Cellular Neurophysiology

Readings

The readings listed below are the foundation of this course. Where available, journal article abstracts from PubMed (an online database providing access to citations from biomedical literature) are included.

Background Reading for Lectures

Section 1: Biophysics of Membrane Ion Channels

Reading to be completed in Section 1.

Books:

• Johnston, and Wu. Chap. 1, 2, 6 - 9, and Appendix A.
• Hille. Chap. 1 - 5.
• Levitan, and Kaczmarek. Chap. 1 - 7.

Journal articles:

• Hoshi, T., Zagotta, W.N., and R. Aldrich. “Biophysical and molecular mechanisms of shaker potassium channel inactivation.” In Science 250, (1990): 533-538.
  • PubMed abstract: The potassium channels encoded by the Drosophila Shaker gene activate and inactivate rapidly when the membrane potential becomes more positive. Site-directed mutagenesis and single-channel patch-clamp recording were used to explore the molecular transitions that underlie inactivation in Shaker potassium channels expressed in Xenopus oocytes. A region near the amino terminus with an important role in inactivation has now been identified. The results suggest a model where this region forms a cytoplasmic domain that interacts with the open channel to cause inactivation.
• Zagotta, W.N., T. Hoshi, and R. Aldrich. “Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB.” In Science 250, (1990): 568-571.
  • PubMed abstract: Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.
• Armstrong, C.M., F. Bezanilla, and E. Rojas. “Destruction of sodium conductance inactivation in squid axons perfused with pronase.” In J. of Gen. Physiol 62, (1973): 375-391.

Section 2: Synaptic Transmission

Reading to be completed in Section 2.

Books:

• Johnston, and Wu. Chap. 11 - 13.
• Hille. Chap. 6.
• Levitan, and Kaczmarek. Chap. 8 - 14.

Journal articles:

• Albright, T.D., T.M. Jessell, et al. “Neural science: a century of progress and the mysteries that remain.” In Neuron 25, Suppl: S1-55 (2000).
• Sudhof, T.C. “The synaptic vesicle cycle: a cascade of protein-protein interactions.” In Nature 375, (1995): 645-53.
  • PubMed abstract: The synaptic vesicle cycle at the nerve terminal consists of vesicle exocytosis with neurotransmitter release, endocytosis of empty vesicles, and regeneration of fresh vesicles. Of all cellular transport pathways, the synaptic vesicle cycle is the fastest and the most tightly regulated. A convergence of results now allows formulation of molecular models for key steps of the cycle. These developments may form the basis for a mechanistic understanding of higher neural function.The synaptic vesicle cycle at the nerve terminal consists of vesicle exocytosis with neurotransmitter release, endocytosis of empty vesicles, and regeneration of fresh vesicles. Of all cellular transport pathways, the synaptic vesicle cycle is the fastest and the most tightly regulated. A convergence of results now allows formulation of molecular models for key steps of the cycle. These developments may form the basis for a mechanistic understanding of higher neural function.
• Stevens. “Quantal release of neurotransmitter and long-term potentiation.” In Cell 72, Suppl: 55-63 (1993).
• Sanes, J.R., and J.W. Lichtman. “Induction, assembly, maturation and maintenance of a postsynaptic apparatus.” In Nat. Rev. Neurosci. 2, (11) (2001): 791-805.

Section 3: Synaptic Plasticity

Reading to be completed in Section 3.

Books:

• Johnston, and Wu. Chap. 15.
• Hille. Chap. 7.
• Levitan, and Kaczmarek. Chap. 16 - 18.

