Alan Fine - Academia.edu (original) (raw)
Papers by Alan Fine
Behavioural brain research, Jan 19, 2016
We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. ... more We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no c...
Neurocomputing, 2004
Intracellular signalling often employs excitable stores of calcium coupled by di usion. We descri... more Intracellular signalling often employs excitable stores of calcium coupled by di usion. We describe here the ability of such a mechanism to generate a complete set of logic gates required for computation, as well as how they can act as coincidence detectors for biological signals.
Biophysical Journal, 1983
To improve the sensitivity of fluorescence measurements of electrical responses from small cells ... more To improve the sensitivity of fluorescence measurements of electrical responses from small cells and their processes, we have optimized the optical measuring system. The fluorescence intensity from a stained cell was increased 40-fold relative to our previous apparatus. The increased fluorescence intensity permits the use of an inexpensive photodiode (or a photodiode array) that has a-10-fold higher quantum efficiency relative to a photomultiplier. Utilizing the improved apparatus, we optically recorded an action potential of a 2 Am wide neuronal process with a signal-to-noise ratio of-50 (root mean square noise) without averaging. We also report the design of an improved fluorescence voltage-sensitive probe; the fractional change of the fluorescence signal under optimal conditions was 21%/100 mV.
![Research paper thumbnail of Cholinergic basal forebrain transplants restore diminished metabolic activity in the somatosensory cortex of rats with acetylcholine depletion [published erratum appears in J Neurosci 1994 Mar;14(3): following table of contents]](https://attachments.academia-assets.com/108269838/thumbnails/1.jpg)
](The Journal of Neuroscience, 1994
It has been known for several years that stimulus-evoked metabolic activity is reduced in the som... more It has been known for several years that stimulus-evoked metabolic activity is reduced in the somatosensory cortex of animals with basal forebrain lesions that deplete the neocortex of acetylcholine (ACh). During 2-deoxyglucose (2-DG) experiments, animals with unilateral basal forebrain lesions demonstrate a decreased response to somatic stimulation, while background metabolic activity in the surrounding cortical regions remains normal. In an attempt to ameliorate these deficits, we examined the ability of embryonic cholinergic basal forebrain transplants inserted into neocortex to innervate surrounding cortical regions and restore functional 2-DG activity in adult host rats previously depleted of ACh by basal forebrain lesions. To accomplish this goal, a series of experiments were conducted in which we (1) depleted the cerebral cortex of ACh by injecting an excitotoxin into the rat basal forebrain, (2) transplanted embryonic basal forebrain or embryonic neocortical (control) tissue...
Biophysical Journal, 2016
F1000 - Post-publication peer review of the biomedical literature, 2010
Protein translation has been implicated in different forms of synaptic plasticity but direct in s... more Protein translation has been implicated in different forms of synaptic plasticity but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based upon incorporation of non-canonical amino acids into proteins followed by chemo-selective fluorescent tagging via click chemistry. Following brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase-like application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single particle tracking and quantum dots. The newly synthesized proteins exhibited a broad range of diffusive behaviors as expected if the pool of labeled proteins was heterogeneous. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
PARALLEL SIMULATIONS OF ences the formation of the dendrites.' We include in our simulation three... more PARALLEL SIMULATIONS OF ences the formation of the dendrites.' We include in our simulation three interacting systems: the cell membrane. the ion distributions within the cell, and the cytoskeletal proteins that define the shape of the cell. A full three-dimensional simulation would represent NEURON GROWTH
Frontiers in Synaptic Neuroscience
Understanding the mechanisms by which long-term synaptic plasticity is expressed remains an impor... more Understanding the mechanisms by which long-term synaptic plasticity is expressed remains an important objective in neuroscience. From a physiological perspective, the strength of a synapse can be considered a consequence of several parameters including the probability that a presynaptic action potential (AP) evokes the release of neurotransmitter, the mean number of quanta of transmitter released when release is evoked, and the mean amplitude of a postsynaptic response to a single quantum. Various methods have been employed to estimate these quantal parameters from electrophysiological recordings; such "quantal analysis" has been used to support competing accounts of mechanisms of expression of long-term plasticity. Because electrophysiological recordings, even with minimal presynaptic stimulation, can reflect responses arising at multiple synaptic sites, these methods are open to alternative interpretations. By combining intracellular electrical recording with optical detection of transmission at individual synapses, however, it is possible to eliminate such ambiguity. Here, we describe methods for such combined optical and electrical monitoring of synaptic transmission in brain slice preparations and illustrate how quantal analyses thereby obtained permit more definitive conclusions about the physiological changes that underlie long-term synaptic plasticity.
