Sunday, 30 December 2012

Synaptic plasticity, memory and the hippocampus: a neural betwork approach to causality

Hippocampus is important in episodic memory. HM suffered from untractable epilepsy. Underwent surgery  Bilateral removal of medial temporal love and large parts of both hippocampi. Could not form new episodic memories. Some loss of old memories. Controlled lesion, pharmacological inactivation or molecular knockouts limited to hippocampus cause failure to learn or loss of spatial memory.

Synaptic plasticity in hippocampus
Use extracellular recording to record synaptic events. LTP was identified in hippocampus.  After learning, studies detected LTP_like synaptic changes in hippocampus. Other activity dependent plasticity found eg LTD, EPSP spike potentiation, etc.  Transverse hippocampus slice preparation enables pharmacological agents to be rapidly washed on and off and allowing intracellular and patch clamp recordings  Culture neurons enabling overexpression or knockdown of spc proteins. Hebb postulated that connections between coactive neurons are strengthened through synaptic plasticity. So subsequent activation by incoming stimulation of only a subcomponent of assembly will activate whole assembly.  LTP requires coincident activity of pre and postsynpatic neurons.
a | Extracellular recordings of LTP induced by tetanic stimulation of the Schaffer-commissural projection (Sch) to CA1 pyramidal cells in a transverse hippocampal slice (shown as a schematic in the top panel). Hippocampal slices can be kept healthy for many hours if a steady flow of oxygen and artificial cerebrospinal fluid is supplied. The laminated organization of the hippocampus lends itself perfectly to extracellular recording techniques, allowing selective pathways to be stimulated and the evoked synaptic responses generated by a population of target neurons to be monitored for prolonged periods of time. The middle panel shows typical synaptic responses recorded from the apical dendritic region of the CA1 subfield following stimulation of the Schaffer-commissural pathway. Two metal stimulating electrodes are placed on either side of the recording electrode to evoke responses in overlapping populations of pyramidal cells through different sets of synapses. A tetanus (a brief, high-frequency train of electrical stimuli) can be used to induce LTP lasting for many hours in the tetanized pathway (bottom panel, closed circles); the second, control pathway (open circles) receives only test stimulation and is not potentiated following the tetanus to the experimental pathway. This demonstrates an important property of LTP, namely input specificity. b | In vivo LTP induction by learning17. Synaptic responses from multiple locations can be recorded in area CA1 of freely moving animals using an array of recording electrodes and a single stimulating electrode (examples in middle panel). Rats were trained in an inhibitory avoidance (IA) task, a hippocampus-dependent form of single-trial learning in which a rodent avoids entering a dark arena where it has received a footshock (top panel). IA training leads to a rapid increase, lasting for hours, in the amplitude of evoked responses in some of the recorded pathways (green circles in lower panel) but not in others (red circles). Training-dependent synaptic enhancement (bottom panel, arrow IA) occludes LTP induced by delivering tetanic stimulation (bottom panel): compare the degree of potentiation induced by tetanic stimulation (arrow Tet) in the pathways that were enhanced by training (green circles) to the pathways that were unchanged (red circles). The numbers 1, 2 and 3 indicate the times at which sample responses were obtained from inputs that were either enhanced (green) or unchanged (red) following learning. Note that post-IA responses are re-normalized before tetanus-induced LTP. Superimposed responses in the middle panel show effects of learning (1+2) and the subsequent effects of delivering three episodes of tetanic stimulation (2+3). These results suggest that experience-dependent synaptic enhancement uses the same molecular mechanisms of expression as tetanus-induced LTP. DG, dentate gyrus; EC, entorhinal cortex; pp, perferant path. Part a modified, with permission, from Ref. 91 © (2003) Blackwell Science. Part b reproduced, with permission, from Ref. 17 © (2006) American Association for the Advancement of Science.

Testing necessity
To establish that synaptic plasticity eg LTP is necessary forinfo storage, block induction or expression of LTP in hippocampus only.  Infusion into hippocampus of selective NMDA blocker APV impairs learning and recall in Morris water maze validates  Drug applies into hippocampus and blocks LTP without affecting basal synaptic transmission. Inhibiting active form of protein kinase PKM by infusing into hippocampus its spec inhibitor ZIP, impairs spatial memory and block LTP even when inhibitor is applied days after memory is acquired or induction of LTP without affecting baseline synaptic transmission..  Drug may have spread outside hippocampus.

