An excitatory cortical feedback loop gates retinal wave transmission in rodent thalamus
Throughout the central nervous system , before the development of sensory input or behavioral experience , specialized circuitry generates spontaneous activity that is required for circuit formation ( Kirkby et al . , 2013 ) . In sensory isocortex the vast majority of spontaneous activity is not generated locally , but rather in the peripheral sense organ ( Khazipov et al . , 2013a ) . For example , prior to vision the retina generates waves of spontaneous firing that refine receptive field characteristics in visual thalamus and cortex ( Huberman et al . , 2008 ) . Retinal waves drive robust firing in LGN , superior colliculus , and VC ( Ackman and Crair , 2014 ) through a complex interaction between these structures ( Weliky and Katz , 1999 ) that remains poorly understood . In the retina , transient circuit properties are responsible for wave propagation , most prominently hyper-connectivity of starburst amacrine cells ( Blankenship and Feller , 2010 ) , but the degree to which the central transmission of retinal waves , or spontaneous activity in other systems , requires similar specializations is unknown . During the period of retinal waves , early ganglion cell activity undergoes both amplification and transformation in the brain in ways substantially different from the adult transmission of retinal activity , suggesting the presence of unique developmental circuitry . For example , the relative amplification of ganglion cell spiking in developing thalamocortical circuits is approximately 40-fold that of the adult ( Colonnese et al . , 2010 ) . Evidence of similar amplification is observed in late second and early third trimester human neonates ( born preterm ) , who show sensory-evoked and spontaneous EEG events that are paradoxically large , reaching amplitudes of over 1 millivolt , compared to microvolts in adults ( Vanhatalo and Kaila , 2006 ) . These strong neonatal activities persist despite low synaptic density ( 5–10% of the adult ) ( Aggelopoulos et al . , 1989; Bourgeois and Rakic , 1993 ) , weak individual synapses ( Chen and Regehr , 2000; Etherington and Williams , 2011 ) , and poor action-potential reliability ( McCormick and Prince , 1987 ) . Not only is retinal wave activity amplified in the thalamocortical pathway , it is also transformed into synchronized rapid oscillations that enhance propagation and synaptic plasticity ( Khazipov et al . , 2013b; Luhmann et al . , 2016 ) . Cortical activity during the period of retinal waves follows nearly exactly the macro-patterning of retinal activity: periods of silence lasting 30–60 s are interrupted by 500 ms–2 s waves that occur in clusters lasting up to 10 s ( Hanganu et al . , 2006; Colonnese and Khazipov , 2010; Ackman et al . , 2012 ) . However , the cortical activity within waves is organized in rapid ‘spindle-burst’ oscillations ( 8–30 Hz ) ( Hanganu et al . , 2006; Kummer et al . , 2016 ) that are not present in retina and not observed in adult cortical activity , suggesting transitory early circuit properties are responsible for their production ( Tolner et al . , 2012 ) . Spindle-bursts are not limited to VC , but are ubiquitous throughout neonatal cortex . They are the rodent homologue of delta-brushes ( Khazipov et al . , 2013a ) , one of the major components of the human preterm EEG ( André et al . , 2010 ) , and their absence in patients indicates a poor prognosis . Thus understanding their central generative mechanisms would be a critical contribution to neonatal health and development . We hypothesized that spindle-burst oscillations and retinal-wave amplification arise in LGN , but depend critically on corticothalamic feedback ( Weliky and Katz , 1999 ) . To test this we recorded simultaneously from LGN and primary VC of head-fixed , unanesthetized rats both during the period of retinal waves ( P5-11 ) , and immediately after ( P13-14 ) , when mature cortical activity patterns have begun to emerge ( Rochefort et al . , 2009; Colonnese , 2014; Hoy and Niell , 2015 ) . We investigated the relative roles of retina , thalamus and cortex by pharmacological or optogenetic modulation of each region . Our results demonstrate a developmentally unique role for the corticothalamic projection that results from the late development of inhibition relative to excitation , and is necessary for the propagation and synchronization of retinal wave-driven activity through thalamus and cortex .
