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granule cell synapses is associated with a post-synaptic induction of proenkephalin gene expression
Neuroscience Letters 227 (1997) 205–208
Long-term potentiation in perforant path/granule cell synapses is associated with a post-synaptic induction of proenkephalin gene expression L.A. Roberts a, C.H. Large b, C.T. O’Shaughnessy b, B.J. Morris a ,* a
Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, West Medical Building, Glasgow University, Glasgow, UK b GlaxoWellcome Research and Development Ltd., Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK Received 12 February 1997; revised version received 6 May 1997; accepted 8 May 1997
Abstract Enkephalin peptides released from hippocampal mossy fibres lower the threshold for the generation of long-term potentiation (LTP) at the mossy fibre synapses. High frequency stimulation of the hippocampal dentate gyrus, sufficient to induce mossy fibre LTP, is associated with increased expression of the proenkephalin gene in the granule cells. We show here that a similar elevation in proenkephalin mRNA levels is observed, in anaesthetised rats, following stimulation of the perforant path sufficient to induce LTP in the perforant path/granule cell synapses. This strengthens the evidence implicating granule cell enkephalins as mediators of functional plasticity in the hippocampus. Furthermore, the results hint at a form of ‘domino plasticity’, where potentiation of transmission at the perforant path/granule cell synapses is subsequently followed by an enkephalin-mediated potentiation of transmission at the mossy fibre synapses. 1997 Elsevier Science Ireland Ltd. Keywords: Long-term potentiation; Enkephalins; Hippocampus; Dentate gyrus; Gene expression; Plasticity
The phenomenon of long-term potentiation (LTP) in the hippocampus has become widely accepted as a possible physiological substrate for memory formation [1]. These long-lasting increases in the efficiency of synapses which have been subjected to high frequency afferent stimulation can be maintained in vivo for days or weeks. There has been considerable interest in trying to identify the cellular mechanisms which allow LTP to occur, and it has now become clear that the most sustained phase of LTP (lasting for 3–6 h or longer) is dependent on de novo protein synthesis [1,4,8,15]. A number of different proteins are likely to be involved in changing the properties of synaptic transmission during this late phase of LTP, and evidence from experiments studying alterations in mRNA levels after high frequency stimulation has provided some clues to their identity. Twenty-four hours following high frequency stimulation of the perforant path, which synapses on to the dendrites of * Corresponding author. Pharmacological laboratories, Institute of Biomedical and Life Sciences, West Medical Building, Glasgow University, Glasgow, UK.
the hippocampal granule cells, changes can be observed in the levels of the mRNAs encoding the a subunit of calcium/ calmodulin-dependent protein kinase II (CamKIIa) [20], and protein kinase C isoforms [20]. Both of these kinases have well-characterised roles in synaptic plasticity [1]. A further series of changes in gene expression, involving CamKIIa [11,18], cyclooxygenase 2 [25], tissue plasminogen activator [16], and arc [9,10] genes, can be detected 1–3 h after the induction of LTP. Similarly, 24 h after induction of LTP in the mossy fibre/CA3 pyramidal cell synapses, the levels of prodynorphin (PDYN) mRNA are reduced in the dentate gyrus, while the levels of proenkephalin (PENK) mRNA are correspondingly increased [13]. Since dynorphins act to suppress transmission at the mossy fibre synapses [21], while enkephalins act to enhance neurotransmission [2,3,22], this switch from dynorphinergic to enkephalinergic activity in the mossy fibres is likely to contribute to the sustained potentiation of the efficiency of the mossy fibre synapses [14]. In contrast to the PDYN mRNA levels, which change relatively slowly [13], the increase in PENK mRNA levels occurs within a few hours of the stimulus (H.M. Johnston
