Directional Summation in Non-direction Selective Retinal Ganglion Cells
[['Regardless of their classification , virtually all ganglion cells receive input from multiple bipolar cells .', 'For example , in the cat and guinea pig retina , as many as 150 bipolar cells synapse onto a single On type ganglion cells [1]–[3] .', 'With the exception of the soma and very proximal dendrites , bipolar cells contact ganglion cells uniformly throughout the dendritic tree [1] , [4] .', 'Thus ganglion cells have the task of integrating synaptic input from multiple bipolar cells whose synapses are distributed throughout the entire dendritic tree before a decision to fire an action potential can be made [5] , [6] .', 'Much of our current knowledge of the dendritic integration of multiple excitatory inputs comes from modeling studies [7]–[10] as well as analysis of conductance changes evoked by illumination of ganglion cell receptive fields [11] .', 'However , these studies do not address the rules regarding summation of closely timed inputs along single or multiple dendrites .', 'Similarities in the local statistics of light in natural scenes [12] result in highly correlated activity of neighboring bipolar cells .', 'Thus , as the eyes sample the visual world , individual ganglion cell dendrites are likely to be activated by cohorts of bipolar cells whose input with spatio-temporally correlated synaptic output .', 'Theoretical work has suggested that there is a directional component to dendritic integration [13] , and this has been confirmed in cortical neurons where activation sequence toward the soma ( centripetal ) produced more summation than the sequence directed away from the soma ( centrifugal ) [14] .', 'However , it is unclear whether the same rules for dendritic integration apply to retinal ganglion cells .', 'To test for direction-dependent summation of excitatory postsynaptic potentials ( EPSPs ) in On ganglion cells , we activated multiple distinct loci along a dendrite with targeted local photolysis of caged glutamate .', 'Surprisingly , we found that summation was supralinear for centrifugal input , while centripetal activation resulted in linear summation .', 'Hyperpolarization activated cyclic nucleotide-gated ( HCN ) channels have been shown to modulate summation and firing in a number of brain regions making them excellent candidates for modulators of dendritic integration in ganglion cells [15]–[20] .', 'We found that blockade of HCN channels increased overall summation , and eliminated the directional component of summation .', 'Further , the effect of blockade was most pronounced in the distal dendrites suggesting that the density of HCN current increases with distance from the soma .', 'Our results show that intrinsic properties of the ganglion cell allow non-direction selective cells to code specific input sequences .', 'This directional summation of EPSP is similar to that seen in starburst amacrine cells where centrifugal stimuli produce larger Ca2+ responses [21] , [22] .', 'Our results suggest that this fundamental mechanism for directional summation is an essential building block across multiple cell types and species for generating a traditionally direction selective cell .'], ['Sprague Dawley rats were purchased from Charles River Laboratories ( Wilmington , PA , U . S . A . ) and housed in the Albert Einstein College of Medicine Animal Care Facility .', 'Rats were subjected to 12 h light/12 h dark cycles , and were fed a standard chow diet .', 'All procedures were performed according to a protocol approved by the Institute for Animal Studies of the Albert Einstein College of Medicine .', 'Whole mounts of rat retina ( P14–18 and P21–35 ) , were prepared from dark adapted ( 1 hr ) animals .', 'Animals were anesthetized with isoflurane applied to gauze attached to the top of a closed chamber .', 'This results in loss of consciousness within a minute .', 'Animals were then decapitated , and subsequent surgery and cell recordings were performed in dim red light .', 'The eyes were enucleated and retinal dissections were performed in oxygenated physiological saline ( in mM: 119 NaCl , 2 . 5 KCl , 1 . 3 MgCl2 , 2 . 5 CaCl2 , 1 NaH2PO4 , 26 . 2 NaHCO3 , 11 glucose ) .', 'The eyecup was cut into two pieces , the retina removed with forceps , and transferred to a recording chamber .', 'Whole-cell recordings were made from ganglion cells using a patch pipette filled with ( in mM ) : 135 KMeSO3 , 5 KCl , 1 CaCl2 , 5 EGTA , 10 glucose , 2 ATP , 0 . 