Review
Long-term potentiation in cultured hippocampal neurons

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Abstract

Studies performed on low-density primary neuronal cultures have enabled dissection of molecular and cellular changes during N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Various electrophysiological and chemical induction protocols were developed for the persistent enhancement of excitatory synaptic transmission in hippocampal neuronal cultures. The characterisation of these plasticity models confirmed that they share many key properties with the LTP of CA1 neurons, extensively studied in hippocampal slices using electrophysiological techniques. For example, LTP in dissociated hippocampal neuronal cultures is also dependent on Ca2+ influx through post-synaptic NMDA receptors, subsequent activation and autophosphorylation of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor insertion at the post-synaptic membrane. The availability of models of LTP in cultured hippocampal neurons significantly facilitated the monitoring of changes in endogenous postsynaptic receptor proteins and the investigation of the associated signalling mechanisms that underlie LTP. A central feature of LTP of excitatory synapses is the recruitment of AMPA receptors at the postsynaptic site. Results from the use of cell culture-based models started to establish the mechanism by which synaptic input controls a neuron's ability to modify its synapses in LTP. This review focuses on key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors.

Highlights

► Models were developed for the investigation of LTP in dissociated hippocampal neurons. ► Key features of LTP induction procedures are reviewed. ► Characteristics of NMDAR-LTP in hippocampal neuronal cultures are discussed. ► Molecular mechanisms underlying synaptic activity-induced changes are considered. ► Advantages and limitations of cell culture-based LTP models are highlighted.

Introduction

Understanding the molecular basis of long-term potentiation (LTP) is of fundamental importance because it has been implicated in learning and memory as well as other physiological and pathological processes. One form of LTP develops through the activation of NMDA receptors (NMDARs) and an increase in AMPA receptor (AMPAR) insertion at the post-synaptic membrane [1]. NMDARs are formed from hetero-tetrameric assemblies of GluN1 (previously NR1) subunits with GluN2A–D and GluN3A/B (previously NR2A–D and NR3A/B) subunits [2]. NMDARs require the binding of l-glutamate and the co-agonist glycine, as well as depolarisation, to become activated and conduct Na+, K+, and Ca2+ ions. The voltage-dependent blockade of the NMDAR pore by Mg2+ produces this additional requirement for depolarisation [2]. AMPARs are composed of four subunits, GluA1–4 (previously GluR1–4) [2]. The edited GluA2 subunit is critical for the biophysical properties of AMPARs producing low conductance, non-rectifying, Ca2+-impermeable AMPARs, and postnatally the great majority of AMPARs contain edited GluA2 [2], [3]. AMPARs are activated directly by l-glutamate binding, and their activation leads to changes in the membrane potential of the postsynaptic neuron [2].

Neuronal synapses are densely packed in the brain, making them difficult to analyse and visualise individually in vivo. The most obvious advantage of dissociated neuronal culture is that it makes individual living cells and their synapses more accessible. Dissociated neurons will grow as a monolayer (Fig. 1A) and the surface-expressed neurotransmitter receptors and other membrane proteins are therefore accessible for both immunocytochemical and biochemical analysis (Fig. 1B). Hippocampal neurons in dissociated cultures retain their characteristics, from the properties of the transmitter receptors and ion channels they express, to the organisation of their cytoskeletal constituents and the characteristics of specific synapses. This is due to the fact that they are postmitotic and are therefore committed in their differentiation at the time they are introduced into culture. Neuronal cultures were extensively used to understand the formation and development of synapses [4] and developmental changes in glutamate receptors [5]. Using immunocytochemical approaches, these studies have also provided morphological evidence for “silent synapses” that physically contain NMDARs but no AMPARs [3], [5]. The established link between synaptic plasticity and dendritic exocytosis, along with the demonstration of the role of silent synapses in LTP provided strong stimulus for the development of cellular and molecular techniques that could monitor and interfere with trafficking of AMPARs to and away from synapses. While sustained depression of glutamatergic transmission has been readily induced and studied in neuronal cultures [6], [7], the development of reliable and robust procedures for the induction LTP in dissociated cultures proved to be more challenging. Conventional electrophysiological LTP induction protocols involve an intensive but transient activation of a small set of synapses. In contrast, continuous chemical activation of neuronal networks, which involves many more synapses, maximizes the likelihood of detection of molecular and morphological changes in cells involved [8], [9]. Over the last ten years various protocols were used for the investigation of NMDAR dependent LTP in primary hippocampal neuronal cultures. These models created new opportunities for the study of LTP-related changes in endogenous native proteins and signalling pathways.

This review focuses on experimental models developed for the investigation of LTP in dissociated hippocampal neurons. After the introduction of various LTP induction protocols, some of their applications and limitations will be discussed.

Section snippets

Protocols for the induction of synaptic potentiation in cultured hippocampal neurons

Standard electrode stimulation activates only a small fraction of synapses, making it very difficult to detect molecular and cellular changes associated with LTP. Most biochemical analysis and imaging studies require high proportion of potentiated synapses. Therefore, a range of strategies were applied to chemically induce LTP (Chem-LTP) at a majority of synapses. Several of the Chem-LTP induction protocols were originally developed for electrophysiological studies of brain slices [10], [11],

Characteristics of LTP in hippocampal neuronal cultures

The characterisation of these hippocampal neuronal culture-based plasticity models confirmed that they share many key properties with the NMDAR-dependent LTP of CA1 neurons in hippocampal slices. Chem-LTP in neuronal cultures also requires NMDAR activation and a rise in postsynaptic intracellular Ca2+ concentration [23], [32], [34], [48], [55]. This is consistent with the idea that APV pre-conditioning of hippocampal neuronal cultures and the use of Mg2+ and APV free medium for LTP induction