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and B.J. Morris, unpublished observations), suggesting that there may be a time after the induction of LTP where the altered PENK expression plays a relatively greater role in sustaining the plasticity. In addition to electrical stimulation, the decrease in PDYN gene expression and the elevation in PENK gene expression in the granule cells of the dentate gyrus can be produced by stimulation of N-methyl-d-aspartate (NMDA) receptors on the granule cells [6,7]. This is of interest, considering that the induction of LTP in the perforant path/ granule cell synapses is dependent on NMDA receptor activation [1]. There is the intriguing possibility that potentiation of the perforant path synapses induces the changes in PENK expression in the post-synaptic granule cells that then, in turn, contribute to potentiation of the mossy fibres. In this study, we have tested the hypothesis that induction of LTP in the perforant path/granule cell synapses results in an elevation in PENK expression in the post-synaptic granule cells. Male Lister hooded rats (250–350 g) were anaesthetised with urethane (1.8 g/kg). Bipolar stimulating electrodes (Rhodes Medical Instruments Inc.) were positioned bilaterally in the medial perforant path (4.2 mm lateral to lambda). A glass microelectrode was positioned in the hilus of the right dentate gyrus (ipsilateral side; 2.5 mm lateral to the midline, 4 mm caudal to bregma). Electrode positions were optimised by maximising the responses to ipsi- and contralateral stimulation. Square wave pulses (120–400 mA, 50 ms) were applied to both stimulating electrodes, and a current which evoked a 1 mV population spike chosen as the test stimulus for both sides. Baseline responses to test stimuli were recorded every 30 s for 30 min prior to induction of LTP. LTP was induced on the ipsilateral side using three high frequency trains of pulses (50 pulses at 250 Hz, at twice test pulse amplitude, inter-train interval was 60 s). A similar number of pulses at low frequency was applied to the contralateral perforant path as a control. Potentiation of excitatory postsynaptic potential (EPSP) slope and population spike amplitude was measured 2 h after the tetani. Some animals received i.p. injections of 5 mg/kg 3-((R)-2carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP; Tocris Cookson), a competitive NMDA antagonist, 90 min prior to the tetanus. Two hours following the stimulus, the brains were removed, and cryostat sections (14 mm) cut through the hippocampal region. The sections were fixed in 4% paraformaldehyde and processed for in situ hybridisation as described. The 45-mer oligonucleotide probes used to detect PENK mRNA and tubulin T26 mRNA were as previously described [7], and were 3′ end-labelled with [35S]dATP. Hybridisation was conducted in buffer containing 50% formamide and 4 × standard saline citrate solution (SSC) [24] at 42°C, while post-hybridisation washes were conducted in 1 × SSC at 55°C. Sections were exposed to X-ray film (Kodak Biomax) before dipping in Ilford K5 liquid photographic emulsion and exposing for 5 weeks. The hybridisa-
tion signal was quantified by computerised image analysis (Image v1.52; NIH). Since NMDA receptor stimulation results in increased numbers of granule cells expressing PENK mRNA [7,14], the results for PENK mRNA were expressed as the percentage increase in the number of labelled cells ipsilateral to the stimulation relative to the contralateral side. Conversely, since the tubulin T26 gene is expressed in all granule cells, the results for tubulin T26 mRNA were expressed as the percentage increase in the autoradiographic grain density over the fascia dentata ipsilateral to the stimulation relative to the contralateral side [7]. High frequency unilateral stimulation of the perforant path in vivo produced a robust potentiation in the ipsilateral dentate gyrus (Fig. 1) of both EPSP slope (+22% of prestimulation size (5%, n = 6) and population spike amplitude (+326% of prestimulation size ±64%, n = 6). In animals pretreated with R-CPP, however, no potentiation of EPSP slope or population spike amplitude was observed. Two hours following low frequency stimulation, the levels of PENK mRNA were equivalent to those in normal rats, with only a small percentage of the granule cells expressing detectable PENK mRNA. However, 2 h following high frequency stimulation, there was an increase in the number of granule cells expressing PENK mRNA in the dentate gyrus (Fig. 2, Table 1). In contrast, high frequency stimulation did not alter the levels of tubulin T26 mRNA in the granule cells (Table 1). No increase in dentate gyrus proenkephalin
Fig. 1. Typical examples of (a) population spike amplitude, and (b) EPSP slope, recorded in the hilus following high frequency stimulation of the perforant path at t = 30 min. The graph shows average data points from a single experiment, each point is the mean ± SEM of four responses.
