosci. 17 Ž1997. 6707–6716. w2x J.T. Bartrup, J.M. Moorman, N.R. Newberry, BDNF enhances neuronal growth and synaptic activity in hippocampal cell cultures, NeuroReport 8 Ž1997. 3791–3794. w3x J.E. Cavazos, T.P. Sutula, Progressive neuronal loss induced by kindling: a possible mechanism for mossy fiber synaptic reorganization and hippocampal sclerosis, Brain Res. 527 Ž1990. 1–6, Published erratum appeared in Brain Res. 541 Ž1. ŽFeb 8, 1991. 179. w4x P. Greengard, F. Valtorta, A.J. Czernik, F. Benfenati, Synaptic vesicle phosphoproteins and regulation of synaptic function, Science 259 Ž1993. 780–785. w5x H. Kang, E.M. Schuman, Long-lasting neurotrophin-induced en-
w10x w11x
w12x
w13x w14x
w15x
w16x
219
hancement of synaptic transmission in the adult hippocampus, Science 267 Ž1995. 1658–1662. J.C. Lauterborn, S. Rivera, C.T. Stinis, V.Y. Hayes, P.J. Isackson, C.M. Gall, Differential effects of protein synthesis inhibition on the activity-dependent expression of BDNF transcripts: evidence for immediate-early gene responses from specific promoters, J. Neurosci. 16 Ž1996. 7428–7436. D.C. Lo, Neurotrophic factors and synaptic plasticity, Neuron 15 Ž1995. 979–981. K.J. Mack, P.A. Mack, Induction of transcription factors in somatosensory cortex after tactile stimulation, Brain Res. Mol. Brain Res. 12 Ž1992. 141–147. K.J. Mack, S.D. Yi, S. Chang, N. Millan, P. Mack, NGFI-C expression is affected by physiological stimulation and seizures in the somatosensory cortex, Brain Res. Mol. Brain Res. 29 Ž1995. 140–146. P.R. Montague, Transforming sensory experience into structural change, Proc. Natl. Acad. Sci. USA 90 Ž1993. 6379–6380. S.L. Patterson, L.M. Grover, P.A. Schwartzkroin, M. Bothwell, Neurotrophin expression in rat hippocampal slices: a stimulus paradigm inducing LTP in CA1 evokes increases in BDNF and NT-3 mRNAs, Neuron 9 Ž1992. 1081–1088. N. Rocamora, E. Welker, M. Pascual, E. Soriano, Upregulation of BDNF mRNA expression in the barrel cortex of adult mice after sensory stimulation, J. Neurosci. 16 Ž1996. 4411–4419. H. Thoenen, Neurotrophins and neuronal plasticity, Science 270 Ž1995. 593–598. T. Timmusk, K. Palm, M. Metsis, T. Reintam, V. Paalme, M. Saarma, H. Persson, Multiple promoters direct tissue-specific expression of the rat BDNF gene, Neuron 10 Ž1993. 475–489. F. Zafra, B. Hengerer, J. Leibrock, H. Thoenen, D. Lindholm, Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors, EMBO J. 9 Ž1990. 3545–3550. F. Zafra, D. Lindholm, E. Castren, J. Hartikka, H. Thoenen, Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes, J. Neurosci. 12 Ž1992. 4793–4799.
