Rescue of synaptic plasticity and spatial learning deficits in the hippocampus of Homer1 knockout mice by recombinant Adeno-associated viral gene delivery of Homer1c

Homer1 belongs to a family of scaffolding proteins that interact with various post-synaptic density proteins including group I metabotropic glutamate receptors (mGluR1/5). Previous research in our laboratory implicates the Homer1c isoform in spatial learning. Homer1 knockout mice (H1-KO) display cognitive impairments, but their synaptic plasticity properties have not been described. Here, we investigated the role of Homer1 in long-term potentiation (LTP) in the hippocampal CA1 region of H1-KO mice in vitro. We found that late-phase LTP elicited by high frequency stimulation (HFS) was impaired, and that the induction and maintenance of theta burst stimulation (TBS) LTP were reduced in H1-KO. To test the hypothesis that Homer1c was sufficient to rescue these LTP deficits, we delivered Homer1c to the hippocampus of H1-KO using recombinant adeno-associated virus (rAAV). We found that rAAV-Homer1c rescued HFS and TBS-LTP in H1-KO animals. Next, we tested whether the LTP rescue by Homer1c was occurring via mGluR1/5. A selective mGluR5 antagonist, but not an mGluR1 antagonist, blocked the Homer1c-induced recovery of late-LTP, suggesting that Homer1c mediates functional effects on plasticity via mGluR5. To investigate the role of Homer1c in spatial learning, we injected rAAV-Homer1c to the hippocampus of H1-KO. We found that rAAV-Homer1c significantly improved H1-KO performance in the Radial Arm Water Maze. These results point to a significant role for Homer1c in synaptic plasticity and learning.     

Reelin supplementation enhances cognitive ability, synaptic plasticity, and dendritic spine density

Apolipoprotein receptors belong to an evolutionarily conserved surface receptor family that has intimate roles in the modulation of synaptic plasticity and is necessary for proper hippocampal-dependent memory formation. The known lipoprotein receptor ligand Reelin is important for normal synaptic plasticity, dendritic morphology, and cognitive function; however, the in vivo effect of enhanced Reelin signaling on cognitive function and synaptic plasticity in wild-type mice is unknown. The present studies test the hypothesis that in vivo enhancement of Reelin signaling can alter synaptic plasticity and ultimately influence processes of learning and memory. Purified recombinant Reelin was injected bilaterally into the ventricles of wild-type mice. We demonstrate that a single in vivo injection of Reelin increased activation of adaptor protein Disabled-1 and cAMP-response element binding protein after 15 min. These changes correlated with increased dendritic spine density, increased hippocampal CA1 long-term potentiation (LTP), and enhanced performance in associative and spatial learning and memory. The present study suggests that an acute elevation of in vivo Reelin can have long-term effects on synaptic function and cognitive ability in wild-type mice.     

Selective modulation of some forms of schaffer collateral-CA1 synaptic plasticity in mice with a disruption of the CPEB-1 gene

CPEB-1 is a sequence-specific RNA binding protein that stimulates the polyadenylation-induced translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). Although CPEB-1 was identified originally in Xenopus oocytes, it has also been found at postsynaptic sites of hippocampal neurons where, in response to N-methyl-D-aspartate receptor activation, it is thought to induce the polyadenylation and translation of alphaCaMKII and perhaps other CPE-containing mRNAs. Because some forms of synaptic modification appear to be influenced by local (synaptic) protein synthesis, we examined long-term potentiation (LTP) in CPEB-1 knockout mice. Although the basal synaptic transmission of Schaffer collateral-CA1 neurons was not affected in the knockout mice, we found that there was a modest deficit in LTP evoked by a single train of 100 Hz stimulation, but a greater deficit in LTP evoked by one train of theta-burst stimulation. In contrast, LTP evoked by either four trains of 100 Hz stimulation or five trains of theta-burst stimulation were not or were only modestly affected, respectively. The deficit in LTP evoked by single stimulation in knockout mice appeared several minutes after tetanic stimulation. Long-term depression (LTD) evoked by 1 Hz stimulation was moderately facilitated; however, a stronger and more enduring form of LTD induced by paired-pulse 1 Hz stimulation was unaffected. These data suggest that CPEB-1 contributes in the translational control of mRNAs that is critical only for some selected forms of LTP and LTD.     