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Fig. 2. Cells in the dentate gyrus expressing PENK mRNA contralateral (a) and ipsilateral (b) to high frequency stimulation of the perforant path 2 h earlier. Note the greater number of labelled neurones observed following the induction of LTP. Scale bar, 40 mm.
expression was observed following high frequency stimulation in animals pretreated with the NMDA receptor antagonist R-CPP (Table 1). These results show that PENK gene expression is markedly enhanced in hippocampal granule cells following the induction of LTP in the afferent synapses. Since LTP induction in this area involves NMDA receptor stimulation, this is consistent with our previous work showing that pharmacological activation of NMDA receptors on the granule cell dendrites produces a similar enhancement of PENK expression [6,7]. There is evidence that the use of anaesthetised, as opposed to conscious, animals can affect the characteristics of LTP induction in vivo [5]. In anaesthetised animals, the most sustained phase of LTP, decaying with a time constant of around 20 days (‘LTP3’), is reportedly not seen, and perforant path LTP fails to induce the immediate early gene c-fos in the granule cells. However, the other phases of LTP, including the late phase that decays with a time constant of around 5 days (‘LTP2’) are still observed in anaesthetised animals, and indeed many changes in gene expression have been reported using anaesthetised animals [5,23]. Both LTP2 and LTP3 are thought to be dependent on de novo protein synthesis [1,15]. It seems likely that the use of anaesthetised animals will selectively reveal those components of the changing pattern of gene expression that contribute to LTP2 rather then LTP3. A number of other changes in gene expression have been detected around 2 h following high frequency stimulation of the perforant path [12]. These include the CamKIIa [11,18], cyclooxygenase 2 [25], tissue plasminogen activator [16], and arc [9,10] genes. In each of these cases, the alteration in gene expression can be predicted to alter the characteristics of neurotransmission at the perforant path/granule cell synapses which have been potentiated. However, the increase in PENK expression we have reported here is unique,
because the enkephalins produced are transported to the terminals of the mossy fibres, and act at the granule cell/ CA3 pyramidal cell synapses. The effect of enhanced proenkephalin gene expression will therefore be manifest at a synapse downstream from the one which has been stimulated. This suggests that a phenomenon of ‘domino plasticity’ occurs, where high frequency activity can affect the characteristics of neurotransmission at a chain of synapses, and not just at the synapse initially stimulated [14]. Additional evidence for this phenomenon comes from studies into the expression of the synaptic vesicle-associated protein syntaxin. Syntaxin expression in the mossy fibre terminals is also increased by the induction of LTP at the perforant path/ granule cell synapses [19]. Our results therefore have implications beyond the role of enkephalins in hippocampal plasticity. Since enkephalins act to potentiate neurotransmission at the mossy fibre/CA3 synapses [2,3,22], the enhanced enkeTable 1 Effect of LTP induction on the expression of PENK mRNA and tubulin T26 mRNA in the dentate gyrus mRNA species
Proenkephalin Tubulin T26
Hybridisation signal (% control) High frequency stimulation (6)
High frequency stimulation + CPP (3)
222.5 ± 31.1* 121.3 ± 11.6
96.6 ± 5.6** ND
Values are the mean ± SEM from the number of animals shown in parentheses. The hybridisation signal was assessed for each probe as described [7], and the signal on the side ipsilateral to the high frequency stimulation expressed as a percentage of the signal on the contralateral (control) side. *Median significantly different from 100% (P , 0.05) by Wilcoxon signed rank test. **Median significantly different from group receiving high frequency stimulation alone (P , 0.05) by Mann–Whitney U-test. ND, not determined.