The Journal of Neuroscience
Journal of Neural Transplantation and Plasticity
F1000 - Post-publication peer review of the biomedical literature
F1000 - Post-publication peer review of the biomedical literature
Long-term plasticity of dendritic integration is induced in parallel with long-term potentiation ... more Long-term plasticity of dendritic integration is induced in parallel with long-term potentiation (LTP) or depression (LTD) based on presynaptic activity patterns. It is, however, not clear whether synaptic plasticity induced by temporal pairing of pre-and postsynaptic activity is also associated with synergistic modification in dendritic integration. We show here that the spike timing-dependent plasticity (STDP) rule accounts for long-term changes in dendritic integration in CA1 pyramidal neurons in vitro. Positively correlated pre-and postsynaptic activity (delay: +5/+50 ms) induced LTP and facilitated dendritic integration. Negatively correlated activity (delay: −5/−50 ms) induced LTD and depressed dendritic integration. These changes were not observed following positive or negative pairing with long delays (> ±50 ms) or when NMDA receptors were blocked. The amplitude-slope relation of the EPSP was facilitated after LTP and depressed after LTD. These effects could be mimicked by voltage-gated channel blockers, suggesting that the induced changes in EPSP waveform involve the regulation of voltage-gated channel activity. Importantly, amplitude-slope changes induced by STDP were found to be input specific, indicating that the underlying changes in excitability are restricted to a limited portion of the dendrites. We conclude that STDP is a common learning rule for long-term plasticity of both synaptic transmission and dendritic integration, thus constituting a form of functional redundancy that insures significant changes in the neuronal output when synaptic plasticity is induced.
Scientific Reports, 2016
Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools ... more Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f u displaying fluorescence rise and decay times (t 1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6f u revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t 1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6f u is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators. Genetically encoded Ca 2+ indicators (GECI) facilitated monitoring Ca 2+ dynamics in intact and even freely moving animals. A limiting factor in the application of GECI has been their high Ca 2+ affinity and slow response kinetics 1. In GCaMP-type GECI 2-11 Ca 2+ binding to calmodulin (CaM) induces the formation of a complex with a target peptide which in turn restores the fluorescence of circularly permutated (cp) EGFP 12. The Ca 2+-induced binding of CaM to a target peptide allows the detection of Ca 2+ signals also by GCaMP-related G-GECO-s 13 , GEM-GECO-s 13 and sub-cellularly targeted CEPIA 14 and GCaMPer 15. Further broadening of the approaches for non-invasive mapping of neuronal circuits is represented by the development of red fluorescent probes for optogenetic stimulation and of photoactivatable derivatives 13,16-18. The most commonly used target peptide is RS20, a smooth muscle myosin light chain kinase-derived peptide and, in the case of RCaMP2, a CaMKKα-derived peptide 16. GCaMP-s reported to have the greatest brightness and Ca 2+ induced fluorescence enhancement to date are variants of a GCaMP6 generation termed GCaMP6 slow, GCaMP6 medium and GCaMP6 fast (GCaMP6s, GCaMP6m and GCaMP6f) 19 as well as GCaMP7 20 and GCaMP8 21. Even though indicators such as GCaMP7 show sufficiently high level of brightness to detect single action potentials (AP-s) 22 , their slow kinetic properties due to the Ca 2+-CaM-RS20 peptide interaction limit achieving temporal fidelity. Typically, Ca 2+-CaM-RS20 interactions have a high affinity (half-maximal brightness concentration, K d values of 100-300 nM) as for GCaMP6s and GCaMP6f 19. Correspondingly, the Ca 2+ dissociation rates and thus the signal decay rates are slow, 1-5 s −1 at 20 °C, comparable to that of GCaMP3 10. By rational design, mutations at the Ca 2+-CaM-RS20 binding interface have produced GCaMP3-and GCaMP6f-derived probes with improved decay kinetics 23,24. The probe with the fastest signal rise (t 1/2 of 1 ms) and decay (t 1/2 of 3 ms) kinetics (37 °C) so far is GCaMP3 fast 25. The mutation strategy that gave rise to GCaMP3 fast can serve as a template for generating fast-response CaM-based GECI.
Data in brief, 2016
This article provides supporting data for the research article "A simple automated system fo... more This article provides supporting data for the research article "A simple automated system for appetitive conditioning of zebrafish in their home tanks" (J.M. Doyle, N. Merovitch, R.C. Wyeth, M.R. Stoyek, M. Schmidt, F. Wilfart, A. Fine, R.P. Croll, 2016) [1]. In that article, we described overall movements of zebrafish toward a food source as a response to auditory or visual cues as conditioned stimuli in a novel learning paradigm. Here, we describe separate analyses of the vertical and horizontal components of the learned response. These data provide evidence that the conditioning might result from both classical conditioning of an innate response of zebrafish to move to the surface in response to food cues and secondary conditioning of the fish to associate a food presentation with a specific location in the tank. Movement data from the twenty trial acquisition period and probe trials from 2-32 days post conditioning are included.
