Role of Ca2+in striatal LTD and LTP

seminars in

THE NEUROSCIENCES, Vol 8, 1996: pp 321–328

Role of Ca2 + in striatal LTD and LTP Paolo Calabresi*, Antonio Pisani*, Diego Centonze* and Giorgio Bernardi*†

glutamate within the striatum.3 They can be subdivided into autoreceptors (metabotropic glutamate receptors, mGluRs) and heteroreceptors (muscarinic, GABA b, dopamine D2 and adenosine A1 receptors).2-4 The short-term regulation of corticostriatal activity is also exerted by postsynaptic voltage-dependent conductances which modulate the synaptic signals either by enhancing the inputs originating in the dendritic region of the neuron or by counteracting them. The ionic mechanisms exerting a negative control on striatal neuronal excitability are mainly represented by various potassium conductances.5-7 In contrast, activation of voltage-dependent sodium and Ca2 + conductances8-11 enhances the electroresponsiveness of striatal neurons. These conductances are probably acting not only in the somatic region of striatal neurons, but also in the dendritic regions. The large majority (more than 95%) of the striatal neurons is represented by spiny neurons.12 These neurons are small GABAergic projecting neurons whose dendritic tree is densely covered with spines (Figure 1). Morphological studies have shown that both glutamatergic and dopaminergic inputs converge on striatal dendritic spines.12 Dendritic spines have been proposed to constitute the main locus of long-term synaptic modifications associated with functional plasticity of central neurons.13 Thus it is possible that the dendrites of striatal spiny cells might represent the cellular substrate for the synaptic plasticity within the basal ganglia. Moreover, biochemical and morphological changes occurring in this region might influence the activity of the basal ganglia in the storage of motor skills.14 In this regard, it is interesting to note that the lesion of dopaminergic inputs to the striatum causes morphological alterations of dendrites of spiny cells15 and alters striatal synaptic plasticity.14 Finally, since the role of the spines in the regulation of the rise of postsynaptic intracellular Ca2 + concentration has been suggested,16,17 we can speculate that this regulatory function is relevant for striatal LTD and LTP (see previously).

The corticostriatal projection has a major function in the control of movements. Alterations of the release of glutamate from corticostriatal terminals have been implicated in disorders of the basal ganglia such as Parkinson’s disease and Huntington’s chorea. The long-term regulation of corticostriatal transmission might require the participation of different forms of striatal synaptic plasticity. In physiological condition (1.2 mM external magnesium) the tetanic stimulation of cortical afferents produces a LTD of excitatory synaptic potentials recorded in the striatum. When the external magnesium is omitted, this tetanus induces LTP. NMDA receptor antagonists prevent the induction of LTP, but not the generation of LTD. LTD is blocked either by BAPTA and EGTA or by thapsigargin suggesting that a rise of intracellular Ca2 + is required for this form of synaptic plasticity. Nifedipine is also able to prevent LTD indicating that high voltage-activated (HVA) Ca2 + channels of the L-type are implicated in the formation of LTD. Moreover, LTD is blocked by inhibitors of Ca2 + -dependent kinases suggesting that a rise in internal Ca2 + is a crucial step for the subsequent activation of a second messenger cascade. Although both striatal LTD and LTP seem to require an increase in intracellular Ca2 + concentration, this change is probably arising from different sources. Key words: striatum / basal ganglia / Parkinson’s disease / dopamine / excitatory amino acids ©1996 Academic Press Ltd

THE STRIATUM plays a key role in the control of cortical inputs originating from the basal ganglia and directed towards their output structures.1 Different mechanisms exert this control. A short-term control of corticostriatal activity is mediated by presynaptic receptors located on the axon terminals of glutamatergic fibres.2 These receptors inhibit the release of

From the *Clinica Neurologica, Dip. Sanita’, Universita’ di Roma Tor Vergata, Via O. Raimondo 8, Rome 00173 and †I.R.C.C.S. Ospedale Santa Lucia, Via Ardeatina, Rome, Italy ©1996 Academic Press Ltd 1044-5765/96/050321 + 08 $25.00/0

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Ca2 + and striatal synaptic plasticity