3 GTP , 10 HEPES ( pH to 7 . 2 ) and 100 µM Oregon-green BAPTA .', 'The retina was superfused at 30°C with oxygenated physiological saline .', 'Recordings were performed under infrared conditions using a CCD video camera fitted with IR filter and macro lenses .', 'A halogen source was used for light stimuli .', 'Cells were selected for patching based on large soma size .', 'This approach yielded ∼85% On cells .', 'At the beginning of each experiment ganglion cell subtype was identified using a light stimulus in the dark adapted retina .', 'Subsequent use of the 405 nm uncaging or 488 imaging laser changed adaptation state .', 'The use of caged compound allowed us to isolate the postsynaptic response independent of presynaptic circuitry .', 'Only one cell was obtained from a preparation insuring all cells were initially dark adapted .', 'Light evoked EPSP responses were kept subthreshold by placing 3 or 4 log unit neutral density filters in the light path , and varying stimulus duration ( range 10–200 ms ) .', 'Individual ganglion cells were then imaged with an Olympus Fluoview 1000 confocal microscope using a 40× water-immersion objective [23] .', 'Laser light was limited to brief exposures sufficient to identify the dendritic target for the uncaging beam .', 'If holding current exceeded 100 pA the experiment was terminated .', 'A Z-series of confocal images were collected at the completion of the experiment .', '200–500 µM MNI-caged-glutamate ( Tocris , Ellisville , MO ) was added to the external solution .', '( 2R ) -amino-5-phosphonovaleric acid ( APV; 50 µM; Tocris , Ellisville , MO ) , and ZD7288 ( 100 µM; Tocris , Ellisville , MO ) were used to block NMDA and HCN channels , respectively .', 'Recordings were accepted only if the holding current was less than −150 pA when ganglion cells were voltage clamped at −67 mV .', 'Electrophysiology data were collected and analyzed off line with pClamp ( Molecular Devices , Sunnyvale , CA ) .', 'The “HCN isolation solution” contained ( in mM ) : TTX 0 . 1 , BaCl2 1 , NiCl2 0 . 1 , D-AP5 0 . 05 , NBQX 0 . 01 , picrotoxin 0 . 05 , TPMPA 0 . 05 , strychnine 0 . 001; NaH2PO4 was omitted when Ba2+ and Ni2+ were present to avoid precipitation .', 'TTX , BaCl2 , NiCl2 , APV , NBQX , picrotoxin , TPMPA , and strychnine were used to block sodium , inward rectifying potassium , calcium , N-methyl-d-aspartate ( NMDA ) , AMPA receptor , GABAA , GABAC and glycine receptors , respectively [24] , [25] .', 'An Olympus Fluoview 1000 confocal microscope equipped with a 405 diode laser ( 1–3 ms pulses ) was used for uncaging MNI-caged glutamate ( 200–500 µM ) at multiple targeted regions of the dendrite ( 0 . 5 ms minimum delay between locations ) .', 'Laser power and uncaging duration were kept constant for the duration of an experiment .', 'For all experiments we obtained 8 trials for each uncaging sequence .', 'Retinas were isolated from newborn ( P0 ) rats after cryoanesthesia and were incubated for 45 min at 37°C in DMEM with HEPES ( Mediatech , Washington , DC ) , supplemented with 6 units/ml papain ( Worthington , Freehold , NJ ) and 0 . 2 mg/ml cysteine .', 'Papain was then inactivated by replacing the enzyme solution with complete medium composed of DMEM , 5 mM HEPES , 0 . 1% Mito+ serum extender ( Collaborative Research , Bedford , MA ) , 5% heat-inactivated fetal calf serum ( HIFCS ) , 0 . 75% penicillin-streptomycin-glutamine mix ( Life Technologies ) .', 'Osmolarity was adjusted to 300 mOsm by addition of distilled water .', 'Retinas were triturated through a fire-polished Pasteur pipette , plated onto glass coverslips pretreated with poly-D-lysine ( 0 . 1 mg/ml ) , and maintained in complete medium supplemented with 15 mM KCl .', '72 hr after plating , cells were treated with the antimitotics 5-fluoro-2-deoxyuridine ( 0 . 01 mg/ml ) and uridine ( 0 . 026 mg/ml ) for 24 hrs .', 'Cells were used for recording or immunohistochemistry at 11–14 days in vitro .', 'Cultured retinal ganglion cells were fixed for 10 min in 4% paraformaldehyde ( PFA ) , rinsed with Tris-buffered saline ( TBS ) , and blocked for one hour with TBS containing 4% bovine serum albumin ( BSA ) , and 0 . 