To quantify the contribution of retinal input to spontaneous activity in LGN ( Figure 2A ) and VC ( Figure 2B ) , we acutely silenced both eyes by intraocular injection of the blood brain barrier-impermeant glutamate receptor antagonists APV and CNQX . Silencing was confirmed by loss of the visual response ( not shown ) . As predicted , retinal silencing dramatically reduced firing rates in LGN ( –86 . 6 ± 3 . 9% , mean ± SEM ) and VC ( –67 . 2 ± 10 . 2% ) . The continuity of activity , measured as the proportion of the recording with spike activity ( periods containing at least two spikes with an interval of less than 500 ms , called 'events' ) , was also significantly reduced ( LGN –88 . 5 ± 6 . 5% , VC –90 . 6 ± 2 . 3% ) . Examination of the distribution of event lengths after retinal silencing showed that events over 5 s in duration were reduced in LGN and eliminated in VC ( Figure 2A4 , B4 ) . Retinal silencing largely eliminated the prominent peak in LGN MUA and VC LFP spectra in the spindle-burst range , leaving only lower frequency activity ( Figure 2A5 , B5 ) . In combination with previous results showing that spindle-burst oscillations are driven by retinal waves ( Colonnese and Khazipov , 2010 ) , the present retinal silencing data confirms that in LGN , as in VC , spontaneous retinal activity is the primary driver of spontaneous activity and spindle-burst oscillations . In the absence of retinal input , spontaneous activity in the intact thalamocortical loop can provide at most 20% of normal LGN firing . 10 . 7554/eLife . 18816 . 004Figure 2 . Spindle-burst oscillations require retinal and thalamic activity . ( A ) Blockade of spontaneous retinal waves reduces thalamic firing and spindle-burst oscillations . ( A1 ) Ocular injection of glutamate receptor antagonists APV and CNQX to silence retinal activity with simultaneous recording in LGN . ( A2 ) Representative LGN MUA ( >300 Hz ) before ( control ) and after retinal silencing . ( A3 ) Percent change in multi-unit spike rate and continuity of activity during retinal blockade ( n = 6 , Wilcoxon signed-rank test for difference from pre-silence , p=0 . 0313 , p=0 . 0313 ) . Spike continuity is calculated as the proportion of periods containing at least two spikes with interval of less than 500 ms . ( A4 ) Analysis of event duration . Cumulative distribution is shown in solid lines ( left y-axis ) . 95% confidence interval ( CI ) is shown as shaded area ( n = 856 and n = 52 , two sample KS test , p=0 . 0193 ) . Proportion distribution is additionally plotted as a bar graph to aid visualization of event duration ( right y-axis ) . ( A5 ) Population mean of normalized spike spectra . Silencing retina reduced spindle-burst frequencies ( n = 6 , permutation test , p<0 . 05 between 23 . 9–25 . 5 Hz , bar = frequencies significantly different by permutation test ) . ( B ) Silencing retinal activity reduces cortical firing and spindle-burst oscillations . ( B1–5 ) As for A1–5 but for L4 of VC ( B2: VC LFP ( 1–150 Hz ) ; B3: n = 6 , Wilcoxon signed-rank test , p=0 . 0313 , p=0 . 0313; B4: n = 1109 and 189 , two sample KS test , p=10–12; B5: n = 6 , permutation test , p<0 . 05 between 14 . 4–54 . 6 Hz ) . ( C ) Silencing thalamic activity with muscimol while recording in VC . ( C1–5 ) As for B1–5 . Larger reduction in spontaneous firing , event duration and spectral power than for retinal silencing suggests residual activity in thalamocortical loop after retinal silencing can generate small amounts of spindle-burst activity . ( C2: VC LFP ( 1–150 Hz ) ; C3: n = 6 , Wilcoxon signed-rank test , p=0 . 0313 , p=0 . 0313; C4: n = 811 and 377 , two sample KS test , p=10–22; C5: n = 6 , permutation test , p<0 . 05 between 8 . 6–42 . 5 Hz ) . All error bars are SEM unless noted . DOI: http://dx . doi . org/10 . 7554/eLife . 18816 . 004 To determine the ability of VC alone to generate spontaneous activity and spindle-burst oscillations , we pharmacologically silenced LGN by local injection of the GABA receptor agonist muscimol ( Figure 2C ) . Silencing LGN significantly reduced VC L4 MUA rate ( –79 . 0 ± 3 . 9% ) and continuity ( –89 . 0 ± 2 . 