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phalinergic gene expression in the granule cells following high frequency synaptic activity is likely to contribute to a sustained elevation in the efficiency of information flow in the trisynaptic pathway through the hippocampus [14]. Elevated expression of the proenkephalin gene in the dentate gyrus has been observed in hippocampal tissue from patients with temporal lobe epilepsy [17]. Our results further emphasise the importance of hippocampal opioid peptides for the physiological and pathological regulation of hippocampal excitability. [1] Bliss, T.V.P. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in the hippocampus, Nature, 361 (1993) 31– 38. [2] Derrick, B.E. and Martinez, J.L., Opioid receptor activation is one factor underlying the frequency dependence of mossy fibre LTP induction, J. Neurosci., 14 (1994) 4359–4367. [3] Derrick, B.E., Rodriguez, S.B., Lieberman, D.N. and Martinez, J.L., Mu opioid receptors are associated with the induction of hippocampal mossy fibre long-term potentiation, J. Pharmacol. Exp. Ther., 263 (1992) 725–733. [4] Fazeli, M.S., Corbet, J., Dunn, M.J., Dolphin, A.C. and Bliss, T.V.P., Changes in protein synthesis accompanying long-term potentiation in the dentate gyrus in vivo, J. Neurosci., 13 (1993) 1346–1353. [5] Jeffrey, K.J., Abraham, W.C., Dragunow, M. and Mason, S.E., Induction of fos-like immunoreactivity and the maintenance of long-term potentiation in the dentate gyrus of unanaesthetised rats, Mol. Brain Res., 8 (1990) 267–284. [6] Johnston, H.M. and Morris, B.J., Increased c-fos expression does not cause the elevated proenkephalin mRNA levels in the dentate gyrus following NMDA receptor stimulation, Mol. Brain Res., 25 (1994) 147–150. [7] Johnston, H.M. and Morris, B.J., Nitric oxide alters proenkephalin and prodynorphin gene expression in hippocampal granule cells, Neuroscience, 61 (1994) 435–439. [8] Krug, M., Lossner, B. and Ott, T., Anisomycin blocks the late phase of long-term potentiation in the dentate gyrus of freely moving rats, Brain Res. Bull., 13 (1984) 39–42. [9] Link, W., Konietzo, U., Kauselmann, G., Krug, M., Schwanke, B., Frey, U. and Kuhl, D., Somatodendritic expression of an immediateearly gene is regulated by synaptic activity, Proc. Natl. Acad. Sci. USA, 92 (1995) 5734–5738. [10] Lyford, G.L., Yamagata, K., Kaufmann, W.E., Barnes, C.A., Sanders, L.K., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Lanahan, A.A. and Worley, P.F., Arc, a growth factor and activityregulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites, Neuron, 14 (1995) 433–445. [11] Mackler, S.A., Brooks, B.P. and Eberwine, J.H., Stimulus-induced coordinate changes in mRNA abundance in single post-synaptic hippocampal neurones, Neuron, 9 (1992) 539–551.
[12] Morris, B.J., Neuronal plasticity. In R.W. Davies and B.J. Morris (Eds.), The Molecular Biology of the Neuron, Bios, Oxford, UK, 1996. [13] Morris, B.J., Feasey, K.J., ten Bruggencate, G., Herz, A. and Ho¨llt, V., Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in rat hippocampal granule cells, Proc. Natl. Acad. Sci. USA, 85 (1988) 3226–3230. [14] Morris, B.J. and Johnston, H.M., A role for hippocampal opioids in long-term functional plasticity, Trends Neurosci., 18 (1995) 350– 355. [15] Otani, S., Marshall, C.J., Tate, W.P., Goddard, G.V. and Abraham, W.C., Maintenance of long-term potentiation in rat dentate gyrus requires protein synthesis but not messenger RNA synthesis immediately post-tetanisation, Neuroscience, 28 (1989) 519–530. [16] Qian, Z., Gilbert, M.E., Colicos, M.A., Kandel, E.R. and Kuhl, D., Tissue plasminogen activator is induced as an immediate-early gene during seizure, kindling and long-term potentiation, Nature, 361 (1993) 453–456. [17] Rees, H., Ang, L.-C., Shul, D.D., George, D.H., Begley, H. and McConnell, T., Increase in enkephalin-like immunoreactivity in hippocampi of adults with generalised epilepsy, Brain Res., 652 (1994) 113–119. [18] Roberts, L.A., Higgins, M.J., O’Shaughnessy, C.T., Stone, T.W. and Morris, B.J., Changes in hippocampal gene expression associated with the induction of long-term potentiation, Mol. Brain Res., 42 (1996) 123–127. [19] Smirnova, T., Laroche, S., Errington, M.L., Hicks, A., Bliss, T.V.P. and Mallet, J., Trans-synaptic expression of a presynaptic glutamate receptor during hippocampal long-term potentiation, Science, 262 (1993) 433–437. [20] Thomas, K.L., Laroche, S., Errington, M.L., Bliss, T.V.P. and Hunt, S.P., Spatial and temporal changes in signal transduction pathways during LTP, Neuron, 13 (1994) 737–745. [21] Weisskopf, M.G., Zalutsky, R.A. and Nicoll, R.A., The opioid peptide dynorphin mediates heterosynaptic depression of hippocampal mossy fibre synapses and modulates long-term potentiation, Nature, 362 (1993) 423–427. [22] Williams, S.H. and Johnston, D., Actions of endogenous opioids on NMDA-receptor-independent long-term potentiation in area CA3 of the hippocampus, J. Neurosci., 16 (1996) 3652–3661. [23] Wisden, W., Errington, M.L., Williams, S., Dunnett, S.B., Waters, C., Hitchcock, D., Evan, G., Bliss, T.V.P. and Hunt, S.P., Differential expression of immediate-early genes in the hippocampus and spinal cord, Neuron, 4 (1990) 603–614. [24] Wisden, W. and Morris, B.J., In Situ Hybridisation Protocols for the Brain, Academic Press, New York, 1994. [25] Yamagata, K., Andreasson, K.I., Kaufmann, W.E., Barnes, C.A. and Worley, P.F., Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity, Neuron, 11 (1993) 371–379.