1% Triton-X 100 .', 'After blocking , cells were incubated with primary antibodies ( chicken anti-MAP2 , 1∶1000 , Millipore , Billerica , MA; rabbit anti-GluR2 , 1∶200 , Millipore; anti-HCN1 , anti-HCN2 , and anti HCN4 raised in mouse , 1∶200 , NeuroMab , Davis , CA ) for 1 hour .', 'Cells were washed in TBS and incubated with fluorescently-conjugated secondary antibodies ( Alexa Fluor 488 goat anti-chicken IgG , 1∶1000 , Life Technologies , Grand Island , NY; Cy-3 conjugated donkey anti-mouse IgG ( H+L ) , 1∶500; Cy-5 conjugated donkey anti-rabbit IgG ( H+L ) , 1∶500 , Jackson ImmunoResearch Laboratories , West Grove , PA ) .', 'After washing , coverslips were mounted for imaging .', 'Retinal ganglion cells were imaged using a Hamamatsu Orca ER camera mounted on an inverted Nikon fluorescent microscope with a 60× Plan Apo lens .', 'The strength of the excitation light and length of exposure time were kept constant for precise comparisons .', 'Images were background-subtracted , and analyzed using Metamorph software ( Molecular Devices , Sunnyvale , CA ) .', 'No labeling was observed in control slices in which the primary antibody was omitted .', 'HCN1 immunoreactivity was quantified by comparing the average intensity value for HCN1 labeling in proximal ( 0–50 µm ) and distal ( >100 µm ) dendrites .', 'Morphologies for ganglion cells were derived from live images of ganglion cells taken after recordings .', 'An image of the dendritic arbor was digitized by tracing each dendrite manually or by a semi-automatic algorithm , similar to previous models [10] , [26] .', 'The dendritic arbor was divided into regions ( proximal , middle , and distal ) that could be assigned different biophysical properties .', 'For some simulations , the cell was given ion channels defined by Markov sequential-state models , with various combinations of densities of 3 channel types ( KHCN , Na , Kdr ) .', 'Channel densities and other biophysical properties ( Rm , Ri ) were specified for each region of the cell .', 'The standard set of biophysical properties is listed in Table 1 .', 'For some models , we set the channel densities uniform across the cell , but for others , we specified a gradient of channel densities as a function of distance from the soma .', 'In addition , an offset voltage and kinetic rate multiplier could be specified for the rate functions for each channel type .', 'The model of the HCN channel was taken from the literature [27] , [28] .', 'Further modifications were performed to match results obtained from voltage step responses and tail current experiments .', 'The rate of activation and deactivation for HCN were decreased by a factor of 0 . 33 and 2 respectively , to approximately match the data in whole cell recording .', 'We simulated the uncaging of glutamate with a synaptic model connected to a presynaptic compartment that was voltage-clamped .', 'The voltage clamp was directly set by the intensity of a light stimulus at the local ( X , Y ) location .', 'The background light “intensity” was set to the resting potential for the pre-synaptic compartment , and the stimulus “contrast” generated a depolarizing pulse of several mV .', 'This voltage controlled an exponential release function with 2 mV per e-fold change .', 'To approximate the time course of the real responses , the released neurotransmitter was passed through a first-order temporal filter with separate time constants for rise and fall , set to 75 ms and 125 ms respectively .', 'The filtered glutamate release bound to post-synaptic AMPA receptors modeled using a markov diagram , derived from [29] .', 'The reversal potential for the AMPA conductance was set to 0 mV .', 'Thus , synaptic conductance was modulated by the “contrast” of the stimulus to the presynaptic compartment , to vary the amplitude of EPSPs recorded in the simulated ganglion cell .', 'Synapses were positioned at regular intervals ( ∼10 µm , with regularity ( m/SD ) =\u200a8 ) over the dendritic tree by an automatic algorithm .', 'Stimulus pulses were given to specific points on a dendrite for a specified duration ( 3 ms ) and inter-pulse interval ( 20 ms ) .', 'Sets of 3 to 5 locations were selected along the major dendrites at different radial distances from the soma .', 'The simulations were performed using the Neuron-C simulation language [26] , [30] .', "For most model runs , we saved the model's state parameters ( voltages , channel occupancies , etc ) after initial equilibrium , just before the first stimulus ( the sequence of “uncaging” in centripetal or centrifugal directions ) , and restored the model parameters after the first stimulus and just before