Short communication
Multiple promoters direct stimulus and temporal specific expression of brain-derived neurotrophic factor in the somatosensory cortex Steven Nanda, Kenneth J. Mack
)
Neuroscience Training Program, UniÕersity of Wisconsin— Madison, Madison, WI 53705, USA 707 Waisman Center, UniÕersity of Wisconsin— Madison, Madison, WI 53705, USA Accepted 25 August 1998
Abstract Neuronal activity rapidly induces expression of brain-derived neurotrophic factor ŽBDNF. in the adult rat cortex. The rat BDNF gene has four differentially regulated promoters, each of which produce an mRNA containing a unique 5X exon ŽI–IV. and a common 3X exon ŽV. that encodes the mature BDNF protein. The present study used an exon-specific RT-PCR analysis to determine the time course of the induction from both seizures and whisker stimulation. Our data show that specific promoters are utilized at different stages of the activity-dependent induction of the BDNF gene. Furthermore, the data show a differential utilization of the four promoters following a specific stimulus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Brain-derived neurotrophic factor; Immediate-early gene; Barrel cortex
Learning and memory are believed to involve changes in synaptic structure w3,10x and the efficiency of communication between neurons w4x. Brain-derived neurotrophic factor ŽBDNF. plays a key role in regulating synaptic function w7,13x. Neuronal activity regulates the expression of BDNF w11,15,16x, and BDNF has been shown to enhance synaptic transmission w1,2,5x. These studies suggest that BDNF may play a significant role in activity-dependent synaptic plasticity. Previous studies have shown an upregulation of BDNF in the hippocampus following kainate acid treatment and stimulation paradigms which elicit long-term potentiation w11,16x. These studies looked at the combined expression from all four promoters, and did not look at expression within the cortex. Additional work has shown an upregulation of BDNF in the cortex following sensory stimulation w12x, but the in situ hybridization technique that was used is not quantitative, and no attempt was made to determine promoter specific expression. The rat BDNF gene has four differentially regulated promoters, each of which produce an mRNA containing a unique 5X exon ŽI–IV. and a common 3X exon ŽV. that
) Corresponding author. 707 Waisman Center, University of Wisconsin —Madison, Madison, WI 53705, USA. Fax: q1-608-265-4103; E-mail: [email protected]
encodes the mature BDNF protein Žsee Fig. 1. w14x. The current study asks which of the specific BDNF promoters are involved in the activity dependent expression of this gene. This study examines the levels of BDNF mRNA in the rat barrel cortex following whisker sensory stimulation and following seizure. Levels of expression from each of BDNF’s four promoters were determined individually, through a quantitative RT-PCR analysis. An understanding of the regulation of the BDNF gene will give us greater insight into BDNF’s role in activity-dependent synaptic plasticity. Two methods of stimulation included a pentylenetetrazol-induced seizure Ž50 mgrkg of pentylenetetrazol. and direct sensory stimulation of the vibrissae Žwhiskers. w8x. The somatosensory cortex was collected from the rats at 0.5, 1, 2, 4, 8, and 24 h post-stimulation. In total, 80 animals were used; six at each time point for both stimulus paradigms, plus eight water injected controls. Total RNA was extracted using the SNAP Total RNA isolation kit ŽInvitrogen; Carlsbad, CA.. The RT-PCR reactions were performed as previously described w9x, with the following modifications. Unique 5X primers were designed within each of BDNF’s first four exons and were paired with a common 3X primer located within the fifth exon. ŽPlease refer to Fig. 1 for BDNF gene diagram. Exon 1: 5X dCAAGACACATTACCTTCC, exon 2: 5X dCTCCGGGTTGGTATACTG, exon 3: 5X dGACCGGTCTTCCCCA-
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 4 2 - 3
S. Nanda, K.J. Mack r Molecular Brain Research 62 (1998) 216–219
217
Fig. 1. Genomic structure of the rat BDNF gene and RT-PCR primer locations. Exons are indicated by boxes, and introns by lines. The hatched portion of exon V indicates the coding region of the BDNF gene. Rightward arrows indicate forward RT-PCR primers, and the leftward arrow indicates the common reverse primer. The BDNF gene is transcribed from four different promoters, which immediately precede each of the first four exons. Each transcript X X contains a unique 5 exon Žexons I–IV. and a common 3 exon Žexon V. which encodes the mature protein.