Journal articles:

• Bliss, T.V., and G.L. Collingridge. “A synaptic model of memory: long-term potentiation in the hippocampus.” In Nature 361, (6407) (1993): 31-9.
• Linden, and Connor. “Long-term synaptic depression.” In Ann. Rev. Neurosci. 18, (1995): 319-357.
• Sanes, J.R., and J.W Lichtman. “Can molecules explain long-term potentiation?” In Nat. Neurosci. 2, (1999): 597-604.
• Malenka, R.C., and R.A. Nicoll. “Long-term potentiation: A decade of progress?” In Science 285, (1999): 1870-4.
  • PubMed abstract: Long-term potentiation of synaptic transmission in the hippocampus is the leading experimental model for the synaptic changes that may underlie learning and memory. This review presents a current understanding of the molecular mechanisms of this long-lasting increase in synaptic strength and describes a simple model that unifies much of the data that previously were viewed as contradictory.
• Zucker R.S. “Calcium- and activity-dependent synaptic plasticity.” In Curr. Opin. Neurobiol 9, (1999): 305-313.
  • PubMed abstract: Calcium ions play crucial signaling roles in many forms of activity-dependent synaptic plasticity. Recent presynaptic [Ca2+]i measurements and manipulation of presynaptic exogenous buffers reveal roles for residual [Ca2+]i following conditioning stimulation in all phases of short-term synaptic enhancement. Pharmacological manipulations implicate mitochondria in post-tetanic potentiation. New evidence supports an influence of Ca2+ in replacing depleted vesicles after synaptic depression. In addition, high-resolution measurements of [Ca2+]i in dendritic spines show how Ca2+ can encode the precise relative timing of presynaptic input and postsynaptic activity and generate long-term synaptic modifications of opposite polarity.

Section 4: Neural Properties and Formation of Functional Neural Networks

Reading to be completed in Section 4.

Books:

• Levitan, and Kaczmarek Chap. 19-20.

Journal articles:

• Lichtman, J.W., and H. Colman. “Synapse elimination and indelible memory.” In Neuron 25, (2) (2000): 269-78.
• Bear, M.F. “A synaptic basis for memory storage in the cerebral cortex.” In Proc. Natl. Acad. Sci. USA 93, (1996): 13453-13459.
  • PubMed abstract: A cardinal feature of neurons in the cerebral cortex is stimulus selectivity, and experience-dependent shifts in selectivity are a common correlate of memory formation. We have used a theoretical “learning rule,” devised to account for experience-dependent shifts in neuronal selectivity, to guide experiments on the elementary mechanisms of synaptic plasticity in hippocampus and neocortex. These experiments reveal that many synapses in hippocampus and neocortex are bidirectionally modifiable, that the modifications persist long enough to contribute to long-term memory storage, and that key variables governing the sign of synaptic plasticity are the amount of NMDA receptor activation and the recent history of cortical activity.
• Zhang, L.I., H.W. Tao, C.E. Holt, W.A. Harris, and M. Poo. “A critical window for cooperation and competition among developing retinotectal synapses.” In Nature 395, (1998): 37-44.
  • PubMed abstract: In the developing frog visual system, topographic refinement of the retinotectal projection depends on electrical activity. In vivo whole-cell recording from developing Xenopus tectal neurons shows that convergent retinotectal synapses undergo activity-dependent cooperation and competition following correlated pre- and postsynaptic spiking within a narrow time window. Synaptic inputs activated repetitively within 20 ms before spiking of the tectal neuron become potentiated, whereas subthreshold inputs activated within 20 ms after spiking become depressed. Thus both the initial synaptic strength and the temporal order of activation are critical for heterosynaptic interactions among convergent synaptic inputs during activity-dependent refinement of developing neural networks.

Papers for Discussion

Section 1: Biophysics of Membrane Ion Channels

Session 1

Background reading:

• Hodgkin, and Katz. “The effect of sodium ions on the electrical activity of the giant axon of the squid.” In J. Physiol 108, (1949): 37-77.
• Hodgkin, Huxley, and Katz. “Measurement of current-voltage relations in the membrane of the giant axon in Loligo.” In J. Physiol 116, (1952): 424-448.

Paper for discussion:

• Hodgkin, and Huxley. “Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo.” In J. Physiol 116, (1952): 449-472.