Long-term potentiation in perforant path/granule cell synapses is associated with a post-synaptic induction of proenkephalin gene expression L.A. Roberts a, C.H. Large b, C.T. O’Shaughnessy b, B.J. Morris a ,* a
Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, West Medical Building, Glasgow University, Glasgow, UK b GlaxoWellcome Research and Development Ltd., Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK Received 12 February 1997; revised version received 6 May 1997; accepted 8 May 1997
Abstract Enkephalin peptides released from hippocampal mossy fibres lower the threshold for the generation of long-term potentiation (LTP) at the mossy fibre synapses. High frequency stimulation of the hippocampal dentate gyrus, sufficient to induce mossy fibre LTP, is associated with increased expression of the proenkephalin gene in the granule cells. We show here that a similar elevation in proenkephalin mRNA levels is observed, in anaesthetised rats, following stimulation of the perforant path sufficient to induce LTP in the perforant path/granule cell synapses. This strengthens the evidence implicating granule cell enkephalins as mediators of functional plasticity in the hippocampus. Furthermore, the results hint at a form of ‘domino plasticity’, where potentiation of transmission at the perforant path/granule cell synapses is subsequently followed by an enkephalin-mediated potentiation of transmission at the mossy fibre synapses. 1997 Elsevier Science Ireland Ltd. Keywords: Long-term potentiation; Enkephalins; Hippocampus; Dentate gyrus; Gene expression; Plasticity
The phenomenon of long-term potentiation (LTP) in the hippocampus has become widely accepted as a possible physiological substrate for memory formation [1]. These long-lasting increases in the efficiency of synapses which have been subjected to high frequency afferent stimulation can be maintained in vivo for days or weeks. There has been considerable interest in trying to identify the cellular mechanisms which allow LTP to occur, and it has now become clear that the most sustained phase of LTP (lasting for 3–6 h or longer) is dependent on de novo protein synthesis [1,4,8,15]. A number of different proteins are likely to be involved in changing the properties of synaptic transmission during this late phase of LTP, and evidence from experiments studying alterations in mRNA levels after high frequency stimulation has provided some clues to their identity. Twenty-four hours following high frequency stimulation of the perforant path, which synapses on to the dendrites of * Corresponding author. Pharmacological laboratories, Institute of Biomedical and Life Sciences, West Medical Building, Glasgow University, Glasgow, UK.