6% ) ( Figure 2C3 ) . The remaining VC activity consisted only of very short bursts ( <1 s ) ( Figure 2C4 ) . Most importantly , LGN silencing completely eliminated oscillatory activity in the VC LFP ( Figure 2C5 ) . All of these activity reductions were more severe than observed following retinal silencing; thus while VC alone can generate very short bursts of activity , as observed in slices ( Allène et al . , 2008 ) , an intact thalamocortical loop is necessary for the generation of activity lasting longer than 1 s that contains any high frequencies . In total these data confirm and extend previous results in somatosensory ( Minlebaev et al . , 2011; Yang et al . , 2013a ) and visual ( Colonnese and Khazipov , 2010 ) cortex that spindle-burst oscillations first emerge in thalamus in response to retinal or sensory input . The thalamic and cortical activity remaining after retinal and LGN silencing suggests that corticothalamic excitation is at least partially functional in neonatal rats , as has been previously demonstrated in ferrets ( Weliky and Katz , 1999 ) . We therefore tested the hypothesis that VC provides critical amplification and transformation of the retinal input in LGN by silencing VC with local application of APV and CNQX ( Figure 3 ) . Silencing VC at P9-11 during the peak of stage III waves reduces LGN spike-rate by 79 . 2 ± 5 . 6% and continuity by 68 . 4 ± 9 . 3% ( Figure 3D2 ) . Remarkably , these activity reductions are of similar magnitude to those caused by retinal silencing . The fact that silencing of either retina or VC results in a greater than 50% reduction in firing indicates that they act synergistically to drive LGN . One explanation is that VC participates in feedback amplification as part of an excitatory loop with LGN that is necessary for the generation of effective thalamic spiking during retinal waves . The effect of VC silencing was limited to the LGN; spike-rates for all contacts from the same shank located 20–100 µm below the last visually responsive contact were not affected ( +3 . 1 ± 17 . 1% , p=1 . 000 , n = 6 , Wilcoxon signed rank test ) , indicating that the pharmacological silencing of VC did not have systemic effects . Event duration was reduced , but not as severely altered as by retinal silencing ( Figure 3D5 ) , indicating that retina initiates and maintains activity in LGN , but VC prolongs it . Silencing VC strongly reduced normalized spike spectral power in the spindle-burst range ( Figure 3D3 ) . VC input appears to be required for normal patterning of the spindle-oscillations , as peak frequency of remaining activity shifted from 24 . 6 ± 0 . 9 to 16 . 2 ± 2 . 0 Hz ( Figure 3D4 ) after VC silencing . These results show that during the period of peak stage III retinal waves , feedback connections from VC to LGN are a critical component of retinal wave transmission , which serve to both amplify and prolong spontaneous activity as well as accelerate spindle-burst oscillations in LGN . 10 . 7554/eLife . 18816 . 005Figure 3 . Corticothalamic feedback amplifies retinal waves in thalamus . ( A ) Experimental set-up: silencing VC with local application of APV and CNQX while recording in LGN . ( B ) Representative spontaneous activity in LGN before ( B1 ) and after VC silencing in a P10 rat ( B2 ) . LGN MUA rasters and spike-rate spectrogram as for Figure 1 . ( C1 ) Representative LGN MUA ( >300 Hz ) before ( control ) and after VC silencing at P5-7 . ( C2 ) Percent change in multi-unit spike rate and continuity of activity after VC silencing during cholinergic retinal waves ( P5-7 ) . VC silencing reduces LGN spiking as early as P5 ( n = 6 each , Wilcoxon signed-rank test for difference from pre-silencing , p=0 . 0313 , p=0 . 0313 ) . ( C3 ) Population mean of normalized LGN spike spectra . Note no change in frequency distribution despite decreased spike rates at P5-7 ( permutation test , p>0 . 05 at all frequency ranges ) . ( C4 ) Peak of spike frequency distribution ( Wilcoxon signed-rank test , p=0 . 8750 ) . ( C5 ) Event duration distributions before and after VC silencing ( n = 1727 and 728 , two sample KS test , p=0 . 1975 ) . ( D ) Effects of VC silencing during glutamatergic waves ( P9-11 ) ( n = 6 ) . ( D1–5 ) as for C1–5 ( D2: Wilcoxon signed-rank test , p=0 . 0313 , p=0 . 0313; D3: permutation test , p<0 . 05 between 19 . 8–27 . 2 Hz; D4: Wilcoxon signed-rank test , p=0 . 