the second .", 'This obviated any interaction between the timing of the first stimulus and the second .', "When we removed the “restore” of the model's state before the second stimulus , we found that within an interval of 500 ms there was a noticeable effect of the first stimulus upon the second , consistent with the slow activation/deactivation of HCN .", 'Because the data from the real cells was obtained with long intervals ( more than 1s ) between directions , we utilized the “save” , “restore” feature of the simulator for all other model runs .', 'Directional summation ( DS ) was calculated for amplitude as ( Vpeak away – Vpeak toward ) / ( Vpeak toward – Vrest ) * 100 , and ( Charge away – Charge toward ) /Charge toward * 100 for charge .'], ['We used local photolysis of caged glutamate to examine location and timing-dependent differences in summation of dendritic input .', 'Whole-cell voltage and current clamp recordings were obtained from On ganglion cells in whole mounts of dark-adapted rat retina .', 'A Fluoview 1000 equipped with a 405 diode laser was used to uncage glutamate over ganglion cell dendrites in whole mount retina ( see Methods ) .', 'Postsynaptic response size was regulated by adjusting laser intensity to insure that the responses were subthreshold ( 1–4 mV ) and did not initiate action potentials .', 'This approach elicited EPSPs with a time course that was similar to the light responses ( Figure 1A ) .', 'Measured decay times were 98 . 0±14 . 9 ms ( light; N\u200a=\u200a10 On cells ) and 109 . 0±14 . 4 ms ( uncaging; N\u200a=\u200a9 On cells ) and the 10–90% rise time was 40 . 3±8 . 2 ms ( light; N\u200a=\u200a10 On cells ) and 48 . 6±5 . 9 ( uncaging; N\u200a=\u200a9 On cells ) .', 'The differences in decay and rise time were not significant ( t-test; p>0 . 05 ) .', 'This suggests that while the uncaging stimulus may not be identical to light stimuli , the EPSPs are in a physiologically relevant range .', 'The size of the uncaging area was measured by uncaging at 2 µm intervals perpendicular to the dendrite ( Figure 1B ) .', 'Current amplitude at each location was plotted against distance from the dendrite and fit with a Gaussian function .', 'The average half-maximal width of that fit was then used as a measure of lateral ( XY ) resolution .', 'On average , the half-maximal width of the Gaussian fit was 5 . 7±0 . 5 µm ( N\u200a=\u200a7 cells; Figure 1C ) .', 'As a result , individual uncaging locations were separated by a minimum of 20 µm , insuring no overlap between sites .', 'To determine the timing rules underlying summation in On ganglion cells we measured the linearity of summation at an individual dendritic location in whole mount retina .', 'To evaluate the timing rules governing summation , trains of three uncaging pulses were delivered at varied delays ( 1–125 ms ) , to the same location ( single pixel ) ( Figure 2A , B ) .', 'Delays were presented in random order to avoid use-dependent artifacts .', 'The amount of summation for each delay was then calculated by dividing the area ( charge ) or amplitude of the summed EPSP response by 3 times the charge or amplitude for a single EPSP response .', 'As a result , a summed response with a total charge equal to 3X that of a single response would have a summation ratio of 1 , indicating linear summation .', 'Ratios larger or smaller than 1 indicated supra or sub linear summation , respectively .', 'Previous work in cortical neurons has suggested that summation is not uniform over the length of the dendrite [31] .', 'To look for local differences in ganglion cell summation we compared summation at proximal locations ( <50 µm ) to summation from distal dendritic locations ( >50 µm; range 80–250 µm; Figure 2C , D ) .', 'Surprisingly , we found no significant differences between the two groups ( paired t-test; p>0 . 05; N\u200a=\u200a9 On cells ) , suggesting summation in ganglion cells is uniform within 250 µm along the length of the dendrites .', 'Delays of 50 ms or less produced supralinear responses while longer delays resulted in near linear summation ( Figure 2E; N\u200a=\u200a11 On cells ) .', 'Summation ratios for short delays ( 1–50 ms ) were not significantly different from each other ( ANOVA; p>0 . 05; N\u200a=\u200a11 On cells ) .', 'However , delays of ≤50 ms resulted in significantly more summation than longer delay times ( >50 ms; ANOVA; p<0 . 