Reversibly inactivated NMDAR subunit NR1 in hippocampus. Gene product induces LTP.  LTP and spatial learning are suppressed. Indicates that NR1 receptors in CA1 is necessary for spatial learning. However LTP may not be necessary for spatial learning. NMDAR affects other processes.

Learning without hippocampal LTP?
LTP may not be necessary for spatial learning, In upstairs downstairs water maze experiment, rats were trained in one maze and could learn and retain info about location of hidden platform in a second upstairs maze even when infused with APV. Conventuainl NMDAR-dependent LT is not always required for acquisition and storage of hippocampus-dependent reference memory.

GluR1 knockout
In these KO moice, total absence of conventional tetanus-induced LTP in CA1 without impairing acquisition or recall in Morris water maze. It is hard to make sure that LTP is abolished. Depends on protocol. LTP could be induced using a theta-burst pairing protocol. Presynaptic stimulation at 5 Hz was paired with synchronous depolarisation of CA1 pyramidal cell. Theta burst stimulation mimics frequency of beta waves generated in hippocampus of rodents as they explore an environment.

Subregion spec deletion of NR1Use Cre recombinase driven by subregion spec promoters to restrict deletion of gene encoding NMDAR-subunit NR1 to spec subfields of hippocampus.  NR1 receptors in pryamidal cells of area CA1 seem to be essential for normal performance on Morris water maze.  Animals can perform tasks as well as controls if deletion is confined to pyramidal cells in CA3. NMDAR-mediated LTP in detate gyrus in CA3 is not necesary for acquisition and storage of spatial memory. This form of LTP is impossible in regions where NR1 is deleted and these animals can learn.

Exploiting immediate early genes
Silencing potentiated neurons at network level. Promoter for plasticity marker gene drives expression of a protein that reduces excitability of cell.  These genes have not been identifies that are expressed when only syanptic plasticity is induced. Expxression of immediate early genes are upregulated by LTP-inducing protocols invitro and in vivo. Arc/ARg3.1 and the TF Zif 268 are required to maintain LTP that lasts for several days and stability of longterm memories. A KI mouse in which coding sequence of Arc/Arg3.1 gene was replace by GFp gene.
The approach depends on genetic constructs in which promoters from activity-dependent genes (such as those that encode Arc/Arg3.1 or Zif268) are used to drive the expression of transgenes specifically in recently potentiated cells. These transgenes can be used to silence activated cell assemblies. a | Arc/Arg3.1 (immunostained in red) is activated in a subset of hippocampal neurons (in this figure, CA1 pyramidal cells) when an animal explores a novel environment92(scale bar 100 μm). b | Green fluroescent protein (GFP) mirrors the endogenous expression of Arc/Arg3.1 in a genetically engineered mouse in which the expression of GFP is controlled by the Arc/Arg3.1 gene promoter59. In this example, expression in the primary visual cortex is upregulated by light exposure. The NMDA (N-methyl-D-aspartate) receptor antagonist MK801 blocks this effect (scale bar 40 μm). c | The left-hand panel shows a confocal micrograph of a section obtained from the spinal cord of a transgenic mouse in which the engrailed gene promoter had been used to drive expression of the allatostatin receptor (AlstR) in a specific subtype of interneuron (labelled in green, reflecting expression of GFP). The right-hand panel shows a current-clamp recording from a labelled interneuron: in the presence of 10 nM allatostatin (Alst), neurons have a higher threshold for triggering action potentials and therefore are effectively inactivated68d | A way in which to abolish specific memories while sparing others. The activity-dependent Arc/Arg3.1 gene promoter is used to drive the expression of the allatostatin receptor. Cells that, as a result of training in Task A, acquire potentiated synapses (green cells in left panel) will express the receptor and can potentially be silenced by perfusion with allatostatin. Silencing is dependent on allatostatin, but also on the presence of the allatostatin receptor on the cell surface. After a certain period of time the receptors will be internalized and degraded (red cells, day 7). Recent memory, activating a different, possibly overlapping, population of cells (Task B, green cells) should therefore be abolished when these cells become silent (black) in the presence of allatostatin, whereas remote memories (red cells) should be spared. Part a reproduced, with permission, from Ref. 92 © (2005) Society for Neuroscience. Partb reproduced, with permission, from Ref. 59 © (2006) Elsevier Science. Part c reproduced, with permission, from Ref. 68 © (2006) Macmillan Publishers Ltd; courtesy of M. Goulding.
Memory erasure
To test necessity if LTP for learning and memory, erase memory b selectively reversing experience dependent plasticity. Perdusion ofZIP erases hippocampus dependent memory. ZIP only targets activated synapses but does not appear to differentiate between recent and old memories. In CA1, synapses that were recently potentiated can be depotentiated by low frequency stimulation.  depotentiation occurs only at synapses that were potentiated in preceding few minutes after which LTP is stabilised and resistant to depotentiation. This could erase LTP specifically in cell assemblies that represent a recently acquired memory.