Ca2 + and glutamate receptor subtypes underlying corticostriatal transmission and plasticity

conditioned pathway, showing that striatal LTD exhibits an input specificity, that is, modifications are confined to the activated inputs. This appears to be a common feature of most of the models of synaptic plasticity. Tetanic stimulation of the corticostriatal pathway induced a long-lasting depression ( > 2 hours) either of intracellularly recorded excitatory postsynaptic potentials (EPSPs), or of extracellular field potentials, without affecting intrinsic membrane properties of the recorded neurons (membrane potential, input resistance, current–voltage relationship). It has to be remarked that LTD was induced only when stimulation intensity was raised to cause suprathreshold EPSP and action potential discharge was produced by the tetanus. In fact, tetanic stimulation of subthreshold EPSP was not able to cause LTD; on the contrary, by coupling membrane depolarization to subthreshold EPSP to determine action potential discharge during the tetanus, a long-lasting depression of synaptic transmission was observed. Moreover, by delivering the tetanus at the hyperpolarized membrane potential, no synaptic depression was detected. Thus, it appears evident that a critical level of depolarization is required for the induction of striatal LTD. It is likely that AMPA receptors may have a role in the induction of striatal LTD, mainly by contributing to the depolarization of the postsynaptic cell. To test whether activation of NMDA receptors could be responsible for the rise in intracellular Ca2 + concentration observed in striatal LTD, we applied APV to the external medium 10–15 minutes before the tetanus was delivered. APV failed to block LTD, showing, like in the cerebellum,20 that Ca2 + entry does not occur through NMDA receptors during the induction of synaptic plasticity. Interestingly, as shown in Figure 2, when magnesium ions were omitted from the perfusing solution to remove NMDA receptor channels voltage-dependent blockade, tetanic stimulation produced an LTP of synaptic transmission in the striatum.21 In this experimental condition, preincubation of slices with APV prevented the post-tetanic potentiation.21 Within the wide group of mGluRs, the subclass of these receptors coupled to PI turnover has been shown to be necessary for the formation of different forms of LTDs.22-25 Its activation leads, through a Ca2 + -dependent enzyme, phospholipase C (PLC), to the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) into two second messengers, dyacilglycerol (DAG), and IP3. DAG acts by stimulating PKC; IP3 induces Ca2 + release from intracellular Ca2 + stores. We tested whether L-AP3, a noncompetitive antagonist of PI-coupled mGluRs,

In-vivo studies have shown that excitatory postsynaptic potentials (EPSPs) recorded from striatal neurons following cortical activation are not altered by NMDA receptor antagonists while they are blocked by AMPA receptor antagonists.18 Similar results were obtained from in-vitro studies.14 In fact, subthreshold striatal EPSPs recorded following cortical activation in corticostriatal slice preparations were blocked by 6-cyano7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of AMPA glutamate receptors, but they were not reduced by D-2-amino-5-phosphonovalerate (APV), an antagonist of NMDA glutamate receptors.14 In a recent study, using the whole-cell patch clamp method, excitatory postsynaptic currents (EPSCs) have been recorded from spiny striatal neurons following intrastriatal synaptic activation.19 This study has suggested that glutamatergic inputs to the striatal spiny neurons are of two types on the basis of their amplitude, presynaptic origin and paired pulse depression.19 The first type of EPSC (termed ‘S’ type) is evoked by the stimulation of single presynaptic fibres and it is probably caused by activation of cortical afferents. The other type of EPSC (the ‘H-type’) is mediated by both non-NMDA and NMDA receptors and a higher intensity of stimulation is required to evoke it. Moreover, the H-type of EPSC is polysynaptically-activated suggesting the participation of glutamatergic interneurons.19 High-frequency (100 Hz, 3 trains, 3 seconds duration, 20 seconds inter-train interval) stimulation of corticostriatal afferents were used as a conditioning tetanus to produce a stable and long-lasting depression of synaptic transmission in a corticostriatal slice preparation. The stimulating electrode was located either in the cortex or in the white matter between the cortex and the striatum. The recording electrode was placed in the striatum, close to the area of stimulation. When two stimulating electrodes were positioned in the cortex, but tetanus was delivered only through one of them, LTD was observed only in the Figure 1. An example of a biocytin-injected striatal spiny projection neuron is shown. This cell displays the characteristic features of spiny neurons including an aspinous soma and proximal dendrites and densely spine-covered distal dendrites. In the lower part of the figure the dendrites of the same neuron are shown at higher magnification. These morphological findings were obtained in collaboration with Dr G. Sancesario and Mrs V. D’Angelo.