0025 ) .', 'Therefore , we used 50 ms delays for all subsequent summation experiments .', 'Theoretical work has suggested that summation would be greater for inputs moving toward the soma than for those moving away from the soma [13] .', 'In support of this hypothesis a recent study of cortical neurons demonstrated that summed EPSPs were larger in amplitude for input sequence directed toward the soma ( centripetal ) [14] .', 'To test for a directional component to summation in ganglion cells , we activated multiple regions of the dendrite in both directions ( Figure 3A ) .', 'We expected that somatic responses elicited from distal dendrites would be smaller and slower than those from proximal dendrites ( as measured at the soma ) .', 'This is due to membrane leak , axial resistance and membrane capacitance , which increases with dendritic length .', 'Surprisingly , we found the glutamate responses were relatively uniform along and between dendrites ( Figure 3B ) .', 'Across all cells , depolarization amplitudes showed no significant location-dependent differences ( ANOVA; p>0 . 05; N\u200a=\u200a15 cells ) .', 'As ganglion cell dendrites are heavily branched it was possible that multiple dendrites could be activated by the uncaging beam , given that measured axial ( Z ) resolution was 40±4 . 7 µm ( N\u200a=\u200a5 cells ) .', 'While this could have been a potential interpretation problem , the uniform response amplitude argues against this possibility as activation of multiple dendrites would produce random and significant irregularity in EPSP magnitude .', 'These results also suggest that ganglion cell dendrites employ mechanisms that compensate for electronic decay to insure input strength is accurately represented at the soma .', 'This is consistent with predictions based on computational modeling which suggest that for a physiologic range of membrane resistance and moderate strength stimuli , inputs are equally effective at all points along the ganglion cell dendritic tree [9] .', 'We found that ganglion cell summation recorded at the soma was directional , but unlike cortical neurons , it was larger for input sequences directed away from soma ( centrifugal; Figure 3C ) .', 'To measure the linearity of summed EPSPs , individual EPSP amplitudes and the integrated charge of each waveform were arithmetically summed and amplitude and charge ratios were calculated for summed EPSPs ( summed EPSP/3x single EPSP; plots of raw amplitude and charge can be found in Figure S1 ) .', 'Response amplitude ratios were significantly larger for inputs moving away from the soma ( Figure 3D ) .', 'This was also true for the charge , which had a significantly greater supralinear ratio for away directed inputs ( Figure 3E ) .', 'Summation experiments were repeated using older animals ( P22–35 ) to examine summation in mature retina .', 'Again , the EPSP amplitude ratio was larger for sequences moving away from the soma ( 0 . 69±0 . 06 away , 0 . 6±0 . 06 toward; paired t-test p<0 . 01; N\u200a=\u200a11 cells ) .', 'The same was true for the charge ratio ( 1 . 32±0 . 37 away , 1 . 16±0 . 36 toward; paired t-test p<0 . 01; N\u200a=\u200a9 cells ) .', 'When summation for multiple dendrites on the same cell was examined we found similar ratios for all dendrites ( N\u200a=\u200a6 cells ) .', 'In contrast , we found that summation in hippocampus oriens lacunosum moleculare interneurons was larger when moving toward the soma ( Figure 4 . ) , similar to what was reported in cortical neurons [14] .', 'These results suggest that retinal ganglion cells have unique integration properties setting them apart from cortical neurons , consistent with their specialized function .', 'This radial directionality is similar to that exhibited by starburst amacrine cells where centrifugal stimulation produces increased dendritic calcium signals [21] , [22] .', 'The directional summation ( DS ) we observe could be a network property rather than an intrinsic property of the ganglion cell .', 'For example , neighboring amacrine cells could be activated by the free glutamate , changing the inhibitory input to ganglion cells .', 'To test this possibility we repeated the experiment in Figure 3 by blocking inhibition .', 'The addition of Picrotoxin ( 100 µM ) and Strychnine ( 1 µM ) to block GABA-A and Glycine receptors , respectively , resulted in unstable spontaneous activity , which rendered the data uninterpretable .', 'Thus , we only included Picrotoxin in the bath solution to block inhibition , and found that peak amplitude and charge increased in both directions ( 28 . 