the hippocampal granule cells, changes can be observed in the levels of the mRNAs encoding the a subunit of calcium/ calmodulin-dependent protein kinase II (CamKIIa) [20], and protein kinase C isoforms [20]. Both of these kinases have well-characterised roles in synaptic plasticity [1]. A further series of changes in gene expression, involving CamKIIa [11,18], cyclooxygenase 2 [25], tissue plasminogen activator [16], and arc [9,10] genes, can be detected 1–3 h after the induction of LTP. Similarly, 24 h after induction of LTP in the mossy fibre/CA3 pyramidal cell synapses, the levels of prodynorphin (PDYN) mRNA are reduced in the dentate gyrus, while the levels of proenkephalin (PENK) mRNA are correspondingly increased [13]. Since dynorphins act to suppress transmission at the mossy fibre synapses [21], while enkephalins act to enhance neurotransmission [2,3,22], this switch from dynorphinergic to enkephalinergic activity in the mossy fibres is likely to contribute to the sustained potentiation of the efficiency of the mossy fibre synapses [14]. In contrast to the PDYN mRNA levels, which change relatively slowly [13], the increase in PENK mRNA levels occurs within a few hours of the stimulus (H.M. Johnston
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and B.J. Morris, unpublished observations), suggesting that there may be a time after the induction of LTP where the altered PENK expression plays a relatively greater role in sustaining the plasticity. In addition to electrical stimulation, the decrease in PDYN gene expression and the elevation in PENK gene expression in the granule cells of the dentate gyrus can be produced by stimulation of N-methyl-d-aspartate (NMDA) receptors on the granule cells [6,7]. This is of interest, considering that the induction of LTP in the perforant path/ granule cell synapses is dependent on NMDA receptor activation [1]. There is the intriguing possibility that potentiation of the perforant path synapses induces the changes in PENK expression in the post-synaptic granule cells that then, in turn, contribute to potentiation of the mossy fibres. In this study, we have tested the hypothesis that induction of LTP in the perforant path/granule cell synapses results in an elevation in PENK expression in the post-synaptic granule cells. Male Lister hooded rats (250–350 g) were anaesthetised with urethane (1.8 g/kg). Bipolar stimulating electrodes (Rhodes Medical Instruments Inc.) were positioned bilaterally in the medial perforant path (4.2 mm lateral to lambda). A glass microelectrode was positioned in the hilus of the right dentate gyrus (ipsilateral side; 2.5 mm lateral to the midline, 4 mm caudal to bregma). Electrode positions were optimised by maximising the responses to ipsi- and contralateral stimulation. Square wave pulses (120–400 mA, 50 ms) were applied to both stimulating electrodes, and a current which evoked a 1 mV population spike chosen as the test stimulus for both sides. Baseline responses to test stimuli were recorded every 30 s for 30 min prior to induction of LTP. LTP was induced on the ipsilateral side using three high frequency trains of pulses (50 pulses at 250 Hz, at twice test pulse amplitude, inter-train interval was 60 s). A similar number of pulses at low frequency was applied to the contralateral perforant path as a control. Potentiation of excitatory postsynaptic potential (EPSP) slope and population spike amplitude was measured 2 h after the tetani. Some animals received i.p. injections of 5 mg/kg 3-((R)-2carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP; Tocris Cookson), a competitive NMDA antagonist, 90 min prior to the tetanus. Two hours following the stimulus, the brains were removed, and cryostat sections (14 mm) cut through the hippocampal region. The sections were fixed in 4% paraformaldehyde and processed for in situ hybridisation as described. The 45-mer oligonucleotide probes used to detect PENK mRNA and tubulin T26 mRNA were as previously described [7], and were 3′ end-labelled with [35S]dATP. Hybridisation was conducted in buffer containing 50% formamide and 4 × standard saline citrate solution (SSC) [24] at 42°C, while post-hybridisation washes were conducted in 1 × SSC at 55°C. Sections were exposed to X-ray film (Kodak Biomax) before dipping in Ilford K5 liquid photographic emulsion and exposing for 5 weeks. The hybridisa-
tion signal was quantified by computerised image analysis (Image v1.52; NIH). Since NMDA receptor stimulation results in increased numbers of granule cells expressing PENK mRNA [7,14], the results for PENK mRNA were expressed as the percentage increase in the number of labelled cells ipsilateral to the stimulation relative to the contralateral side. Conversely, since the tubulin T26 gene is expressed in all granule cells, the results for tubulin T26 mRNA were expressed as the percentage increase in the autoradiographic grain density over the fascia dentata ipsilateral to the stimulation relative to the contralateral side [7]. High frequency unilateral stimulation of the perforant path in vivo produced a robust potentiation in the ipsilateral dentate gyrus (Fig. 1) of both EPSP slope (+22% of prestimulation size (5%, n = 6) and population spike amplitude (+326% of prestimulation size ±64%, n = 6). In animals pretreated with R-CPP, however, no potentiation of EPSP slope or population spike amplitude was observed. Two hours following low frequency stimulation, the levels of PENK mRNA were equivalent to those in normal rats, with only a small percentage of the granule cells expressing detectable PENK mRNA. However, 2 h following high frequency stimulation, there was an increase in the number of granule cells expressing PENK mRNA in the dentate gyrus (Fig. 2, Table 1). In contrast, high frequency stimulation did not alter the levels of tubulin T26 mRNA in the granule cells (Table 1). No increase in dentate gyrus proenkephalin
Fig. 1. Typical examples of (a) population spike amplitude, and (b) EPSP slope, recorded in the hilus following high frequency stimulation of the perforant path at t = 30 min. The graph shows average data points from a single experiment, each point is the mean ± SEM of four responses.