Alpha3-integrins are required for hippocampal long-term potentiation and working memory

Integrins comprise a large family of heterodimeric, transmembrane cell adhesion receptors that mediate diverse neuronal functions in the developing and adult CNS. Recent pharmacological and genetic studies have suggested that beta1-integrins are critical in synaptic plasticity and memory formation. To further define the role of integrins in these processes, we generated a postnatal forebrain and excitatory neuron-specific knockout of alpha3-integrin, one of several binding partners for beta1 subunit. At hippocampal Schaffer collateral-CA1 synapses, deletion of alpha3-integrin resulted in impaired long-term potentiation (LTP). Basal synaptic transmission and paired-pulse facilitation were normal in the absence of alpha3-integrin. Behavioral studies demonstrated that the mutant mice were selectively defective in a hippocampus-dependent, nonmatch-to-place working memory task, but were normal in other hippocampus-dependent spatial tasks. The impairment in LTP and working memory is similar to that observed in beta1-integrin conditional knockout mice, suggesting that alpha3-integrin is the functional binding partner for beta1 for these processes in the forebrain.     

Enriching the environment of alphaCaMKIIT286A mutant mice reveals that LTD occurs in memory processing but must be subsequently reversed by LTP

alphaCaMKII(T286A) mutant mice lack long-term potentiation (LTP) in the hippocampal CA1 region and are impaired in spatial learning. In situ hybridization confirms that the mutant mice show the same developmental expression of alphaCaMKII as their wild-type littermates. A simple hypothesis would suggest that if LTP is a substrate for learning, then enriching the environment should cause learning-dependent changes in wild-type mice that have LTP. Such changes would not be seen in LTP-deficient alphaCaMKII(T286A) mutants. Excitatory synaptic currents in CA1 neurons, recorded with patch clamp in brain slices, revealed that enrichment induces an increase in glutamate release probability and a decreased miniature current amplitude. Confocal microscopy also showed dendritic spine density to be reduced. However, contrary to the hypothesis above, these enrichment-induced changes occur only in the mutant mice and are not detectable in wild-type littermates. We suggest that enrichment induces alphaCaMKII-independent changes in both wild-type and mutant mice. Such changes may be subsequently reversed in wild-type animals via alphaCaMKII-dependent mechanisms, such as LTP. Reversal of plasticity has long been hypothesized to be essential for the hippocampus to maintain its role in memory processing. The inability to reverse plasticity in alphaCaMKII(T286A) mutant mice would then result in impairment of spatial learning.     

Impaired Memory Retention and Decreased Long-Term Potentiation in Integrin-Associated Protein-Deficient Mice

Previously, we have demonstrated that integrin-associated protein (IAP) mRNA level is approximately fourfold higher in rats showing good retention performance (600 sec) than rats showing poor retention performance (<80 sec) in an inhibitory avoidance learning paradigm. In the present study, we have used the gene-targeted IAP-deficient mice to further investigate the role of IAP involved in memory formation and hippocampal long-term potentiation (LTP) in vivo. Results revealed that there was a significant impairment in memory retention and a significant reduction in the magnitude of LTP in IAP-deficient mice when compared with the wild-type and heterozygote mice, whereas the wild-type and heterozygote animals did not show marked differences on these measures. Furthermore, the impairment in retention performance of IAP-deficient mice was not due to different sensitivities of these animals to the electric shock. When we examined locomotor activity and rotarod treadmill performance, no differences were observed among these three groups of animals either. Western blot analysis confirmed the lack of IAP protein in IAP-deficient mice, whereas IAP expression was similar in both the wild-type and heterozygote controls. These results together demonstrate that IAP plays an important role in the process of memory formation and synaptic plasticity in mice.     

Fragile X mental retardation protein regulates heterosynaptic plasticity in the hippocampus