2±5% ( away ) and 19 . 6±3% ( toward ) ) .', 'However , picrotoxin had no effect on DS with an amplitude ratio of 1 . 27±0 . 08 ( away ) vs . 1 . 10±0 . 05 ( toward ) and charge ratios of 1 . 2±0 . 05 ( away ) vs . 1 . 12±0 . 04 ( toward; t-test; p<0 . 05; N\u200a=\u200a7 cells ) , suggesting inhibitory inputs alone are not responsible for DS .', 'As an additional test of network participation in DS we looked at summation in primary retina cultures .', 'Whole-cell current clamp recordings were obtained from ganglion cells 8–21 days in culture and summation was measured in both directions .', 'Figure 5A shows representative summed EPSPs confirming that DS was reconstituted in the cultures .', 'Amplitude and charge ratios for culture experiments were calculated as away/toward .', 'This was necessary because cultured neurons were difficult to hold for the lengthy time needed to acquire single and summed responses .', 'For peak amplitude , summed EPSPs were larger moving away from the soma ( 1 . 18±0 . 06 vs . 1; paired t-test; p<0 . 05; N\u200a=\u200a10 cells; Figure 5B ) .', 'Charge was also higher moving away from the soma ( 1 . 29±0 . 14 vs . 1; paired t-test; p<0 . 05; N\u200a=\u200a11 cells; Figure 5C ) .', 'These results suggest that sequence-dependent summation is an intrinsic property of the ganglion cell rather than the network .', 'Summation moving away from the soma was 26% larger than summation moving toward the soma in whole-mount retina .', 'While this is a significant difference , the physiological importance of this increase is unclear .', 'To determine whether this increase in summation translates to an increase in excitability we examined spike probability as a function of activation sequence .', 'While we chose uncaging parameters intended to elicit subthreshold glutamate responses , a subset of experiments exhibited suprathreshold responses .', 'For all cells exhibiting suprathreshold activity , we counted the number of suprathreshold trials for centripetal and centrifugal activation and calculated spike probability .', 'Overall , spike probability was significantly higher when moving away from the soma ( paired t-test; p<0 . 05; N\u200a=\u200a9 cells; Figure 6A , B ) .', 'Spike probability for activation moving away from the soma was 0 . 28±0 . 05 and 0 . 1±0 . 04 when moving toward the soma ( Figure 6C ) .', 'These results suggest that input sequence can have a significant impact on cell excitability and function for near threshold conditions .', 'While the mechanisms responsible for creating DS are unclear , a likely possibility is the non-uniform expression of ion channels along dendrites .', 'For example , the NMDA receptor has slow decay kinetics which can lengthen the integration time window and increase summation .', 'If these channels were not uniformly distributed , location and direction-dependence of summation could result .', 'In fact , when NMDA receptors were blocked with APV , EPSP summation was reduced and directionality eliminated in cortical neurons [14] .', 'To determine the role of NMDA receptors in ganglion cell summation we compared summation before and after blockade of NMDA receptors with the antagonist APV ( 50 µM ) in whole mount retina .', 'APV reduced mean charge and amplitude for individual EPSPs , however , this difference was not significant ( paired t-test; p>0 . 05; N\u200a=\u200a9 cells ) .', 'APV also reduced the summation ratio for charge from 1 . 51 ( away ) and 1 . 27 ( toward ) to 1 . 27 ( away ) and 1 . 05 ( toward ) .', 'While modest ( 15 . 6 and 17 . 3% ) , this reduction was significant ( Figure 7A , B; paired t-test; p<0 . 025; N\u200a=\u200a9 cells ) .', 'Although APV reduced overall summation , it did not eliminate the directional component as summation remained significantly larger for activation sequences moving away from the soma ( Figure 7 ) .', 'These results suggest that while NMDA receptors can enhance summation , they do not play a substantial role in establishing the direction-dependence of summation .', 'We observed no difference in the effect of APV on proximal ( <50 µm ) vs . distal ( >50 µm; range 80–250 µm ) regions of dendrite , suggesting that NMDA receptor density was uniform .', 'Hyperpolarization activated cyclic nucleotide-gated ( HCN ) channels are partially open at typical resting potentials where