GAG, exon 4: 5X dGACCAGGAGCGTGACAAC, exon 5: 5X dGGCCCATTCACGCTCTCC.. PCR primers were designed to amplify b-actin as well. A multiplex PCR reaction was performed so that levels of BDNF expression were normalized to the level of b-actin expression in order to eliminate tube to tube variations. The radioactive signals were quantified using a phosphorimager from Molecular Dynamics ŽSunnyvale, CA. and analyzed with their Imagequant software. The data was presented as a percentage of the control level of expression. Rats given injections of water served as controls for the seizure animals, and the somatosensory cortex ipsilat-
eral to the whisker stimulation served as controls in the sensory stimulation experiments. The RT-PCR results show a differential utilization of the four promoters for both the type of stimulation and the pattern of activation. Expression of transcripts containing exon I start at levels similar to control, rise above levels of control at 4 h post-stimulus, peaking at 8 h post-stimulus, and remain elevated out to 24 h post-stimulus Žsee Fig. 2 for whisker stimulation data and Fig. 3 for seizure data.. Expression of transcripts containing exon II are not significantly different from control levels at any time point. Expression of transcripts containing exons III and IV both
Fig. 2. Expression of BDNF mRNA in the contralateral somatosensory cortex following unilateral whisker stimulation. Expression of mRNAs specific to each promoter were normalized to expression of b-actin, and the results were presented as a percentage of control Ži.e., the ipsilateral or unstimulated somatosensory cortex.. q Indicates values that were significant at P - 0.01 at this time point, relative to control.
218
S. Nanda, K.J. Mack r Molecular Brain Research 62 (1998) 216–219
Fig. 3. Expression of BDNF mRNA in the somatosensory cortex following seizure. Expression of mRNAs specific to each promoter after induction by a pentylenetetrazol-induced seizure Ž50 mgrkg of pentylenetetrazol. were normalized to expression of b-actin, and the results were presented as a percentage of control ŽH 2 O injected rats.. q Indicates values that were significant at P - 0.01 at this time point, relative to control.
rise immediately, peak at 1 h post-stimulus, then return to levels of control by 8 h post-stimulus. The data indicates quantitative differences between the two forms of stimulation. Peak induction of the first promoter transcripts is greater after whisker stimulation than after seizure Ž P - 0.01.. In contrast, transcripts from the third and fourth promoters show a higher peak expression after seizures than whisker stimulation Ž P - 0.01.. These data indicate that the third and fourth promoters are responsible for an early phase of activity dependent regulation of the BDNF gene, while the first promoter appears to involve a later phase of expression. Additionally, the peak level of induction for promoter 1 expression is greater following whisker stimulation, and the peak level of expression for promoter 3 is greater following seizure Ž P - 0.01.. In short, our results show a differential utilization of the four promoters following specific stimuli. The present studies were undertaken to determine whether neuronal activity differentially regulated the expression of cortical BDNF mRNA through the four separate promoters. Timmusk et al. w14x have found that BDNF’s promoters collectively directed the expression of the BDNF gene. While that study determined that a regional distribution of the transcripts from the four promot-
ers existed, our goal was to quantify Žrelative to basal levels. this expression within the somatosensory cortex, a model of experience dependent plasticity used in our laboratory w8x. To this end, we have determined the time course and magnitude of BDNF expression relative to basal levels. Our data illustrate that the four promoters are differentially activated by type of stimulation and express a different temporal pattern of activation. Previous work has determined that activity induces the expression of BDNF exons III and IV containing transcripts as immediate-early gene ŽIEG. type responses w6x. This work showed that the time course of expression for BDNF exons III and IV transcripts in cultured hippocampal slices was not altered by anisomycin treatment, whereas BDNF exons I and II transcript expression was decreased. It is comforting that our results confirm that expression of exons III and IV transcripts have the time course of IEGs, but by additionally examining expression out to 24 h post-stimulus, we were able to notice a late phase of expression mediated through the first promoter. By determining the time course and magnitude of BDNF expression, we have gained additional understanding of the regulation of the BDNF gene. One may postulate that the early-phase of induction, mediated through the third and
S. Nanda, K.J. Mack r Molecular Brain Research 62 (1998) 216–219
fourth promoter, may be responsible for the initiation of BDNF expression. The late-phase of induction, mediated through the first promoter, may be responsible for the long-term maintenance of changes observed with BDNF expression. Furthermore, the quantitative differences between the two stimuli add yet another degree of complexity to BDNF regulation. We speculate that the four promoters of the BDNF gene allow for a highly orchestrated temporal response to a specific stimulus.
w6x
w7x w8x
w9x
Acknowledgements This research was supported by NIH grant NS33913 to KJM.