0313; D5: n = 1549 and 773 , two sample KS test , p=10–9 ) . Note large decrease in spindle-oscillation power and peak frequency in LGN following VC silencing . ( E ) Effects of VC silencing after retinal wave period ( P13-14 ) ( n = 6 ) . ( E1–5 ) as for C1–5 ( E2: Wilcoxon signed-rank test , p=0 . 0313 p=0 . 0313; E3: permutation test , p>0 . 05 at all frequency ranges; E4: Wilcoxon signed-rank test , p=0 . 8750; E5: n = 1785 and 1129 , two sample KS test , p=0 . 0029 ) . Note absence of effect on spike spectra , and increase , in contrast to the decreases until P11 , in spike rate , continuity and event duration in LGN following VC silencing at P13-14 . DOI: http://dx . doi . org/10 . 7554/eLife . 18816 . 005 Retinothalamic innervation precedes cortical innervation of the thalamus ( Seabrook et al . , 2013 ) . To determine whether corticothalamic feedback is also critical for the stage II retinal waves ( cholinergic ) present during the first post-natal week , we examined the effect of VC silencing at P5-7 ( Figure 3C ) . Spindle-burst oscillations are slower at this age , resulting in a peak frequency of the L4 LFP spectra of 12 . 2 ± 1 . 7 Hz ( data not shown ) . Spontaneous LGN spike-rate frequency is similarly down-shifted ( 16 . 0 ± 1 . 2 Hz ) . Silencing VC at this age significantly reduced spike-rates ( −52 . 0 ± 7 . 4% ) and continuity ( −49 . 8 ± 10 . 7% ) ( Figure 3C2 ) , showing that corticothalamic amplification is present at this age as well . However , there was no significant effect of VC silencing on either spike spectra or event duration ( Figure 3C3–5 ) . Thus during the first post-natal week , the role of VC is limited to amplification . At this age , LGN is able to generate spindle-oscillations in response to retinal waves , albeit with a lower frequency than during the second postnatal week . The difference between P5-7 and P9-11 demonstrates a shift in the role of corticothalamic feedback from simple amplifier to active participant in the shaping of LGN oscillations . Spontaneous activity in VC dramatically changes between P11 and P13 . Cortex no longer exhibits the long silent periods of immaturity; activity becomes continuous and loses the prominent spindle-oscillations , which are replaced by adult-like , broad-band activity ( Colonnese and Khazipov , 2010 ) . To determine if the LGN requirement for corticothalamic feedback is restricted to the period of retinal waves , and what role VC plays in the maturation of spontaneous activity , we silenced VC while recording from LGN at P13-14 . VC silencing at this age does not decrease LGN activity , but rather increases spike rates ( +26 . 2 ± 5 . 4% ) and there is slight but not significant increase in continuity ( +13 . 5 ± 5 . 0% ) ( Figure 3E2 ) . VC silencing increases the proportion of very long duration events ( >10 s ) ( Figure 3E5 ) , but does not change the LGN MUA spectrum ( Figure 3E3 ) . Thus the excitatory role of corticothalamic feedback is limited to the period of retinal waves , and cortex becomes inhibitory to thalamus around eye-opening . Together the VC silencing experiments indicate a continually changing role for corticothalamic feedback in the transmission of retinal activity during development . During the period of retinal waves , VC essentially gates LGN output , first as a simple amplifier of LGN , and later in the second post-natal week , by amplifying and patterning oscillations in LGN . This amplification role ends before eye-opening , after which corticothalamic feedback plays a less prominent role in the generation and patterning of retinal transmission through thalamus . To determine the mechanism of early corticothalamic feedback excitation and rhythmogenesis , we took advantage of the fast kinetics and high sensitivity of the blue light drivable channelrhodopsin Chronos ( Klapoetke et al . , 2014 ) to gain control of VC neurons within a few days of birth . Injection of AAV-Chronos-GFP into VC at P0-1 yielded strong expression in VC neurons in all layers and axon terminals by P6 . In neonatal mice ( Seabrook et al . , 2013; Grant et al . , 2016 ) cortical axons avoid LGN relative to surrounding thalamic nuclei . We observed the same pattern in VC axons expressing Chronos-GFP ( Figure 4B ) , although cortical axon arbors can be seen within LGN even at P6 . A 10 ms pulse of 470 nm LED on VC was sufficient to activate deep VC layers at all ages with similar delay ( P6-7 3 . 7 ± 1 . 8 ms; P9-11 3 . 4 ± 1 . 5 ms; P13-14 3 . 5 ± 1 . 