they depolarize the cell , decrease input resistance , and shorten the decay of synaptic signals , effectively reducing summation .', 'Previous work has shown that these channels modulate EPSP summation and initiation of action potentials in a broad range of brain structures including hippocampus , brain stem , inferior colliculus and cortex [15]–[20] .', 'In the hippocampus HCN current density is not uniformly distributed , increasing in density with distance from the soma [15] .', 'Although mammalian retinal ganglion cells are thought to express these channels [26] , [32] , their distribution pattern has yet to be determined .', 'If channel density is location-dependent , it could influence the observed directionality of summation in ganglion cells .', 'To determine HCN channel density and their role in summation , we measured EPSP charge from whole mount retinal ganglion cells in the presence and absence of the HCN channel blocker ZD7288 ( 100 µM ) .', 'Blockade of HCN channels increased single trial EPSP charge and amplitude ( Figure 8A , B ) .', 'We hypothesized that under control conditions HCN should reduce summation by decreasing input resistance and decay time , reducing the integration time window .', 'Regions of the dendritic arbor with higher HCN conductance should be more strongly affected than regions with reduced HCN conductance .', 'Consistent with this possibility we found the effect of HCN blockade was largest in the distal dendrites ( >50 µm; range 75–275 µm; N\u200a=\u200a8 cells ) .', 'Following HCN blockade with ZD7288 , charge increased by 33 . 3±7 . 7% vs . 12 . 5±6 . 0% at proximal sites ( Figure 8B; <50 µm; paired t-test; p<0 . 025; N\u200a=\u200a8 cells ) .', 'Although we found that summation is uniform over the length of the dendrite under control conditions ( Figure 2 ) , we revisited this question to determine whether HCN channels contributed to this uniformity .', 'As described for Figure 2 , three uncaging pulses were delivered to a single location with varying interstimulus intervals ( 1–125 ms ) .', 'For proximal dendritic locations ( <50 µm ) the range of intervals over which supralinear summation occurred was increased from <50 ms to >75 ms as might be expected when summation is increased ( Figure 8C; N\u200a=\u200a8 cells ) .', 'However , this effect was much larger in the distal dendrites where all interstimulus intervals produced supralinear summation ( Figure 8C ) .', 'To estimate the amount of HCN active at rest we applied voltage steps from −40 to −100 mV in the absence or presence of ZD7288 .', 'IV curves were then created by subtracting the ZD7288 traces from controls and measuring tail current amplitude ( Figure 9A , B; N\u200a=\u200a11 cells ) .', 'We found that about 25% of HCN channels are open at −65 mV .', 'These results suggest that HCN acts to compensate for dendritic filtering , imposing the uniformity in summation seen under control conditions .', 'To determine which HCN channel subtype is expressed in the retina , we used fluorescent antibodies to label isoform 1 , 2 and 4 .', 'We did not test for the presence HCN3 because it is thought to be restricted to cone pedicles [33] .', 'To isolate signals from ganglion cells we immunolabeled HCN channels on cultured ganglion cells .', 'Fluorescently labeled HCN1 channel protein was readily detected in the soma and dendrites of retinal ganglion cells .', 'When overlaid with a dendritic marker antibody , microtubule-associated protein 2 ( MAP-2 ) , HCN1 immunoreactivity could be seen throughout the dendritic tree ( Figure 9C ) .', 'Co-labeling for GluR2 subunits allowed us to identify ganglion cells [34] , [35] .', 'HCN4 immunoreactivity was also present in the soma and dendrites ( Figure 9C ) .', 'These results agree with earlier findings that both channel subtypes are found in retina [36] .', 'We did not detect immunoreactivity for HCN2 subtype , consistent with previous reports [33] , [36] , [37] .', 'To quantify expression levels of HCN1 for proximal and distal dendrites we measured pixel intensity in two bins , <50 µm and >100 µm from the soma .', 'We found no clear difference in expression levels for these regions of dendrite ( Figure 9D; paired t-test; p>0 . 