Silencing of a single gene, FMR1, is linked to a highly prevalent form of mental retardation, characterized by social and cognitive impairments, known as fragile X syndrome (FXS). The FMR1 gene encodes fragile X mental retardation protein (FMRP), which negatively regulates translation. Knockout of Fmr1 in mice results in enhanced long-term depression (LTD) induced by metabotropic glutamate receptor (mGluR) activation. Despite the evidence implicating FMRP in LTD, the role of FMRP in long-term potentiation (LTP) is less clear. Synaptic strength can be augmented heterosynaptically through the generation and sequestration of plasticity-related proteins, in a cell-wide manner. If heterosynaptic plasticity is altered in Fmr1 knockout (KO) mice, this may explain the cognitive deficits associated with FXS. We induced homosynaptic plasticity using the β-adrenergic receptor (β-AR) agonist, isoproterenol (ISO), which facilitated heterosynaptic LTP that was enhanced in Fmr1 KO mice relative to wild-type (WT) controls. To determine if enhanced heterosynaptic LTP in Fmr1 KO mouse hippocampus requires protein synthesis, we applied a translation inhibitor, emetine (EME). EME blocked homo- and heterosynaptic LTP in both genotypes. We also probed the roles of mTOR and ERK in boosting heterosynaptic LTP in Fmr1 KO mice. Although heterosynaptic LTP was blocked in both WT and KOs by inhibitors of mTOR and ERK, homosynaptic LTP was still enhanced following mTOR inhibition in slices from Fmr1 KO mice. Because mTOR will normally stimulate translation initiation, our results suggest that β-AR stimulation paired with derepression of translation results in enhanced heterosynaptic plasticity.     

Phase-dependent synaptic changes in the hippocampal CA1 field underlying extinction processes in freely moving rats

Recent studies focus on the functional significance of a novel form of synaptic plasticity, low-frequency stimulation (LFS)-induced synaptic potentiation in the hippocampal CA1 area. In the present study, we elucidated dynamic changes in synaptic function in the CA1 field during extinction processes associated with context-dependent fear memory in freely moving rats, with a focus on LFS-induced synaptic plasticity. Synaptic transmission in the CA1 field was transiently depressed during each extinction trial, but synaptic efficacy was gradually enhanced by repeated extinction trials, accompanied by decreases in freezing. On the day following the extinction training, synaptic transmission did not show further changes during extinction retrieval, suggesting that the hippocampal synaptic transmission that underlies extinction processes changes in a phase-dependent manner. The synaptic potentiation produced by extinction training was mimicked by synaptic changes induced by LFS (0.5 Hz) in the group that previously received footshock conditioning. Furthermore, the expression of freezing during re-exposure to footshock box was significantly reduced in the LFS application group in a manner similar to the extinction group. These results suggest that LFS-induced synaptic plasticity may be associated with the extinction processes that underlie context-dependent fear memory. This hypothesis was supported by the fact that synaptic potentiation induced by extinction training did not occur in a juvenile stress model that exhibited extinction deficits. Given the similarity between these electrophysiological and behavioral data, LFS-induced synaptic plasticity may be related to extinction learning, with some aspects of neuronal oscillations, during the acquisition and/or consolidation of extinction memory.     

Subregion-specific dendritic spine abnormalities in the hippocampus of Fmr1 KO mice

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by the lack of fragile X mental retardation protein (FMRP). In the brain, spine abnormalities have been reported in both patients with FXS and Fmr1 knockout mice. This altered spine morphology has been linked to disturbed synaptic transmission related to altered signaling in the excitatory metabotropic glutamate receptor 5 (mGluR5) pathway. We investigated hippocampal protrusion morphology in adult Fmr1 knockout mice. Our results show a hippocampal CA1-specific altered protrusion phenotype, which was absent in the CA3 region of the hippocampus. This suggests a subregion-specific function of FMRP in synaptic plasticity in the brain.     

Mice lacking the ERK1 isoform of MAP kinase are unimpaired in emotional learning

The extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase (MAPK) superfamily of enzymes and have recently garnered considerable attention in the field of learning and memory. ERK activation has been shown to be required for the induction of long-term potentiation (LTP) in the rat hippocampus and for the formation of associative and spatial memories in both the rat and the mouse. However, the individual roles for the two isoforms of ERK have yet to be deciphered. To investigate the specific contribution of the ERK1 (p44) isoform of MAPK to mammalian learning, we performed a general behavioral and physiological characterization of mice lacking the ERK1 gene. The ERK1-null animals demonstrated significantly higher levels of activity in the open field test. However, we observed no other discernible deficits in the ERK1 knockout mice in our behavioral testing. Specifically, no differences were observed in the acquisition or retention (24 h and 2 wk after training) of either contextual or cue fear conditioning between the ERK1^−/− and their wild-type littermate controls. In addition, no learning phenotype was observed in the passive avoidance test. When hippocampal slices were analyzed, we found no deficits in baseline synaptic transmission or in tetanus-induced LTP in hippocampal area CA1. We found no apparent compensatory changes in the expression of ERK2 (p42 MAPK). We conclude that hippocampus- and amygdala-dependent emotional learning does not depend critically on the activity of ERK1.     

