Neurobiology 2008 Micha Spira Learning and Memory The

Neurobiology 2008 Micha Spira Learning and Memory The Aplysia Model STF 1

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• Homosynaptic depression is the electrophysiological equivalent of habituation • Dishabituation leads to increased Rin (~140%) of the sensory neuron • WHAT IS THE MECHANISM OF INCREASED Rin ? ? ? 7

A New concept- neurotransmitter that modulate channels conductances rather than turn on ionic channels 8

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13 Figure 15 -30 Molecular Biology of the Cell (© Garland Science 2008)

The structure of an inactive G protein The units are covalently linked to lipids. When activated it operates as a guanine nucleotide exchange factor (GEF) 14 Figure 15 -31 Molecular Biology of the Cell (© Garland Science 2008)

The new concept is that binding to the receptor expose buried surfaces rather than dissociate between the subunits The a subunit function as a GTPase The target or specific proteins regulate by enhancing the GTPase activity of the a subunit 15 Figure 15 -32 Molecular Biology of the Cell (© Garland Science 2008)

c. AMP concentration is 10 -7, an extracellular signal can increase the concentration within seconds by a factor of 20. 16 Figure 15 -33 Molecular Biology of the Cell (© Garland Science 2008)

17 Figure 15 -34 Molecular Biology of the Cell (© Garland Science 2008)

18 Table 15 -1 Molecular Biology of the Cell (© Garland Science 2008)

• Cholera toxin- inhibit the GTPase activity, leads to elevated c. AMP levels , that cause release of Cl and water from epithelial cells to the gut. • Pertussis toxin prevents the protein fro interacting with the receptors 19

The activation of PKA (cyclic AMP 20 Figure 15 -35 Molecular Biology of the Cell (© Garland Science 2008)

21 Figure 15 -36 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

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Neurobiology 2008 Micha Spira PKC and STF Aplysia Model 24

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28 Non depressed synapse. Short term facilitation 5 minutes after 5 HT application

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Inositol Phospholipid signaling pathway • GPCRs (Gq) also exerts their effects by activation of the membrane bound Phospholipids • The substrate is a phosphorylated inositol phospholipids PIP 2 which is presented in small amounts on the inner half of the plasma membrane • Cleavage of PIP 2 gives diacylglycerol and Inositol trisphosphate 31

32 Figure 15 -37 Molecular Biology of the Cell (© Garland Science 2008)

33 Figure 15 -38 Molecular Biology of the Cell (© Garland Science 2008)

Initiation and termination of the signal • IP 3 is water soluble • IP 3 binds to IP 3 gated calcium channels of the ER and releases calcium. • IP 3 is inactivated by: Dephosphorylation to IP 2 IP 3 is phosphorylated by a specific kinase to IP 4 Ca is pumped out of the cell 34

Diacylglycerol • Lipid soluble operate at the interface of the membrane • Ca induce traslocation of PKC from the cytosol to the plasma membrane. The calcium in combination with diacylglycerol activate PKC 35

36 Figure 15 -39 Molecular Biology of the Cell (© Garland Science 2008)

Fertilization of an egg by a sperm The penetrating sperm releases PLC which induces release of calcium 37 Figure 15 -40 Molecular Biology of the Cell (© Garland Science 2008)

38 Figure 15 -41 a Molecular Biology of the Cell (© Garland Science 2008)

39 Figure 15 -41 b Molecular Biology of the Cell (© Garland Science 2008)

Calmodulin acts as a multipurpose intracellular calcium receptor 40 Figure 15 -43 Molecular Biology of the Cell (© Garland Science 2008)

Calmodulin activates Ca. M- Kinase II 41 Figure 15 -44 Molecular Biology of the Cell (© Garland Science 2008)

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Alterations at the post synaptic level 43

44 Figure 15 -45 Molecular Biology of the Cell (© Garland Science 2008)

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Amplification and time scales • A single molecule of rhodopsin activated by a single photon catalyze the activation of hundreds of transducine at a rate of about 1000 transducins / sec • Each transducin activate phosphodiesterase that hydrolyze 4000 c. GMP/ sec. • The cascade lasts 1 sec • This leads to closure of hundreds of cataion channels in the membrane 48