References w1x Y. Akaneya, T. Tsumoto, S. Kinoshita, H. Hatanaka, Brain-derived neurotrophic factor enhances long-term potentiation in rat visual cortex, J. Neurosci. 17 Ž1997. 6707–6716. w2x J.T. Bartrup, J.M. Moorman, N.R. Newberry, BDNF enhances neuronal growth and synaptic activity in hippocampal cell cultures, NeuroReport 8 Ž1997. 3791–3794. w3x J.E. Cavazos, T.P. Sutula, Progressive neuronal loss induced by kindling: a possible mechanism for mossy fiber synaptic reorganization and hippocampal sclerosis, Brain Res. 527 Ž1990. 1–6, Published erratum appeared in Brain Res. 541 Ž1. ŽFeb 8, 1991. 179. w4x P. Greengard, F. Valtorta, A.J. Czernik, F. Benfenati, Synaptic vesicle phosphoproteins and regulation of synaptic function, Science 259 Ž1993. 780–785. w5x H. Kang, E.M. Schuman, Long-lasting neurotrophin-induced en-
w10x w11x
w12x
w13x w14x
w15x
w16x
219
hancement of synaptic transmission in the adult hippocampus, Science 267 Ž1995. 1658–1662. J.C. Lauterborn, S. Rivera, C.T. Stinis, V.Y. Hayes, P.J. Isackson, C.M. Gall, Differential effects of protein synthesis inhibition on the activity-dependent expression of BDNF transcripts: evidence for immediate-early gene responses from specific promoters, J. Neurosci. 16 Ž1996. 7428–7436. D.C. Lo, Neurotrophic factors and synaptic plasticity, Neuron 15 Ž1995. 979–981. K.J. Mack, P.A. Mack, Induction of transcription factors in somatosensory cortex after tactile stimulation, Brain Res. Mol. Brain Res. 12 Ž1992. 141–147. K.J. Mack, S.D. Yi, S. Chang, N. Millan, P. Mack, NGFI-C expression is affected by physiological stimulation and seizures in the somatosensory cortex, Brain Res. Mol. Brain Res. 29 Ž1995. 140–146. P.R. Montague, Transforming sensory experience into structural change, Proc. Natl. Acad. Sci. USA 90 Ž1993. 6379–6380. S.L. Patterson, L.M. Grover, P.A. Schwartzkroin, M. Bothwell, Neurotrophin expression in rat hippocampal slices: a stimulus paradigm inducing LTP in CA1 evokes increases in BDNF and NT-3 mRNAs, Neuron 9 Ž1992. 1081–1088. N. Rocamora, E. Welker, M. Pascual, E. Soriano, Upregulation of BDNF mRNA expression in the barrel cortex of adult mice after sensory stimulation, J. Neurosci. 16 Ž1996. 4411–4419. H. Thoenen, Neurotrophins and neuronal plasticity, Science 270 Ž1995. 593–598. T. Timmusk, K. Palm, M. Metsis, T. Reintam, V. Paalme, M. Saarma, H. Persson, Multiple promoters direct tissue-specific expression of the rat BDNF gene, Neuron 10 Ž1993. 475–489. F. Zafra, B. Hengerer, J. Leibrock, H. Thoenen, D. Lindholm, Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors, EMBO J. 9 Ž1990. 3545–3550. F. Zafra, D. Lindholm, E. Castren, J. Hartikka, H. Thoenen, Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes, J. Neurosci. 12 Ž1992. 4793–4799.