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Fig. 2. Cells in the dentate gyrus expressing PENK mRNA contralateral (a) and ipsilateral (b) to high frequency stimulation of the perforant path 2 h earlier. Note the greater number of labelled neurones observed following the induction of LTP. Scale bar, 40 mm.
expression was observed following high frequency stimulation in animals pretreated with the NMDA receptor antagonist R-CPP (Table 1). These results show that PENK gene expression is markedly enhanced in hippocampal granule cells following the induction of LTP in the afferent synapses. Since LTP induction in this area involves NMDA receptor stimulation, this is consistent with our previous work showing that pharmacological activation of NMDA receptors on the granule cell dendrites produces a similar enhancement of PENK expression [6,7]. There is evidence that the use of anaesthetised, as opposed to conscious, animals can affect the characteristics of LTP induction in vivo [5]. In anaesthetised animals, the most sustained phase of LTP, decaying with a time constant of around 20 days (‘LTP3’), is reportedly not seen, and perforant path LTP fails to induce the immediate early gene c-fos in the granule cells. However, the other phases of LTP, including the late phase that decays with a time constant of around 5 days (‘LTP2’) are still observed in anaesthetised animals, and indeed many changes in gene expression have been reported using anaesthetised animals [5,23]. Both LTP2 and LTP3 are thought to be dependent on de novo protein synthesis [1,15]. It seems likely that the use of anaesthetised animals will selectively reveal those components of the changing pattern of gene expression that contribute to LTP2 rather then LTP3. A number of other changes in gene expression have been detected around 2 h following high frequency stimulation of the perforant path [12]. These include the CamKIIa [11,18], cyclooxygenase 2 [25], tissue plasminogen activator [16], and arc [9,10] genes. In each of these cases, the alteration in gene expression can be predicted to alter the characteristics of neurotransmission at the perforant path/granule cell synapses which have been potentiated. However, the increase in PENK expression we have reported here is unique,
because the enkephalins produced are transported to the terminals of the mossy fibres, and act at the granule cell/ CA3 pyramidal cell synapses. The effect of enhanced proenkephalin gene expression will therefore be manifest at a synapse downstream from the one which has been stimulated. This suggests that a phenomenon of ‘domino plasticity’ occurs, where high frequency activity can affect the characteristics of neurotransmission at a chain of synapses, and not just at the synapse initially stimulated [14]. Additional evidence for this phenomenon comes from studies into the expression of the synaptic vesicle-associated protein syntaxin. Syntaxin expression in the mossy fibre terminals is also increased by the induction of LTP at the perforant path/ granule cell synapses [19]. Our results therefore have implications beyond the role of enkephalins in hippocampal plasticity. Since enkephalins act to potentiate neurotransmission at the mossy fibre/CA3 synapses [2,3,22], the enhanced enkeTable 1 Effect of LTP induction on the expression of PENK mRNA and tubulin T26 mRNA in the dentate gyrus mRNA species
Proenkephalin Tubulin T26
Hybridisation signal (% control) High frequency stimulation (6)
High frequency stimulation + CPP (3)
222.5 ± 31.1* 121.3 ± 11.6
96.6 ± 5.6** ND
Values are the mean ± SEM from the number of animals shown in parentheses. The hybridisation signal was assessed for each probe as described [7], and the signal on the side ipsilateral to the high frequency stimulation expressed as a percentage of the signal on the contralateral (control) side. *Median significantly different from 100% (P , 0.05) by Wilcoxon signed rank test. **Median significantly different from group receiving high frequency stimulation alone (P , 0.05) by Mann–Whitney U-test. ND, not determined.
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