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P. Calabresi et al dampened down by lithium treatment. In addition, it is interesting to note that motor behavior is affected by both lithium or compounds which compete with PI-coupled mGluRs activation.29

Ca2 + ions are necessary for LTD induction Experimental evidence supports the idea that a rise in free Ca2 + concentration in the postsynaptic neuron represents a fundamental event for LTD induction in different areas of the brain. In order to define the role of Ca2 + in striatal plasticity, cells were injected either with 200 mM bis(2-aminophenoxy)ethane-N,N,N'N'tetraacetate (BAPTA) or with 500 mM EGTA, two Ca2 + chelators, 20–30 minutes before LTD induction. In this condition, the development of synaptic depression was prevented30 (Figure 3A). Both EGTA and BAPTA, by themselves, caused no changes of EPSPs amplitude and of intrinsic membrane properties of the recorded cells. In an attempt to clarify the source responsible for the increase in intracellular free Ca2 + , the role of L-type HVA Ca2 + channels was also investigated. The dihydropyridine derivative, nifedipine (10 µM), a selective antagonist of L-type Ca2 + channels, was applied in the bathing solution 15 minutes before delivering tetanic stimulation. Nifedipine affected neither intrinsic membrane properties, nor synaptic potential amplitude. However, in the presence of nifedipine tetanic stimulation did not cause LTD30 (Figure 3B). This finding suggests the involvement of L-type Ca2 + channels in the formation of striatal LTD. At present, however, we are unable to establish whether either pre- or postsynaptic sites are involved in this effect of nifedipine on LTD. Microfluorimetric imaging studies have demonstrated that L-type channels are located on the somata and proximal dendrites of hippocampal neurons, clustering at the base of the major dendrites,17,31 and have been proposed as devices for synaptic input integration.16,17 In striatal neurons, it has been shown that HVA Ca2 + are present both in the cell body and proximal dendrites.10 In addition, electron microscopy studies in the hippocampus have shown that during LTP structural changes, such as the morphology and number of dendritic spines, occur.13,32 Thus, an interesting field of future studies will be the analysis of the distribution of Ca2 + accumulation as well as possible conformational modifications of dendrites during LTD in the striatum. An involvement of intracellular Ca2 + stores may be

Figure 2. Tetanic stimulation of corticostriatal fibres induces either LTD or LTP in striatal neurons. Under control condition (1.2 mM external magnesium) repetitive cortical activation produces LTD of excitatory transmission (open circles). When magnesium is omitted from the external medium (filled circles) the tetanic stimulation induces LTP.

could affect striatal LTD. L-AP3 (10–30 µM) was added to the perfusing medium 10–15 minutes before tetanic stimulation. No significant changes of intrinsic membrane properties were observed during recordings. Likewise, L-AP3 did not induce modifications of synaptic potential amplitude, but when tetanus was delivered, a dose-dependent inhibition of striatal LTD was detected.14 L-AP3, however, is a compound of poorly defined specificity, and this finding will need confirmation as soon as more selective pharmacological tools are developed. Thus, a further experimental approach was carried out, by utilizing lithium, which is known to reduce the supply of inositol, the key substrate of PI metabolism, by inhibiting some of the enzymes which hydrolyse the inositol phosphates.26 Rats were chronically treated with increasing doses of lithium chloride, reaching, after 10 days treatment, plasma levels which are in the therapeutic range. Intracellularly recorded neurons from lithiumtreated slices did not show any significant change of membrane potential, input resistance and firing properties.27 Moreover, in this condition repetitive activation of the corticostriatal pathway failed to induce LTD.27 It is interesting to note that lithium action is directed to those receptors which are ‘overactive’, and not to those which operate ‘normally’;28 mGluRs involved in striatal LTD might be rendered ‘overactive’ by the tetanus, thus being 324