05; N\u200a=\u200a115 dendrites; 50 cells ) .', 'However , this type of assay is not highly sensitive and to reliably detect a difference in proximal and distal staining may require a substantial differential , perhaps as high as 50% .', 'Blockade of HCN increased summed EPSP charge in both directions ( Figure 10A ) .', 'However , this effect was larger for centripetal than for centrifugal summation , effectively eliminating the directional component of summation .', 'Summation moving toward the soma was increased by 33 . 8±8 . 5% , while summation moving away from the soma was only increased by 16 . 5±5 . 2% ( Figure 10C; paired t-test; p<0 . 05; N\u200a=\u200a8 ) .', 'Under control conditions , the charge ratio was larger for input moving away from the soma ( 1 . 24±0 . 08 away vs . 1 . 03±0 . 07 toward; paired t-test , p<0 . 01; N\u200a=\u200a8 ) .', 'After application of ZD7288 the charge ratios were 1 . 21±0 . 04 ( away ) vs . 1 . 19±0 . 0 . 05 ( toward; paired t-test; p>0 . 05; N\u200a=\u200a8; Figure 10B ) .', 'ZD7288 application hyperpolarized resting membrane potential by an average change of −2 . 4±1 mV which was not significant ( paired t-test; p>0 . 05; N\u200a=\u200a8; range 6 . 5–1 mV ) .', 'However , it should be noted that small potential changes may be difficult to detect under current clamp conditions due to fluctuation in resting potential .', 'As a result , we conclude that HCN plays an important role in establishing DS .'], ['The unique integration properties of On ganglion cells differentiate them from cortical pyramidal and hippocampal neurons ( [14] and Figure 4 ) .', 'Glutamate responses recorded at the soma are summed in a supralinear manner for centrifugal input arriving at frequencies greater than 20 Hz ( 1–50 ms delays ) .', 'Lower frequency input ( 75
['Introduction', 'Methods', 'Results', 'Discussion']
['Retinal ganglion cells receive inputs from multiple bipolar cells which must be integrated before a decision to fire is made .', 'Theoretical studies have provided clues about how this integration is accomplished but have not directly determined the rules regulating summation of closely timed inputs along single or multiple dendrites .', 'Here we have examined dendritic summation of multiple inputs along On ganglion cell dendrites in whole mount rat retina .', 'We activated inputs at targeted locations by uncaging glutamate sequentially to generate apparent motion along On ganglion cell dendrites in whole mount retina .', 'Summation was directional and dependent13 on input sequence .', 'Input moving away from the soma ( centrifugal ) resulted in supralinear summation , while activation sequences moving toward the soma ( centripetal ) were linear .', 'Enhanced summation for centrifugal activation was robust as it was also observed in cultured retinal ganglion cells .', 'This directional summation was dependent on hyperpolarization activated cyclic nucleotide-gated ( HCN ) channels as blockade with ZD7288 eliminated directionality .', 'A computational model confirms that activation of HCN channels can override a preference for centripetal summation expected from cell anatomy .', 'This type of direction selectivity could play a role in coding movement similar to the axial selectivity seen in locust ganglion cells which detect looming stimuli .', 'More generally , these results suggest that non-directional retinal ganglion cells can discriminate between input sequences independent of the retina network .']
['Visual information is coded by the output of retinal ganglion cells .', 'Through evolution retinal ganglion cells acquired unique properties that allowed them to transmit to the brain such signals as direction of movement .', 'The quest for the cellular mechanism of the detection of movement by retinal ganglion cells has been the holy grail of research on direction selectivity .', 'In this study we have found a mechanism that allows individual non-direction selective On retinal ganglion cells to code sequences of excitatory inputs moving either away or toward the soma .', 'We observed that inputs moving away from the soma resulted in enhanced , supralinear EPSP summation .', 'Evidence from computational modeling suggests that expression of a specific set of voltage-dependent channels in dendrites introduces nonlinearities that could give a ganglion cell the ability to code looming motion .', 'We predict that in a retinal network , such a directional tuning mechanism , together with asymmetric presynaptic inhibition , could be the building block for the development of more complex detection of visual motion .']
['visual system', 'biology', 'sensory systems', 'neuroscience']