NT-3 facilitates hippocampal plasticity and learning and memory by regulating neurogenesis

In the adult brain, the expression of NT-3 is largely confined to the hippocampal dentate gyrus (DG), an area exhibiting significant neurogenesis. Using a conditional mutant line in which the NT-3 gene is deleted in the brain, we investigated the role of NT-3 in adult neurogenesis, hippocampal plasticity, and memory. Bromodeoxyuridine (BrdU)-labeling experiments demonstrated that differentiation, rather than proliferation, of the neuronal precursor cells (NPCs) was significantly impaired in DG lacking NT-3. Triple labeling for BrdU, the neuronal marker NeuN, and the glial marker GFAP indicated that NT-3 affects the number of newly differentiated neurons, but not glia, in DG. Field recordings revealed a selective impairment in long-term potentiation (LTP) in the lateral, but not medial perforant path-granule neuron synapses. In parallel, the NT-3 mutant mice exhibited deficits in spatial memory tasks. In addition to identifying a novel role for NT-3 in adult NPC differentiation in vivo, our study provides a potential link between neurogenesis, dentate LTP, and spatial memory.     

Transgenic mice expressing a truncated form of CREB-binding protein (CBP) exhibit deficits in hippocampal synaptic plasticity and memory storage

Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII alpha promoter drives expression of an inhibitory truncated CBP protein in forebrain neurons. Examination of hippocampal long-term potentiation (LTP), a form of synaptic plasticity thought to underlie memory storage, revealed significantly reduced late-phase LTP induced by dopamine-regulated potentiation in hippocampal slices from CBP transgenic mice. However, four-train induced late-phase LTP is normal. Behaviorally, CBP transgenic mice exhibited memory deficits in spatial learning in the Morris water maze and deficits in long-term memory for contextual fear conditioning, two hippocampus-dependent tasks. Together, these results demonstrate that CBP is involved in specific forms of hippocampal synaptic plasticity and hippocampus-dependent long-term memory formation.     

Reorganization of learning-associated prefrontal synaptic plasticity between the recall of recent and remote fear extinction memory

We have previously shown that fear extinction is accompanied by an increase of synaptic efficacy in inputs from the ventral hippocampus (vHPC) and mediodorsal thalamus (MD) to the medial prefrontal cortex (mPFC) and that disrupting these changes to mPFC synaptic transmission compromises extinction processes. The aim of this study was to examine whether these extinction-related changes undergo further plasticity as the memory of extinction becomes more remote. Changes in synaptic efficacy in both vHPC-mPFC and MD-mPFC inputs were consequently analyzed when the memory was either 1 d or 7 d old. Increases of synaptic efficacy in the vHPC-mPFC pathway were observed when the memory was 1 d old, but not 7 d after initial extinction. In contrast, potentiation of synaptic efficacy in the MD-mPFC pathway increased over time. In rats that received low-frequency vHPC stimulation immediately after extinction, both vHPC-mPFC and MD-mPFC inputs failed to develop potentiation, and the recall of extinction (both recent and remote memories) was impaired. These findings suggest that post-extinction potentiation in vHPC-mPFC inputs may be necessary for both the recall of recent memory and post-extinction potentiation in the MD-mPFC inputs. This late potentiation process may be required for the recall of remote extinction memory.     

Impairment of spatial learning and hippocampal synaptic potentiation in c-kit mutant rats

The c-kit receptor tyrosine kinase encoded by the white-spotting (W) gene is highly expressed in rat hippocampal CA1–CA4 regions. We found an impaired spatial learning and memory in homozygous c-kit (Ws/Ws) mutant rats that have a 12-base deletion in the tyrosine kinase domain of the c-kit gene and a very low kinase activity. Electrophysiological studies in hippocampal slices revealed that the long-term potentiation (LTP) induced by the tetanic stimulation (100 Hz, 1 sec) in the mossy fiber (MF)–CA3 pathway, but not in the Schaffer collaterals/commissural–CA1 pathway, was significantly reduced in c-kit mutants compared with wild-type (+/+) rats. The paired-pulse facilitation (PPF) was measured before the tetanus and after the establishment of the LTP in each slice. The initial PPF in the MF–CA3 pathway positively correlated with the amplitude of the LTP in the wild-type rats but not in the c-kit mutant rats. Furthermore, they failed to show the normal characteristics observed in the MF–CA3 pathway of +/+ rats; that is, the negative correlation between the initial PPF and the changes in PPF measured after the LTP. These findings suggest an involvement of SCF/c-kit signaling in hippocampal synaptic potentiation and spatial learning and memory.     