3 ms ) . At all ages , 10 ms light-pulses onto VC evoked short duration ( ~30 ms ) initial spiking in LGN ( Figure 4C , D ) , although the latency to activate LGN following VC activation decreased between P6-7 ( 34 . 2 ± 2 . 7 ms ) and P9-11 ( 21 . 5 ± 0 . 5 ms ) , stabilizing by P13-14 ( 20 . 0 ± 0 . 9 ms ) ( Figure 4E ) . The probability of generating a spiking response in LGN with each VC activation increased from 39 . 7 ± 7 . 0% at P6-7 to 92 . 4 ± 3 . 6% at P13-14 ( Figure 4F ) . The spike rate increase was similar in all three ages ( Figure 4G ) . 10 . 7554/eLife . 18816 . 006Figure 4 . Increasing reliability and recruitment of inhibition transforms corticothalamic function . ( A ) Experimental setup: Optogenetic stimulation of VC neurons expressing Chronos-GFP while simultaneously recording in LGN and VC . AAV-Chronos-GFP was injected into VC at P0-1 . ( B ) Representative images of Chronos-GFP expression in the VC and Chronos-GFP expressing corticothalamic axons in LGN at P6 . Scale bars: 100 , 100 and 10 µm . ( C ) Representative MUA rasters for 10 trials with LGN responses at each age . ( D ) Population mean post-stimulus time histogram of LGN spiking ( n = 6 for each age group ) . At P6-11 VC optogenetic stimulation evokes purely excitatory responses in LGN , while in P13-14 animals initial excitation is followed by inhibition . ( E ) Population mean delay between LGN and VC spike onset ( Kruskal-Wallis test , P6-7 vs P9-11 p=0 . 0020 , P6-7 vs P13-14 p=0 . 0058 , P9-11 vs P13-14 p=0 . 6247 ) . ( F ) Population mean reliability: percentage of trials in which VC spike activity produced LGN spike activity ( Kruskal-Wallis test , p=0 . 1744 , 0 . 0057 , 0 . 3960 ) . ( G ) Population mean of spike-rate change in LGN following VC stimulation ( 1–100 ms after stim ) ( Wilcoxon signed-rank test vs baseline , P6-7: p=0 . 0313 , P9-11: p=0 . 0313 , P13-14: p=0 . 0313 ) . ( H ) Population mean spike-rate change in LGN during inhibitory period ( 100–250 ms after stim ) . Significant inhibition is observed only at P13-14 ( Wilcoxon signed-rank test vs baseline , P6-7: p=0 . 6563 , P9-11: p=0 . 8438 , P13-14: p=0 . 0313 ) . ( I ) Cortical stimulation at spindle-burst frequencies ( 20 Hz ) induces oscillations in LGN only at P9-11 . VC stimulation at 20 Hz caused firing in LGN at all ages , but entrained LGN only at P9-11 . DOI: http://dx . doi . org/10 . 7554/eLife . 18816 . 006 The major developmental change in the LGN response occurred between P9-11 and P13-14 . It consisted of the development of post-stimulus inhibition following the initial evoked response ( Figure 4H ) . The relative increase in LGN spike-rates at 100–250 ms after VC stimulation switched from neutral or positive ( P6-7 −0 . 1 ± 36 . 7%; P9-11 +63 . 4 ± 61 . 0% ) to negative at P13-14 ( −89 . 2 ± 8 . 8% ) indicating the recruitment of functional feedforward inhibition into the corticothalamic circuit between P11 and P13 . To determine how the increasing speed , reliability and inhibition of corticothalamic feedback could contribute to the ability of VC to generate spindle-burst oscillations at P9-11 , but not P5-7 or P13-14 , we examined the LGN response to patterned VC stimulation at these ages . Five 10 ms light pulses at 20 Hz evoked different responses in LGN at each age ( Figure 4I ) . At P6-7 , 20 Hz VC stimulation increased LGN spike rates but did not induce oscillatory activity . In contrast , the same stimulation at P9-11 induced 20 Hz oscillatory firing in LGN . At P13-14 , despite reliable LGN responses to the first one or two pulses , induced inhibition prevented further oscillation . Together these cortical stimulation experiments provide a mechanistic explanation for the changing role of corticothalamic feedback in shaping LGN activity . During cholinergic retinal waves , connectivity is sufficient for VC to amplify retinal waves in LGN but its reliability and velocity are insufficient to synchronize LGN firing . Then during the second postnatal week when glutamatergic waves predominate ( P9-11 ) , increasing speed and reliability of the feedback loop enables cortex to synchronize LGN at higher frequencies than LGN alone would naturally oscillate . Finally , at the transition to the third week ( after P13 ) , VC afferents recruit an inhibitory circuit , which reduces their ability to synchronize activity at high frequencies and makes the net effect of corticothalamic feedback inhibitory , as