Ca2 + and striatal synaptic plasticity postulated in the generation of intracellular Ca2 + rise in the course of striatal LTD. It is important to note that in many cell types, the increase in cytosolic free Ca2 + concentration caused by Ca2 + influx through HVA channels is greatly magnified by Ca2 + release from internal stores. As mentioned above, IP3, generated as a result of the mGluR-mediated activation of phospholipase C, determines release of Ca2 + from internal stores. In addition to this mechanism, Ca2 + may also be released from ryanodine-sensitive reservoirs. In order to evaluate the possible contribution of Ca2 + release from internal stores in striatal plasticity, we used thapsigargin, which aspecifically empties various Ca2 + stores by blocking Ca2 + uptake. Preliminary experiments in our laboratory have shown that bath-applied thapsigargin (300 nM, 15 min) prevented the generation of LTD in the striatum (Figure 3C). No changes of the EPSP amplitude or of intrinsic membrane properties were caused by preincubation with thapsigargin. As shown in Figure 3C, immediately after tetanic stimulation and up to 10 min later, a transient, but significant potentiation of EPSP amplitude was observed. However, no LTD was observed following the tetanus. This finding seems to further confirm the idea that a rise in intracellular Ca2 + concentration is required for LTD induction. In various forms of synaptic plasticity it has been demonstrated that the rise in intracellular Ca2 + concentration is followed by the activation of a complex cascade of biochemical events.33,34 Ca2 + rise promotes activation of Ca2 + -dependent enzymes leading to phosphorylation processes that, in turn, are thought to enable long-lasting changes of the neuronal activity. We investigated the role of Ca2 + dependent protein kinases by using three kinase blockers: H-7, staurosporine and calphostin C. H-7 (10–50 µM), staurosporine (50–100 nM) or calphostin C (1 µM), the latter representing a selective blocker of protein kinase C, were bath-applied 15 min before tetanic stimulation. None of these drugs caused modifications in the EPSP amplitude or of the membrane properties of the recorded cells before the tetanus. However, in the presence of each of these kinase blockers, tetanic stimulation failed to induce LTD30 (Figure 4). Interestingly, pretreatment with these inhibitors did not affect presynaptic inhibition of corticostriatal transmission induced either by mGluR agonists or by muscarine (data not shown) suggesting that the mechanisms activated by the presynaptic receptors located on the cortical axon terminals do not require activation of Ca2 + -dependent kinases. Moreover, these results demonstrate that

Figure 3. Calcium is required for striatal LTD. (A) The graph shows that under control condition (open circles), tetanic stimulation induces LTD. In contrast, when the cell is loaded with BAPTA (filled circles), the same tetanus fails to produce this form of synaptic plasticity. (B) The graph shows that when the slice is incubated in nifedipine (filled circles) the repetitive activation does not induce LTD. (C) Thapsigargin (filled circles) prevents the generation of striatal LTD.