Session 2

• Hodgkin, and Huxley. “The components of membrane conductance in the giant axon of Loligo.” In J. Physiol 116, (1952): 473-496.
• Hodgkin, and Huxley. “The dual effect of membrane potential on sodium conductance in the giant axon of Loligo.” In J. Physiol 116, (1952): 497-506.

Session 3

• Hodgkin, and Huxley. “A quantitative description of membrane current and its application to conduction and excitation in nerve.” In J. Physiol 116, (1952): 500-544.

Session 4

• Fox, Nowycky, and Tsien. “Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones.” In J. Physiol 394, (1987): 149-172.
  • PubMed abstract: 1. Calcium currents in cultured dorsal root ganglion (d.r.g.) cells were studied with the whole-cell patch-clamp technique. Using experimental conditions that suppressed Na+ and K+ currents, and 3-10 mM-external Ca2+ or Ba2+, we distinguished three distinct types of calcium currents (L, T and N) on the basis of voltage-dependent kinetics and pharmacology. 2. Component L activates at relatively positive test potentials (t.p. greater than -10 mV) and shows little inactivation during a 200 ms depolarization. It is completely reprimed at a holding potential (h.p.) of -60 mV, and can be isolated by using a more depolarized h.p. (-40 mV) to inactivate the other two types of calcium currents. 3. Component T can be seen in isolation with weak test pulses. It begins activating at potentials more positive than -70 mV and inactivates quickly and completely during a maintained depolarization (time constant, tau approximately 20-50 ms). The current amplitude and the rate of decay increase with stronger depolarizations until both reach a maximum at approximately -40 mV. Inactivation is complete at h.p. greater than -60 mV and is progressively removed between -60 and -95 mV. 4. Component N activates at relatively strong depolarizations (t.p. greater than -20 mV) and decays with time constants ranging from 50 to 110 ms. Inactivation is removed over a very broad range of holding potentials (h.p. between -40 and -110 mV). 5. With 10 mM-EGTA in the pipette solution, substitution of Ba2+ for Ca2+ as the charge carrier does not alter the rates of activation or relaxation of any component. However, T-type channels are approximately equally permeable to Ca2+ and Ba2+, while L-type and N-type channels are both much more permeable to Ba2+. 6. Component N cannot be explained by current-dependent inactivation of L current resulting from recruitment of extra L-type channels at negative holding potentials: raising the external Ba2+ concentration to 110 mM greatly increases the amplitude of L current evoked from h.p. = -30 mV but produces little inactivation. 7. Cadmium ions (20-50 microM) virtually eliminate both N and L currents (greater than 90% block) but leave T relatively unaffected (less than 50% block). 200 microM-Cd2+ blocks all three components. 8. Nickel ions (100 microM) strongly reduce T current but leave N and L current little changed. 9. The dihydropyridine antagonist nifedipine (10 microM) inhibits L current (approximately 60% block) at a holding potential that inactivates half the L-type channels.
• Fox, Nowycky, and Tsien. “Single-channel recordings of three types of calcium channels in chick sensory neurones.” In J. Physiol 394, (1987): 173-200.
  • PubMed abstract: 1. T-, and L-type Ca2+ channels were studied in cell-attached patch recordings from the cell bodies of chick dorsal root ganglion neurones. All experiments were performed with isotonic BaCl2 (110 mM) in the recording pipette and with isotonic potassium aspartate in the bathing solution to zero the cell membrane potential. 2. L-type channels are distinguished by a unitary slope conductance of 25 pS, activation over the range of membrane potentials between 0 and +40 mV, little inactivation over the course of a 136 ms depolarization, and availability for opening even at depolarized holding potentials (h.p. greater than -40 mV). L channels show a predominant mode of gating (mode 1) characterized by brief openings (approximately 1 ms), occasionally interspersed with another pattern of gating characterized by much longer openings (mode 2). 3. The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 promotes mode 2 activity and shifts the voltage dependence of L-type channel activation towards more negative potentials. It leaves the unitary current-voltage relation unchanged. 4. Nifedipine, a DHP Ca2+ antagonist, strongly inhibits L-type channel activity through an increase in the proportion of blank sweeps. 5. T-type Ca2+ channels are distinguished by a much smaller unitary slope conductance (8 pS) and by activation and inactivation over relatively negative ranges of potential. Inactivation is complete by the end of 136 ms pulses to test potentials beyond -20 mV. 6. N-type Ca2+ channels are distinguished by an intermediate unitary slope conductance (13 pS), and by activation over a range of potentials between those of T- and L-type channels. Inactivation of N-type channels takes place over an exceptionally broad range of holding potentials (-80 to -20 mV). 7. Cell-attached patch data on the voltage dependence of activation and inactivation of T- and N-type channels are in excellent agreement with results from whole-cell recordings (Fox, Nowycky & Tsien, 1987) if allowances are made for variations in external surface potential. 8. Patches containing one or two channels of a single type were used for analysis of gating kinetics. The predominant pattern of activity for each of the channel types is an exponential distribution of relatively brief (approximately 1 ms) openings, and a bi-exponential distribution of short and long closings. 9. Patches containing all possible combinations of channel types were observed. However, preliminary evidence suggests that channels are distributed unevenly over the cell body; clustering of N-type channels is particularly prominent.