Alcoholism Clinical and Experimental Research, 1998
Behavioural brain research, Jan 19, 2016
We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. ... more We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no c...
Neurocomputing, 2004
Intracellular signalling often employs excitable stores of calcium coupled by di usion. We descri... more Intracellular signalling often employs excitable stores of calcium coupled by di usion. We describe here the ability of such a mechanism to generate a complete set of logic gates required for computation, as well as how they can act as coincidence detectors for biological signals.
Biophysical Journal, 1983
To improve the sensitivity of fluorescence measurements of electrical responses from small cells ... more To improve the sensitivity of fluorescence measurements of electrical responses from small cells and their processes, we have optimized the optical measuring system. The fluorescence intensity from a stained cell was increased 40-fold relative to our previous apparatus. The increased fluorescence intensity permits the use of an inexpensive photodiode (or a photodiode array) that has a-10-fold higher quantum efficiency relative to a photomultiplier. Utilizing the improved apparatus, we optically recorded an action potential of a 2 Am wide neuronal process with a signal-to-noise ratio of-50 (root mean square noise) without averaging. We also report the design of an improved fluorescence voltage-sensitive probe; the fractional change of the fluorescence signal under optimal conditions was 21%/100 mV.
The Journal of Neuroscience, 1994
It has been known for several years that stimulus-evoked metabolic activity is reduced in the som... more It has been known for several years that stimulus-evoked metabolic activity is reduced in the somatosensory cortex of animals with basal forebrain lesions that deplete the neocortex of acetylcholine (ACh). During 2-deoxyglucose (2-DG) experiments, animals with unilateral basal forebrain lesions demonstrate a decreased response to somatic stimulation, while background metabolic activity in the surrounding cortical regions remains normal. In an attempt to ameliorate these deficits, we examined the ability of embryonic cholinergic basal forebrain transplants inserted into neocortex to innervate surrounding cortical regions and restore functional 2-DG activity in adult host rats previously depleted of ACh by basal forebrain lesions. To accomplish this goal, a series of experiments were conducted in which we (1) depleted the cerebral cortex of ACh by injecting an excitotoxin into the rat basal forebrain, (2) transplanted embryonic basal forebrain or embryonic neocortical (control) tissue...
Biophysical Journal, 2016
F1000 - Post-publication peer review of the biomedical literature, 2010
Protein translation has been implicated in different forms of synaptic plasticity but direct in s... more Protein translation has been implicated in different forms of synaptic plasticity but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based upon incorporation of non-canonical amino acids into proteins followed by chemo-selective fluorescent tagging via click chemistry. Following brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase-like application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single particle tracking and quantum dots. The newly synthesized proteins exhibited a broad range of diffusive behaviors as expected if the pool of labeled proteins was heterogeneous. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
PARALLEL SIMULATIONS OF ences the formation of the dendrites.' We include in our simulation three... more PARALLEL SIMULATIONS OF ences the formation of the dendrites.' We include in our simulation three interacting systems: the cell membrane. the ion distributions within the cell, and the cytoskeletal proteins that define the shape of the cell. A full three-dimensional simulation would represent NEURON GROWTH
Frontiers in Synaptic Neuroscience
Understanding the mechanisms by which long-term synaptic plasticity is expressed remains an impor... more Understanding the mechanisms by which long-term synaptic plasticity is expressed remains an important objective in neuroscience. From a physiological perspective, the strength of a synapse can be considered a consequence of several parameters including the probability that a presynaptic action potential (AP) evokes the release of neurotransmitter, the mean number of quanta of transmitter released when release is evoked, and the mean amplitude of a postsynaptic response to a single quantum. Various methods have been employed to estimate these quantal parameters from electrophysiological recordings; such "quantal analysis" has been used to support competing accounts of mechanisms of expression of long-term plasticity. Because electrophysiological recordings, even with minimal presynaptic stimulation, can reflect responses arising at multiple synaptic sites, these methods are open to alternative interpretations. By combining intracellular electrical recording with optical detection of transmission at individual synapses, however, it is possible to eliminate such ambiguity. Here, we describe methods for such combined optical and electrical monitoring of synaptic transmission in brain slice preparations and illustrate how quantal analyses thereby obtained permit more definitive conclusions about the physiological changes that underlie long-term synaptic plasticity.