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P. Calabresi et al tribution, the amount and the duration of the changes in internal concentration of this ion. LTD and LTP might also be linked to different physiopathological conditions of the basal ganglia. It is possible that under control conditions corticostriatal transmission is mainly mediated by the activation of AMPA glutamate receptors. Thus, we presume that usually, under physiological situations, repetitive activation of cortical afferents to the striatum leads to LTD. This form of synaptic plasticity should result in a disinhibition of the output structures of the basal ganglia and, possibly, in motor activation. In the rat the unilateral lesion of the nigral dopaminergic system by 6-hydroxy-dopamine causes motor abnormalities which have been considered an experimental correlate of Parkinson’s disease.35 Interestingly, this lesion prevents the formation of striatal LTD suggesting that endogenous dopamine is necessary not only for motor activation, but also for the generation of this form of synaptic plasticity within the striatum.14 Striatal LTP might have various physiological meanings. In a physiological condition, it could be activated only in the presence of a strong depolarizing driving force on spiny striatal neurons. We presume that this situation can be achieved when cortical inputs arising from different areas of the cortex converge on striatal cells causing both spatial and temporal summation of these excitatory inputs. This large membrane depolarization deinactivates the NMDA receptor channel complex leading to Ca2 + influx and subsequent LTP generation. However, we believe that several physiological factors contribute to exert a negative control on this long-term excitatory mechanism: presynaptic inhibition of glutamate release via autoreceptors and heteroreceptors,2-4 postsynaptic potassium conductances,5,6 intracellular Ca2 + buffering systems36 and Na + /K + ATP-dependent pump.37 These inhibitory mechanisms which counteract the activation of the NMDA receptor channel complex might be impaired in particular pathological conditions such as Huntington’s chorea. This disease is an autosomal dominant neurodegenerative disease that is characterized by psychiatric disorders, dementia and involuntary movements.38 In Huntington’s disease a selective degeneration of striatal spiny neurons has been reported.38 This degeneration has been related to excitotoxic mechanisms and to selective deficits in energetic metabolism of spiny cells or both.39 Accordingly, experimental models of this disease have been obtained either by using agonists of excitatory amino acid receptors or by administrating mitochondrial toxins which block energetic metabolism (3-nitropro-

Figure 4. The formation of striatal LTD is prevented by inhibitors of calcium-dependent kinases. The histograms show the EPSP amplitude measured 15 min after the tetanic stimulation. Note that under control conditions the EPSP amplitude is clearly reduced. The depression of the EPSP is prevented by three different inhibitors of protein kinase C: staurosporine (50 nM), H-7 (50 µM) and calphostin C (1 µM).

Ca2 + -dependent kinases following tetanic stimulation play a critical role in the formation of striatal LTD.

Ca2 + influx and synaptic plasticity in striatal spiny neurons: physiological and pathological implications The finding that repetitive activation of corticostriatal fibres is able to generate both LTD and LTP of excitatory transmission indicates that the striatum, as well as the cerebellum, possesses cellular substrates for the storage of motor skills. Membrane depolarization and Ca2 + influx are required for the generation of both of these forms of striatal synaptic plasticity. Ca2 + influx via NMDA receptors is required for striatal LTP. In contrast, activation of NMDA receptors is not necessary for the generation of LTD. Our experimental findings suggest that possible sources of intracellular Ca2 + in LTD might be represented by: (i) activation of AMPT glutamate receptors, (ii) activation of L-type HVA Ca2 + channels and (iii) mobilization of intracellular Ca2 + following mGluR activation. Thus, it is possible that, although both LTD and LTP require a rise of intracellular Ca2 + to initiate a cascade of biochemical events, crucial differences occur between these forms of synaptic plasticity concerning the subcellular dis326

