Sara E. Mason-Parker

Heterosynaptic metaplasticity in the hippocampus in vivo: a BCM-like modifiable threshold for LTP.

The homeostatic maintenance of the “modification threshold” for inducing long-term potentiation (LTP) is a fundamental feature of the Bienenstock, Cooper, and Munro (BCM) model of synaptic plasticity. In the present study, two key features of the modification threshold, its heterosynaptic expression and its regulation by postsynaptic neural activity, were tested experimentally in the dentate gyrus of awake, freely moving rats. Conditioning stimulation ranging from 10 to 1,440 brief 400-Hz trains, when applied to medial perforant path afferents, raised the threshold for LTP induction heterosynaptically in the neighboring lateral perforant path synapses. This effect recovered slowly over a 7- to 35-day period. The same conditioning paradigms, however, did not affect the reversal of long-term depression. The inhibition of LTP by medial-path conditioning stimulation was N-methyl-D-aspartate (NMDA) receptor-dependent, but antidromic stimulation of the granule cells could also inhibit lateral path LTP induction, independently of NMDA receptor activation. Increased calcium buffering is a potential mechanism underlying the altered LTP threshold, but the levels of two important calcium-binding proteins did not increase after conditioning stimulation, nor was de novo protein synthesis required for generating the threshold shift. These data confirm, in an in vivo model, two key postulates of the BCM model regarding the LTP threshold. They also provide further evidence for the broad sensitivity of synaptic plasticity mechanisms to the history of prior activity, i.e., metaplasticity.

Long-term regulation of N-methyl-D-aspartate receptor subunits and associated synaptic proteins following hippocampal synaptic plasticity.

Synaptic plasticity in the dentate gyrus is dependent on activation of the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors. In this study, we show that synaptic plasticity in turn regulates NMDA receptors, since subunits of the NMDA receptor complex are bidirectionally and independently regulated in the dentate gyrus following activation of perforant synapses in awake animals. Low-frequency stimulation that produced a mild synaptic depression resulted in a decrease in the NMDA receptor subunits NR1 and NR2B 48 h following stimulation. High-frequency stimulation that produced long-term potentiation resulted in an increase in NR1 and NR2B at the same time point. Further investigations revealed that in contrast to NR2B, NR1 levels increased gradually after long-term potentiation induction, reaching a peak level at 48 h, and were insensitive to the competitive NMDA receptor antagonist 3-3(2-carboxypiperazin-4-yl) propyl-1-phosphate. The increased levels of NR1 and NR2B at 48 h were found associated with synaptic membranes and with increased NMDA receptor-associated proteins, postsynaptic density protein 95, neuronal nitric oxide synthase and Ca(2+)/calmodulin-dependent protein kinase II, alpha subunit. These data suggest that the persistence of long-term potentiation is associated with an increase in the number of NMDA receptor complexes, which may be indicative of an increase in synaptic contact area.

Low-frequency stimulation does not readily cause long-term depression or depotentiation in the dentate gyrus of awake rats

The ability of low-frequency stimulation (LFS) to induce either long-term depression (LTD) or depotentiation was assessed for perforant path synapses in the dentate gyrus of awake, adult rats. Neither LFS at 1 Hz (100 or 900 pulses) nor LFS at 3 Hz (900 pulses) was sufficient to produce either LTD or depotentiation. LFS at 3 Hz did produce a transient response depression of previously potentiated synapses, but this lasted less than 24 h and was secondary to seizure-like afterdischarges. We conclude that the LFS protocols so effective at eliciting LTD and depotentiation in area CA1 are ineffective for perforant path synapses in the dentate gyrus.

Biphasic changes in the levels of N-methyl-d-aspartate receptor-2 subunits correlate with the induction and persistence of long-term potentiation

N-Methyl-D-aspartate glutamate receptors (NMDAR) form ion channels made up of polypeptides from two classes of subunits; NR1 is obligatory for function whereas members of the NR2 class regulate the properties of the channel. Long-term potentiation (LTP) of synaptic transmission is an event largely dependent on NMDAR activation, and is studied as the primary cellular model of memory in the mammalian brain. While there has been a focus on non-NMDARs in mediating the expression of LTP, we report here biochemical evidence for plasticity of the NMDAR that is associated with LTP persistence in awake animals. Following the establishment of LTP in perforant path synapses of the dentate gyrus, we observed a rise in NR2B protein levels 48 h post-tetanus which was dependent upon activation of NMDARs during the tetanization, and which strongly correlated with the degree of LTP measured at this time-point. We also observed a transient increase in both NR2B and NR2A protein levels 20 min post-tetanus that returned to control levels by 4 h. These early increases were not observed in anaesthetized animals which do not sustain persistent LTP. Our data demonstrate a marked plasticity of NMDAR subunit expression, which may affect LTP persistence, as well as the subsequent ability to induce LTP at previously activated synapses.

Differential Trafficking of AMPA and NMDA Receptors during Long-Term Potentiation in Awake Adult Animals

Despite a wealth of evidence in vitro that AMPA receptors are inserted into the postsynaptic membrane during long-term potentiation (LTP), it remains unclear whether this occurs in vivo at physiological concentrations of receptors. To address the issue of whether native AMPA or NMDA receptors undergo such trafficking during LTP in the adult brain, we examined the synaptic and surface expression of glutamate receptor subunits during the early induction phase of LTP in the dentate gyrus of awake adult rats. Induction of LTP was accompanied by a rapid NMDA receptor-dependent increase in surface expression of glutamate receptor 1–3 (GluR1–3) subunits. However, in the postsynaptic density fraction only GluR1 accumulated. GluR2/3-containing AMPA receptors, in contrast, were targeted exclusively to extrasynaptic sites in a protein synthesis-dependent manner. NMDA receptor subunits exhibited a delayed accumulation, both at the membrane surface and in postsynaptic densities, that was dependent on protein synthesis. These data suggest that trafficking of native GluR1-containing AMPA receptors to synapses is important for early-phase LTP in awake adult animals, and that this increase is followed homeostatically by a protein synthesis-dependent trafficking of NMDA receptors.