a | The first thought-experiment requires a very large array of metal electrodes to monitor the spike activity of each cell in areas CA3 and CA1 (connectivity is unidirectional, from CA3 to CA1); the same electrodes can be used to stimulate each cell individually. Connectivity is sparse, and the great majority of CA3–CA1 cell pairs are not connected. b | Cross-correlation of spontaneous activity will identify connected pairs of CA3 and CA1 cells (here, A to 1, B to 3 and C to 2). The cross-correlogram plots the number of spikes emitted by a given CA1 cell during a given time interval (τ, τ + δτ) after each action potential of a given CA3 cell; a peak at a delay τ of a few milliseconds suggests that the two cells are monosynaptically connected. Following learning, the SPM hypothesis predicts that a subset of synapses will be potentiated (some perhaps will be depressed); these pairs will be identified by an increase (or decrease) in the peak of the cross-correlogram. Each of these affected synapses can be either depotentiated by low-frequency stimulation (which has no effect on unpotentiated synapses) or re-potentiated by appropriately timed spike-timing-dependent potentiation. Returning all synaptic strengths to baseline by depotentiation should abolish the memory. At an arbitrary later time, the memory can be reinstalled by re-potentiating or re-depressing the affected synapses by appropriately timed spike-timing-dependent plasticity. c | An attempt to use molecular genetics to achieve the same aim. With currently available technology, the best way to gain access to potentiated synapses is by first training the animal to form a memory that is transient. One way to achieve this is to use mutant animals that fail to form long-term memory, termed here 'forgetful mice', such as mice in which Arc/Arg 3.1 (Ref. 58), Zif268 (Ref. 60) or α/δCREB93 have been knocked out. An immediate-early gene promoter can be used to drive transcription of a molecular LTP device in recently activated synapses (shown in red in e). The transcript could encode, for instance, an exogenous ligand-gated Ca2+ channel (d). Infusion of the exogenous ligand would activate the Ca2+ channel (free-standing LTP device) in only those synapses that had recently been potentiated, inducing further potentiation in those synapses and thus re-installation of a memory in an animal in which memories were normally only transient. e | An important development that existing technologies do not yet allow is the targeting of transgenes to the specific synaptic sites that have undergone plasticity. Arc mRNA94 and protein95 are selectively transported to dendritic regions containing recently potentiated synapses, and possibly to the potentiated synapses themselves. The molecular mechanics behind the putative 'tagging' of synapses that allows them to capture recently synthesized proteins remains elusive. When we learn how synapses do this, we may be in a position to target exogenous proteins, including free-standing LTP devices, specifically to synapses that are activated during learning.

Is synaptic plasticity sufficient for memory?
Make memory without need for learning. Hippocampus dependent memory is formed. Memory is erased and reinstalled by exploiting knowledge about synaptic changes that occured during original learning.

Multi-electrode array
Access assembly of pre- and postsynpatic neurons to monitor plasticity between pairs of cells participating in encoding a new memory. Each has one partner in CA3 and one in Ca1. Cross correlation of action potentials from each CA3-CA1 pair before learning will detect rare pairs of cells that are connected. Amplitude of cross-correlogram measures strength of synapse that links both cells  Comparing peak before and after learning indicate which of connected pairs are part of cell assembly that encodes memory. Depotentation by immediate posttraining application of low-frequency stimulation erases memory. Spoke-timing-depedent plasticity in which LTP or LTD is induced by ordering sequence of pre and postsynaptic spiking is applied to return synapses to memory sate. Selectively reverse synaptic changes that underlie storage on one memory without affecting basal transmission or synaptic changes that subserve storage or other memories.

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