Ca2 + and striatal synaptic plasticity 4. Calabresi P, Mercuri NB, DeMurtas M, Bernardi G (1991) Involvement of GABA systems in the feed-back regulation of glutamate- and GABA-mediated synaptic potentials in rat neostriatum. J Physiol (London) 440:581-599 5. Nisembaum ES, Wilson CJ (1995) Potassium currents responsible for inward and outward rectification in rat neostriatal spiny projection neurons. J Neurosci 15:4449-4463 6. Surmeier DJ, Wilson CJ, Eberwine J (1994) Patch-clamp techniques for studying potassium currents in mammalian brain neurons, in Methods in Neuroscience: Methods for the Study of Ion Channels, pp 39-67. Academic Press, San Diego 7. Calabresi P, Misgeld U, Dodt UH (1987) Intrinsic membrane properties of neostriatal neurons can account for their low levels of spontaneous activity. Neuroscience 20:293-303 8. Surmeier DJ, Eberwine J, Wilson CJ, Stefani A, Kitai ST (1992) Dopamine receptor co-localize in acutely-isolated rat striatonigral neurons. Proc Natl Acad Sci USA 89:10178-10182 9. Cepeda C, Chandler SH, Shumate LW, Levine MS (1995) Persistent Na + conductance in medium-sized neostriatal neurons: characterization using infrared videomicroscopy in whole cell patch-clamp recordings. J Neurophysiol 74:1343-1348 10. Hoehn K, Watson TWJ, MacVicar BA (1993) Multiple types of calcium channels in acutely isolated rat neostriatal neurons. J Neurosci 13:1244-1257 11. Stefani A, Pisani A, Mercuri NB, Bernardi G, Calabresi P (1994) Activation of metabotropic glutamate receptors inhibits calcium currents and GABA-mediated synaptic potentials in striatal neurons. J Neurosci 14:6734-6743 12. Graybiel AM (1990) Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci 13:244-254 13. Desmond NL, Levy WB (1990) Morphological correlates of long-term potentiation imply the modification of existing synapses, not synaptogenesis, in the hippocampal dentate gyrus. Synapse 5:139-143 14. Calabresi P, Maj R, Pisani A, Mercuri NB, Bernardi G (1992) Long-term synaptic depression in the striatum: physiological and pharmacological characterization. J Neurosci 12:4224-4233 15. Nitsch C, Riesenberg R (1995) Synaptic reorganisation in the rat striatum after dopaminergic deafferentation: an ultrastructural study using glutamate decarboxylase immunocytochemistry. Synapse 19:247-263 16. Konnerth A, Dreessen J, Augustine G (1992) Brief dendritic calcium signals initiate long-lasting synaptic depression in cerebellar Purkinje cells. Proc Natl Acad Sci USA 89:7051-7055 17. Regehr WG, Connor JA, Tank DW (1989) Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation. Nature 341:533-536 18. Herrling PL (1985) Pharmacology of the corticocaudate excitatory postsynaptic potential in the cat: evidence for its modulation by quisqualate- or kainate receptors. Neuroscience 14:417-426 19. Mori A, Takahashi T, Miyashita Y, Kasai H (1994) Two distinct glutamatergic synaptic inputs to striatal medium spiny neurones of neonatal rats and paired pulse depression. J Physiol (London) 476:217-228 20. Ito M (1989) Long-term depression. Annu Rev Neurosci 12:85-102 21. Calabresi P, Pisani A, Mercuri NB, Bernardi G (1992) Longterm potentiation in the striatum is unmasked by removing the voltage-dependent blockade of NMDA receptor channel. Eur J Neurosci 4:929-935 22. Kano M, Kano N (1987) Quisqualate receptors are specifically involved in cerebellar synaptic plasticity. Nature 325:276-279 23. Stanton PK, Chattarji S, Sejnowski TJ (1991) 2-Amino-3-phosphonopropionic acid, an inhibitor of glutamate-stimulated phosphoinositide turnover, blocks induction of homosynaptic

pionic acid, malonic acid, aminooxyacetic acid). These toxins impair oxidative phosphorylation and disturb the function of the mitochondrial respiratory chain.40 The consequent results are the intracellular energy deprivation and the reduction of ATP stores in mitochondria. These events, in turn, interfere with Na + /K + ATPase activity leading to disturbances in the maintenance of the membrane potential. Alterations of energy metabolism will also induce a reduction of the voltage-dependent magnesium block of NMDA channels.41 As the number of activated NMDA channels increases, a higher amount of Ca2 + will enter the cell. The resulting rapid increase in Ca2 + concentration in the neuron induces the storage of this ion in the mitochondria, which further compromises energy supply. In fact, Ca2 + overload of mitochondria inhibits ATP synthesis.42 The latter event irreversibly blocks the respiratory chain leading to activation of phospholipases and neuronal death.43 Thus it is possible that in Huntington’s disease the altered metabolic activity of striatal neurons by deinactivating the NMDA receptors induces LTP in striatal neurons. In this pathological condition, a NMDA-dependent longterm enhancement of corticostriatal transmission will increase the Ca2 + influx and will favour the consequent neuronal death. Further studies are required to elucidate the possible role of striatal LTD and LTP in the physiological control of motor activity and their implications in neurodegenerative disorders of the basal ganglia.

Acknowledgements We wish to thank Dr G. Sancesario and Mrs V. D’Angelo for the morphological analysis of the stained cells. We also thank Mr G. Gattoni, Mr M. Tolu and Mr M. Federici for the excellent technical assistance provided.

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