The Journal of Neuroscience
Journal of Neural Transplantation and Plasticity
F1000 - Post-publication peer review of the biomedical literature
F1000 - Post-publication peer review of the biomedical literature
Long-term plasticity of dendritic integration is induced in parallel with long-term potentiation ... more Long-term plasticity of dendritic integration is induced in parallel with long-term potentiation (LTP) or depression (LTD) based on presynaptic activity patterns. It is, however, not clear whether synaptic plasticity induced by temporal pairing of pre-and postsynaptic activity is also associated with synergistic modification in dendritic integration. We show here that the spike timing-dependent plasticity (STDP) rule accounts for long-term changes in dendritic integration in CA1 pyramidal neurons in vitro. Positively correlated pre-and postsynaptic activity (delay: +5/+50 ms) induced LTP and facilitated dendritic integration. Negatively correlated activity (delay: −5/−50 ms) induced LTD and depressed dendritic integration. These changes were not observed following positive or negative pairing with long delays (> ±50 ms) or when NMDA receptors were blocked. The amplitude-slope relation of the EPSP was facilitated after LTP and depressed after LTD. These effects could be mimicked by voltage-gated channel blockers, suggesting that the induced changes in EPSP waveform involve the regulation of voltage-gated channel activity. Importantly, amplitude-slope changes induced by STDP were found to be input specific, indicating that the underlying changes in excitability are restricted to a limited portion of the dendrites. We conclude that STDP is a common learning rule for long-term plasticity of both synaptic transmission and dendritic integration, thus constituting a form of functional redundancy that insures significant changes in the neuronal output when synaptic plasticity is induced.
Scientific Reports, 2016
Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools ... more Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f u displaying fluorescence rise and decay times (t 1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6f u revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t 1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6f u is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators. Genetically encoded Ca 2+ indicators (GECI) facilitated monitoring Ca 2+ dynamics in intact and even freely moving animals. A limiting factor in the application of GECI has been their high Ca 2+ affinity and slow response kinetics 1. In GCaMP-type GECI 2-11 Ca 2+ binding to calmodulin (CaM) induces the formation of a complex with a target peptide which in turn restores the fluorescence of circularly permutated (cp) EGFP 12. The Ca 2+-induced binding of CaM to a target peptide allows the detection of Ca 2+ signals also by GCaMP-related G-GECO-s 13 , GEM-GECO-s 13 and sub-cellularly targeted CEPIA 14 and GCaMPer 15. Further broadening of the approaches for non-invasive mapping of neuronal circuits is represented by the development of red fluorescent probes for optogenetic stimulation and of photoactivatable derivatives 13,16-18. The most commonly used target peptide is RS20, a smooth muscle myosin light chain kinase-derived peptide and, in the case of RCaMP2, a CaMKKα-derived peptide 16. GCaMP-s reported to have the greatest brightness and Ca 2+ induced fluorescence enhancement to date are variants of a GCaMP6 generation termed GCaMP6 slow, GCaMP6 medium and GCaMP6 fast (GCaMP6s, GCaMP6m and GCaMP6f) 19 as well as GCaMP7 20 and GCaMP8 21. Even though indicators such as GCaMP7 show sufficiently high level of brightness to detect single action potentials (AP-s) 22 , their slow kinetic properties due to the Ca 2+-CaM-RS20 peptide interaction limit achieving temporal fidelity. Typically, Ca 2+-CaM-RS20 interactions have a high affinity (half-maximal brightness concentration, K d values of 100-300 nM) as for GCaMP6s and GCaMP6f 19. Correspondingly, the Ca 2+ dissociation rates and thus the signal decay rates are slow, 1-5 s −1 at 20 °C, comparable to that of GCaMP3 10. By rational design, mutations at the Ca 2+-CaM-RS20 binding interface have produced GCaMP3-and GCaMP6f-derived probes with improved decay kinetics 23,24. The probe with the fastest signal rise (t 1/2 of 1 ms) and decay (t 1/2 of 3 ms) kinetics (37 °C) so far is GCaMP3 fast 25. The mutation strategy that gave rise to GCaMP3 fast can serve as a template for generating fast-response CaM-based GECI.
Data in brief, 2016
This article provides supporting data for the research article "A simple automated system fo... more This article provides supporting data for the research article "A simple automated system for appetitive conditioning of zebrafish in their home tanks" (J.M. Doyle, N. Merovitch, R.C. Wyeth, M.R. Stoyek, M. Schmidt, F. Wilfart, A. Fine, R.P. Croll, 2016) [1]. In that article, we described overall movements of zebrafish toward a food source as a response to auditory or visual cues as conditioned stimuli in a novel learning paradigm. Here, we describe separate analyses of the vertical and horizontal components of the learned response. These data provide evidence that the conditioning might result from both classical conditioning of an innate response of zebrafish to move to the surface in response to food cues and secondary conditioning of the fish to associate a food presentation with a specific location in the tank. Movement data from the twenty trial acquisition period and probe trials from 2-32 days post conditioning are included.
Alcoholism Clinical and Experimental Research, 1998