Synaptic plasticity in hippocampal CA1 neurons of mice lacking type 1 inositol-1,4,5-trisphosphate receptors

In hippocampal CA1 neurons of wild-type mice, delivery of a standard tetanus (100 pulses at 100 Hz) or a train of low-frequency stimuli (LFS; 1000 pulses at 1 Hz) to a naive input pathway induces, respectively, long-term potentiation (LTP) or long-term depression (LTD) of responses, and delivery of LFS 60 min after tetanus results in reversal of LTP (depotentiation, DP), while LFS applied 60 min before tetanus suppresses LTP induction (LTP suppression). To evaluate the role of the type 1 inositol-1,4,5-trisphosphate receptor (IP3R1) in hippocampal synaptic plasticity, we studied LTP, LTD, DP, and LTP suppression of the field excitatory postsynaptic potentials (EPSPs) in the CA1 neurons of mice lacking the IP3R1. No differences were seen between mutant and wild-type mice in terms of the mean magnitude of the LTP or LTD induced by a standard tetanus or LFS. However, the mean magnitude of the LTP induced by a short tetanus (10 pulses at 100 Hz) was significantly greater in mutant mice than in wild-type mice. In addition, DP or LTP suppression was attenuated in the mutant mice, the mean magnitude of the responses after delivery of LFS or tetanus being significantly greater than in wild-type mice. These results suggest that, in hippocampal CA1 neurons, the IP3R1 is involved in LTP, DP, and LTP suppression but is not essential for LTD. The facilitation of LTP induction and attenuation of DP and LTP suppression seen in mice lacking the IP3R1 indicates that this receptor plays an important role in blocking synaptic potentiation in hippocampal CA1 neurons.     

Removal of S6K1 and S6K2 leads to divergent alterations in learning, memory, and synaptic plasticity

Protein synthesis is required for the expression of enduring memories and long-lasting synaptic plasticity. During cellular proliferation and growth, S6 kinases (S6Ks) are activated and coordinate the synthesis of de novo proteins. We hypothesized that protein synthesis mediated by S6Ks is critical for the manifestation of learning, memory, and synaptic plasticity. We have tested this hypothesis with genetically engineered mice deficient for either S6K1 or S6K2. We have found that S6K1-deficient mice express an early-onset contextual fear memory deficit within one hour of training, a deficit in conditioned taste aversion (CTA), impaired Morris water maze acquisition, and hypoactive exploratory behavior. In contrast, S6K2-deficient mice exhibit decreased contextual fear memory seven days after training, a reduction in latent inhibition of CTA, and normal spatial learning in the Morris water maze. Surprisingly, neither S6K1- nor S6K2-deficient mice exhibited alterations in protein synthesis-dependent late-phase long-term potentiation (L-LTP). However, removal of S6K1, but not S6K2, compromised early-phase LTP expression. Furthermore, we observed that S6K1-deficient mice have elevated basal levels of Akt phosphorylation, which is further elevated following induction of L-LTP. Taken together, our findings demonstrate that removal of S6K1 leads to a distinct array of behavioral and synaptic plasticity phenotypes that are not mirrored by the removal of S6K2. Our observations suggest that neither gene by itself is required for L-LTP but instead may be required for other types of synaptic plasticity required for cognitive processing.     