Section 2: Synaptic Transmission

Session 6

• Castillo, and Katz. “Quantal components of end-plate potential.” In J. Physiol 124, (1954): 560-573.

Session 7

• Dodge, and Rahamimoff. “Co-operative action of calcium ions in transmitter release at the neuromuscular junction.” In J. Physiol 193, (1967): 419-432.
• Katz, and Miledi. “The timing of calcium action during neuromuscular transmission.” In J. Physiol 189, (1967): 535-544.

Session 8

• Heuser et al. “Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.” In J. Cell. Biol 81: 275-300.
  • PubMed abstract: We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the “active zones” that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the “temporal resolution” of our quick-freezing technique is 2 ms or better.

Section 3: Synaptic Plasticity

Session 10

• Bliss and Lomo. “Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path.” In J. Physiol (London) 232, (1973): 331-356.
• Mayer, and Westbrook. “The action of N-methyl-d-aspartic acid on mouse spinal neurons in culture.” In J. Physiol 361, (1985): 65-90.

Session 11

• Liao et. al. “Activation of postsynaptically silent synapses during pairing-induced LTP in CA1 region of hippocampal slice.” In Nature 375, (1995): 400-404.
  • PubMed abstract: Long-term potentiation (LTP) is an enhancement of synaptic strength that can be produced by pairing of presynaptic activity with postsynaptic depolarization. LTP in the hippocampus has been extensively studied as a cellular model of learning and memory, but the nature of the underlying synaptic modification remains elusive, partly because our knowledge of central synapses is still limited. One proposal is that the modification is postsynaptic, and that synapses expressing only NMDA (N-methyl-D-aspartate) receptors before potentiation are induced by LTP to express functional AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors. Here we report that a high proportion of synapses in hippocampal area CA1 transmit with NMDA receptors but not AMPA receptors, making these synapses effectively non-functional at normal resting potentials. These silent synapses acquire AMPA-type responses following LTP induction. Our findings challenge the view that LTP in CA1 involves a presynaptic modification, and suggest instead a simple postsynaptic mechanism for both induction and expression of LTP.
• Renger, J.J., C. Egles, et al. “A developmental switch in neurotransmitter flux enhances synaptic efficacy by affecting AMPA receptor activation.” In Neuron 29, (2) (2001): 469-84.
• Science. 284: 1811-1816.
  • PubMed abstract: Formation of glutamatergic synapses entails development of “silent” immature contacts into mature functional synapses. To determine how this transformation occurs, we investigated the development of neurotransmission at single synapses in vitro. Maturation of presynaptic function, assayed with endocytotic markers, followed accumulation of synapsin I. During this period, synaptic transmission was primarily mediated by activation of NMDA receptors, suggesting that most synapses were functionally silent. However, local glutamate application to silent synapses indicated that these synapses contained functional AMPA receptors, suggesting a possible presynaptic locus for silent transmission. Interference with presynaptic vesicle fusion by exposure to tetanus toxin reverted functional to silent transmission, implicating SNARE-mediated fusion as a determinant of the ratio of NMDA:AMPA receptor activation. This work reveals that functional maturation of synaptic transmission involves transformation of presynaptic silent secretion into mature synaptic transmitter release.