TrkB expression in dentate granule cells is associated with a late phase of long-term potentiation

Recent studies have demonstrated that the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are induced in hippocampal neurons following the induction of long-term potentiation (LTP), a model of memory, and that BDNF and NT-3 (but not NGF) can induce LTP-like increases in synaptic efficacy. Receptors for these neurotrophins have been cloned and characterized and we investigated whether LTP alters the expression of two neurotrophin receptors, trkB (BDNF receptor) and trkC (NT-3 receptor) in dentate granule neurons of the hippocampus using in situ hybridization analysis. Results show that trkB is strongly induced in these neurons in an N-methyl-D-aspartate (NMDA) receptor-dependent manner. Moreover, the induction of trkB and trkC mRNAs was attenuated by sodium pentobarbital, which interferes with the durability of LTP. Low-frequency stimulation of the perforant path had no effect on trkB mRNA levels but significantly reduced trkC mRNA in dentate granule cells. Thus, both BDNF and its receptor trkB are induced in granule cells by stimulation that produces durable LTP, suggesting that this neurotrophin and its receptor play an important role in memory formation and may be suitable targets for the development of cognitive-enhancing drugs in the treatment of diseases, such as Alzheimers.

Synaptic activity-dependent modulation of mitochondrial gene expression in the rat hippocampus.

In order to identify genes that may underlie the maintenance of long-term potentiation (LTP) at perforant path synapses, complementary DNA libraries were synthesised from dentate gyrus total RNA extracts prepared 48 h after the induction of LTP and from control dentate gyrus extracts. Through differential screening of the LTP library we have identified the mitochondrial 12S rRNA (mt12SrRNA) as a transcript that was elevated at this late time. Northern blot analyses showed that the elevation in mt12SrRNA expression began around 8 h and persisted for at least 2 weeks post-tetanus. We then examined the expression patterns of other mitochondrially-encoded genes and demonstrated a similar elevation in their expression. mt12SrRNA levels were also elevated in other hippocampal regions, including areas CA3 and CA1 and were elevated following low-frequency stimulation or in the presence of an N-methyl-D-aspartate receptor antagonist where induction of LTP was precluded. Taken together, these observations suggest that a long-lasting up-regulation of energy production may be triggered by synaptic activity and this activity need not be of sufficient strength to induce LTP, but may be related to the induction of a metaplastic state.

Secreted amyloid precursor protein-α upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism

Secreted amyloid precursor protein-alpha (sAPPalpha) is a neuroprotective and neurotrophic protein derived from the parent APP molecule. We have shown that sAPPalpha enhances long-term potentiation in vivo and can restore spatial memory in rats whose endogenous sAPPalpha production is impaired. These observations imply that the reduction of sAPPalpha levels seen in Alzheimers disease, which occurs alongside increased levels of toxic amyloid-beta, may be aetiologically significant. The mechanism by which sAPPalpha brings about changes in plasticity at synapses remains unresolved. We hypothesised that sAPPalpha may stimulate changes in synaptodendritic protein synthesis, an important mechanism for normal plasticity. To test this hypothesis, we investigated the effect of sAPPalpha on protein synthesis in synaptoneurosomes prepared from the hippocampi of adult male Sprague-Dawley rats. sAPPalpha (10nM) significantly increased de novo protein synthesis as measured by the incorporation of [(35)S]-methionine into acid-insoluble proteins. This was dose-dependent and blocked completely by inhibitors of protein synthesis (cycloheximide) and of cGMP-dependent protein kinase (KT5823). Inhibitors of calcium/calmodulin-dependent protein kinases (KN62) and mitogen-activated protein kinase (PD98059) partially blocked the response. Further, the sAPPalpha-induced increase in protein synthesis was significantly attenuated when measured in synapses isolated from aged rats. These observations imply de novo protein synthesis at synapses may contribute to the long-lasting modulatory effects of sAPPalpha on synaptic plasticity.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation.

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-1 DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP.

Rapidly induced gene networks following induction of long-term potentiation at perforant path synapses in vivo.

The canonical view of the maintenance of long‐term potentiation (LTP), a widely accepted experimental model for memory processes, is that new gene transcription contributes to its consolidation; however, the gene networks involved are unknown. To address this issue, we have used high‐density Rat 230.2 Affymetrix arrays to establish a set of genes induced 20‐min post‐LTP, and using Ingenuity Pathway network analysis tools we have investigated how these early responding genes are interrelated. This analysis identified LTP‐induced regulatory networks in which the transcription factors (TFs) nuclear factor‐KB and serum response factor, which, to date, have not been widely recognized as coordinating the early gene response, play a key role alongside the more well‐known TFs cyclic AMP response element‐binding protein, and early growth response 1. Analysis of gene‐regulatory promoter sites and chromosomal locations of the genes within the dataset reinforced the importance of these molecules in the early gene response and predicted that the coordinated action might arise from gene clustering on particular chromosomes. We have also identified a transcription‐based response that affects mitogen‐activated protein kinase signaling pathways and protein synthesis during the stabilization of the LTP response. Furthermore, evidence from biological function, networks, and regulatory analyses showed convergence on genes related to development, proliferation, and neurogenesis, suggesting that these functions are regulated early following LTP induction. This raises the interesting possibility that LTP‐related gene expression plays a role in both synaptic reorganization and neurogenesis.