Impaired long-term potentiation and enhanced neuronal excitability in the amygdala of CaV1.3 knockout mice

Previously, we demonstrated that mice in which the gene for the L-type voltage-gated calcium channel Ca_V1.3 is deleted (Ca_V1.3 knockout mice) exhibit an impaired ability to consolidate contextually-conditioned fear. Given that this form of Pavlovian fear conditioning is critically dependent on the basolateral complex of the amygdala (BLA), we were interested in the mechanisms by which Ca_V1.3 contributes to BLA neurophysiology. In the present study, we used in vitro amygdala slices prepared from Ca_V1.3 knockout mice and wild-type littermates to explore the role of Ca_V1.3 in long-term potentiation (LTP) and intrinsic neuronal excitability in the BLA. We found that LTP in the lateral nucleus (LA) of the BLA, induced by high-frequency stimulation of the external capsule, was significantly reduced in Ca_V1.3 knockout mice. Additionally, we found that BLA principal neurons from Ca_V1.3 knockout mice were hyperexcitable, exhibiting significant increases in firing rates and decreased interspike intervals in response to prolonged somatic depolarization. This aberrant increase in neuronal excitability appears to be at least in part due to a concomitant reduction in the slow component of the post-burst afterhyperpolarization. Together, these results demonstrate altered neuronal function in the BLA of Ca_V1.3 knockout mice which may account for the impaired ability of these mice to consolidate contextually-conditioned fear.     

Signaling Mechanisms Mediating BDNF Modulation of Synaptic Plasticity in the Hippocampus

Although recent studies indicate that brain-derived neurotrophic factor (BDNF) plays an important role in hippocampal synaptic plasticity, the underlying signaling mechanisms remain largely unknown. Here, we have characterized the signaling events that mediate the BDNF modulation of high-frequency synaptic transmission. Mitogen-associated protein kinase (MAPK), phosphotidylinositol-3 kinase (PI3K), and phospholipase C-γ (PLC-γ) are the three signaling pathways known to mediate neurotrophin signaling in other systems. In neonatal hippocampal slices, application of BDNF rapidly activated MAPK and PI3K but not PLC-γ. BDNF greatly attenuated synaptic fatigue at CA1 synapses induced by a train of high-frequency, tetanic stimulation (HFS). Inhibition of the MAPK and PI3K, but not PLC-γ, prevented the BDNF modulation of high-frequency synaptic transmission. Neurotrophin-3 (NT-3), a close relative of BDNF, did not activate MAPK or PI3K and had no effect on synaptic fatigue in the neonatal hippocampus. Neither forskolin, which activated MAPK but not PI3 kinase, nor ciliary neurotrophic factor (CNTF), which activated PI3K but not MAPK, affected HFS-induced synaptic fatigue. Treatment of the slices with forskolin together with CNTF still had no effect on synaptic fatigue. Thus, although the activation of MAPK and PI3K is required, the two together are not sufficient to mediate the BDNF effect. Inhibition of new protein synthesis by anisomycin or cycloheximide did not prevent the BDNF effect. These data suggest that BDNF modulation of high-frequency transmission is independent of protein synthesis but requires MAPK and PI3K and yet another signaling pathway to act together in the hippocampus.     

Coordinate high-frequency pattern of stimulation and calcium levels control the induction of LTP in striatal cholinergic interneurons

Current evidence appoints a central role to cholinergic interneurons in modulating striatal function. Recently, a long-term potentiation (LTP) of synaptic transmission has been reported to occur in these neurons. The relationship between the pattern of cortico/thalamostriatal fibers stimulation, the consequent changes in the intracellular calcium concentration ([Ca2+]i), and the induction of synaptic plasticity was investigated in striatal cholinergic interneurons from a rat corticostriatal slice preparation by means of combined electrophysiological intracellular recordings and microfluorometric techniques. Different protocols of stimulation were considered, varying both the frequency and the duration of the train of stimuli. High-frequency stimulation (HFS) (three trains at 100 Hz for 3 sec, 20-sec interval) induced a rise in [Ca2+]i, exceeding by fivefold the resting level, and caused a LTP of synaptic transmission. Tetanic stimulation delivered at lower frequencies (5-30 Hz) failed to induce long-term changes of synaptic efficacy. The observed elevation in [Ca2+]i during HFS was primarily mediated by L-type high-voltage activated (HVA)-Ca2+ channels, as it was fully prevented by nifedipine. Conversely, blockade of NMDA and AMPA glutamate receptor did not affect either LTP or the magnitude of the [Ca2+]i rise. Interestingly, the pharmacological analysis of the post-tetanic depolarizing postsynaptic potential (DPSP) revealed that LTP was attributable, to a large extent, to the potentiation of the GABA(A)-mediated component. In conclusion, the expression of LTP in striatal cholinergic interneurons is a selective response to a precise stimulation pattern of induction requiring a critical rise in [Ca2+]i.