Long term potentiation 49

Micha Spira Neurobiology 2008 • Long term 50 55

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59 57 Figure 15 -36 Molecular Biology of the Cell (© Garland Science 2008)

58 59 Figure 15 -36 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)

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Activation of nuclear transcription factor • c. AMP responsive element • CRE –binding protein (CREB 1) phosphorylated by PKA, MAPK or Cam kinase • CREB 1 function as transcriptional activator • Leads to LTF • Injection of CRE oligonucleotides binds to CREB 1 and block LTF but not STF • Concomitantly CREB 2 is down regulated by MAPK • Injection of anti. CREB 2 antibodies is sufficient to trigger facilitation 60 58

Aplysia sensory neurons Fig. 2. Time course and functional contribution of two distinct presynaptic structural changes associated with intermediate- and long-term facilitation in Aplysia. Repeated pulses of 5 -HT in sensory to motor neuron co-cultures trigger two distinct classes of presynaptic structural changes: (1) the rapid clustering of synaptic vesicles to pre-existing silent sensory neuron varicosities (3– 6 h) and (2) the slower generation of new sensory neuron synaptic varicosities (12– 18 h). The resultant newly filled and newly formed varicosities are functionally competent (capable of evoked transmitter release) and contribute to the synaptic enhancement that underlies LTF. The rapid filling and activation of silent presynaptic terminals at 3 h suggests that, in addition to its role 61 in LTF, this modification of pre-existing varicosities may also contribute to the intermediate phase of synaptic plasticity. Red trianglesrepresent transmitter release sites (active zones). (Modified from Kim et al. , 2003. ) (See Color Plate 10. 2 in color plate

Fig. 3. Regional specific down-regulation of the transmembrane isoform of ap. CAM. This model is based on the assumption that the relative concentration of the GPI-linked versus transmembrane isoforms of ap. CAM is highest at points of synaptic contact between the sensory neuron and motor neuron and reflects the results of studies done in dissociated cell culture. Thus, previously established connections might remain intact following exposure to 5 -HT since they would be held in place by the adhesive, homophilic interactions of the GPIlinked isoforms and the process of outgrowth from sensory neuron axons would be initiated by down-regulation of the transmembrane form at extrasynaptic sites of membrane apposition. In the intact ganglion, the axons of sensory neurons are likely to fasciculate not only with other sensory neurons but also with the processes of other neurons and perhaps even glia. One of the attractive features of this model is that the mechanism for down-regulation is intrinsic to the sensory neurons. Thus, even if some of the sensory neuron axonal contacts in the intact ganglion were heterophilic in nature, i. e. , with other neurons or glia, we would still expect the selective internalization of ap. CAM at the sensory neuron surface membrane at these sites of heterophilic apposition to destabilize adhesive contacts and to facilitate disassembly. (From Bailey et al. , 1997. ) (See Color Plate 10. 3 in color plate section. ) 62

Hippocampus • 1 - The neurotransmitter is glutamate • Glutamate activate NMDAR leading to increased free intracellular calcium concentration. • Ca /calmodulin stimulate postsynaptically adenylyl cyclase. • Increased AMPAR 63

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Synaptic capture of learning induces gene products • The activation of a specific synapse tag the synapse • Covalent modification • Local protein synthesis to stabilize the mark 62 67

Local protein synthesis at active synapse • m. RNA species are transported by special mechanisms to the synaptic sites. They are translated only after docking and in response to a specific signal. • 5 x 5 HT at one synapse activate local protein synthesis. If inhibited no LTF. 63 68

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Imaging of Alexa, synaptotagmin – GFP, and synaptop. Hluorins • 1 - rapid activation of silent synapses by filling empty varicosities by vesicles. The process requires translation but not transcription. The process is seen within 3 -6 hr =intermediate memory accounts for approximately 32% of the new synapses. • 2 - generation of new varicosities requires transcription and translation. Is seen only 12 -18 hr after 5 HT and accounts for 68% of the new synapses 71 66

Interesting to note • Intermediate- uses pre existing structure in which the presynaptic elements may be already in register with post synaptic receptors • Long term requires more time to develop the structure and to assemble both new pre and post synaptic structures 72

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End of Aplysia Model 76

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