Recruitment of AMPARs to the cell surface following LTP induction in neuronal cultures

While NMDARs are controlling synaptic plasticity, AMPARs mediate the vast majority of fast excitatory synaptic transmission in the mammalian brain and they are key components of the modifiable synaptic response. A central feature of LTP of excitatory synapses is the recruitment of AMPARs at the postsynaptic site [1]. The availability of antibodies that recognise epitopes of glutamate receptors on living neurons [73], [74], [75] combined with Chem-LTP models in dissociated hippocampal neurons in

Conclusions

Several studies have proved the possibility to chemically promote a lasting increase in synaptic transmission though the activation of NMDARs in hippocampal neuronal cultures. These cell culture-based Chem-LTP models offer several advantages: (1) Hippocampal neurons in culture simplify complex circuitry compare to the considerably more complex brain slice preparations. (2) Relatively simple induction procedures have been developed and characterised. (3) Following induction, the activity of a

Acknowledgements

This work was supported by the Medical Research Council UK [Grant 80049] and the Biotechnology and Biological Sciences Research Council UK [Grant BB/F011326/1].

References (84)

  • H. Lin et al.

    Temporal dynamics of NMDA receptor-induced changes in spine morphology and AMPA receptor recruitment to spines

    Biochem Biophys Res Commun

    (2004)
  • M. Park et al.

    Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes

    Neuron

    (2006)
  • E. Korkotian et al.

    Morphological constrains on calcium dependent glutamate receptor trafficking into individual dendritic spine

    Cell Calcium

    (2007)
  • H.Y. Man et al.

    Activation of PI3-kinase is required for AMPA receptor insertion during LTP of mEPSCs in cultured hippocampal neurons

    Neuron

    (2003)
  • Y. Huang et al.

    S-nitrosylation of N-ethylmaleimide sensitive factor mediates surface expression of AMPA receptors

    Neuron

    (2005)
  • K. Sharma et al.

    Postsynaptic protein mobility in dendritic spines: long-term regulation by synaptic NMDA receptor activation

    Mol Cell Neurosci

    (2006)
  • E.M. Petrini et al.

    Endocytotic trafficking and recycling maintain a pool of mobile surface AMPA receptors required for synaptic potentiation

    Neuron

    (2009)
  • S.M. Fitzjohn et al.

    An electrophysiological characterisation of long-term potentiation in cultured dissociated hippocampal neurones

    Neuropharmacology

    (2001)
  • L. Pickard et al.

    Transient synaptic activation of NMDA receptors leads to the insertion of native AMPA receptors into hippocampal neuronal plasma membrane

    Neuropharmacology

    (2001)
  • K. Fukunaga et al.

    Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II

    J Biol Chem

    (1993)
  • S. Strack et al.

    Translocation of autophosphorylated calcium/calmodulin-dependent protein kinase II to the postsynaptic density

    J Biol Chem

    (1997)
  • N. Inagaki et al.

    Activation of Ca2+/calmodulin-dependent protein kinase II within post-synaptic dendritic spines of cultured hippocampal neurons

    J Biol Chem

    (2000)
  • B. Bingol et al.

    Autophosphorylated CaMKIIa acts as a scaffold to recruit proteasomes to dendritic spine

    Cell

    (2010)
  • A. Barria et al.

    NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII

    Neuron

    (2005)
  • J.E. Lisman et al.

    A model of synaptic memory: a CaMKII/PP1 switch that potentiates transmission by organizing an AMPA receptor anchoring assembly

    Neuron

    (2001)
  • P. Opazo et al.

    CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazing

    Neuron

    (2010)
  • S.A. Richmond et al.

    Localization of the glutamate receptor subunit GluR1 on the surface of living and within cultured hippocampal neurons

    Neuroscience

    (1996)
  • J. Noel et al.

    Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF-dependent mechanism

    Neuron

    (1999)
  • Z. Wang et al.

    Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity

    Cell

    (2008)
  • Y. Serulle et al.

    A GluA1-cGKII interaction regulates AMPA receptor trafficking

    Neuron

    (2007)
  • S. Shi et al.

    Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons

    Cell

    (2001)
  • S. Cull-Candy et al.

    Regulation of Ca2+-permeable AMPA receptors: synaptic plasticity and beyond

    Curr Opin Neurobiol

    (2006)
  • J.T.R. Isaac et al.

    The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity

    Neuron

    (2007)
  • S.J. Liu et al.

    Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death

    Trends Neurosci

    (2007)
  • G.L. Collingridge et al.

    Receptor trafficking and synaptic plasticity

    Nat Rev Neurosci

    (2004)
  • S.F. Traynelis et al.

    Glutamate receptor ion channels: structure, regulation, and function

    Pharmacol Rev

    (2010)
  • E. Molnár et al.

    Developmental and activity dependent regulation of ionotropic glutamate receptors at synapses

    ScientificWorldJournal

    (2002)
  • E. Molnár et al.

    Developmental changes in ionotropic glutamate receptors: lessons from hippocampal synapses

    Neuroscientist

    (2002)
  • C.M. Gladding et al.

    Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms

    Pharmacol Rev

    (2009)
  • G.L. Collingridge et al.

    Long-term depression in the CNS

    Nat Rev Neurosci

    (2010)
  • J.F. MacDonald et al.

    LTP in a culture dish

    TheScientificWord

    (2001)
  • T. Hosokawa et al.

    Repeated confocal imaging of individual dendritic spines in the living hippocampal slice: evidence for changes in length and orientation associated with chemically induced LTP

    J Neurosci

    (1995)
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