Session 12

• Malenka et al. “Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission.” In Science 242, (1988): 81-84.
  • PubMed abstract: Long-term potentiation of synaptic transmission in the hippocampus is the leading experimental model for the synaptic changes that may underlie learning and memory. This review presents a current understanding of the molecular mechanisms of this long-lasting increase in synaptic strength and describes a simple model that unifies much of the data that previously were viewed as contradictory.
• Shi, S.H., Y. Hayashi, et al. “Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.” In Science 284, (5421) (1999): 1811-6.
  • PubMed abstract: To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.

Section 4: Neural Properties and Formation of Functional Neural Networks

Session 13

• Bi, G.Q., and M.M. Poo. “Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type.” In J. Neurosci. 18, (24) (1998): 10464-72.
  • PubMed abstract: In cultures of dissociated rat hippocampal neurons, persistent potentiation and depression of glutamatergic synapses were induced by correlated spiking of presynaptic and postsynaptic neurons. The relative timing between the presynaptic and postsynaptic spiking determined the direction and the extent of synaptic changes. Repetitive postsynaptic spiking within a time window of 20 msec after presynaptic activation resulted in long-term potentiation (LTP), whereas postsynaptic spiking within a window of 20 msec before the repetitive presynaptic activation led to long-term depression (LTD). Significant LTP occurred only at synapses with relatively low initial strength, whereas the extent of LTD did not show obvious dependence on the initial synaptic strength. Both LTP and LTD depended on the activation of NMDA receptors and were absent in cases in which the postsynaptic neurons were GABAergic in nature. Blockade of L-type calcium channels with nimodipine abolished the induction of LTD and reduced the extent of LTP. These results underscore the importance of precise spike timing, synaptic strength, and postsynaptic cell type in the activity-induced modification of central synapses and suggest that Hebb’s rule may need to incorporate a quantitative consideration of spike timing that reflects the narrow and asymmetric window for the induction of synaptic modification.
• Markram, H., J. Lubke, et al. “Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs.” In Science 275, (5297) (1997): 213-5.
  • PubMed abstract: Activity-driven modifications in synaptic connections between neurons in the neocortex may occur during development and learning. In dual whole-cell voltage recordings from pyramidal neurons, the coincidence of postsynaptic action potentials (APs) and unitary excitatory postsynaptic potentials (EPSPs) was found to induce changes in EPSPs. Their average amplitudes were differentially up- or down-regulated, depending on the precise timing of postsynaptic APs relative to EPSPs. These observations suggest that APs propagating back into dendrites serve to modify single active synaptic connections, depending on the pattern of electrical activity in the pre- and postsynaptic neurons.

Session 14

• Zhang, L.I., H.W. Tao, et al. “Visual input induces long-term potentiation of developing retinotectal synapses.” In Nat Neurosci. 3, (7) (2000): 708-15.
  • PubMed abstract: Early visual experience is essential in the refinement of developing neural connections. In vivo whole-cell recording from the tectum of Xenopus tadpoles showed that repetitive dimming-light stimulation applied to the contralateral eye resulted in persistent enhancement of glutamatergic inputs, but not GABAergic or glycinergic inputs, on tectal neurons. This enhancement can be attributed to potentiation of retinotectal synapses. It required spiking of postsynaptic tectal cells as well as activation of NMDA receptors, and effectively occluded long-term potentiation (LTP) of retinotectal synapses induced by direct electrical stimulation of retinal ganglion cells. Thus, LTP-like synaptic modification can be induced by natural visual inputs and may be part of the underlying mechanism for the activity-dependent refinement of developing connections.