in the adult . During the period of stage III retinal waves , visual responses can be evoked in retina ( through closed eyelids ) and these are transmitted to VC ( Colonnese et al . , 2010 ) . Until just before eye-opening , visual responses have an immature structure that consists of two distinct phases: an initial response composed of 'early gamma oscillations' ( EGOs ) ( Khazipov et al . , 2013b ) followed by a secondary response consisting of spindle-bursts ( Colonnese et al . , 2010 ) . We used this pattern to examine the differential role of corticothalamic feedback on these two prominent early activity patterns , as well as in the transition to the adult pattern . P9-11 LGN visual responses also consisted of early-gamma ( 30–50 Hz ) followed by spindle-burst oscillations ( 8–30 Hz ) ( Figure 5B ) . Silencing VC reduced spike rates in LGN during both the primary and secondary responses ( −46 . 2 ± 4 . 8% and −48 . 7 ± 5 . 8% respectively ) ( Figure 5D , F ) , but reduced the power of spike-rate oscillations only for spindle-burst oscillations ( Figure 5G ) . These results confirm the strong amplification role of early corticothalamic feedback , and specifically implicate corticothalamic excitation in the generation of spindle-bursts , but not EGOs . 10 . 7554/eLife . 18816 . 007Figure 5 . Corticothalamic feedback required for spindle-burst oscillations but not early-gamma oscillations . ( A ) Visually evoked MUA responses were measured in LGN before ( A1 ) and after ( A2 ) VC silencing . Visual stimulus was 100 ms flash . ( B ) Representative evoked LGN MUA before ( B1 ) and after VC silencing ( B2 ) in a P9 rat . Before VC silencing , visual responses consists of an initial ( first ) early-gamma oscillation ( EGO ) , followed by a spindle-burst ( second ) , as observed in VC ( Colonnese et al . , 2010 ) . After VC silencing , EGOs remain , but spindle-bursts oscillations are reduced . ( C ) Representative visual responses at P13 before ( C1 ) and after VC silencing ( C2 ) . Note disappearance of EGOs and spindle-burst in LGN after P13 as observed in VC ( Colonnese et al . , 2010 ) . ( D ) Population mean post-stimulus spike rate histogram for P9-11 ( n = 6 ) . Spike rates are reduced by VC silencing both during primary ( EGO ) and secondary ( spindle-burst ) responses . ( E ) Population mean post-stimulus spike rate histogram for P13-14 ( n = 6 ) . LGN spike-rates are increased by VC silencing at P13-14 , in contrast to decrease after VC silencing at P9-11 . ( F ) Population mean change in LGN spike-rate caused by VC silencing in the baseline ( left ) , primary visual ( first , middle ) and secondary visual ( second , right ) responses for both age groups . ( P9-11 , n = 6 , Wilcoxon signed-rank test for difference from pre-silencing , baseline: p=0 . 0313 , first: 0 . 0313 , second: 0 . 0313; P13-14 , n = 6 , p=0 . 0625 , 0 . 4375 , 0 . 0313 ) . ( G ) Normalized LGN spike spectra for first ( left ) and second ( right ) visual response at P9-11 . Despite reductions in spike rate for both responses , only spindle-burst frequencies ( second ) are reduced by VC silencing ( n = 6 , bar = frequencies significantly different by permutation test , first: p>0 . 05 at all frequency ranges , second: p<0 . 05 at 26 . 7–31 . 7 Hz ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18816 . 007 At P13-14 visual responses in LGN were shorter and consisted of a rapid , non-oscillatory primary response , and greatly reduced secondary response ( Figure 5C ) . This is identical to changes observed in VC ( Colonnese et al . , 2010 ) . As observed for spontaneous activity , silencing VC at this age no longer reduced LGN activity . Instead , it caused a non-significant increase in the evoked spike-rate during the primary response ( +15 . 1 ± 13 . 3% ) and a significant increase in the secondary response ( +48 . 5 ± 9 . 0% ) ( Figure 5F ) . These results show that the switch in corticothalamic feedback from functionally excitatory to inhibitory is not limited to spontaneous activity . They further show that corticothalamic feedback is not necessary for the loss of evoked gamma and spindle-bursts from visual responses at P13 , which instead appear to depend on altered circuitry in LGN and retina .
The present work reinforces previous findings ( Weliky and Katz , 1999; Colonnese and Khazipov , 2010; Ackman et al . , 2012 ) that sustained ( >2 s ) thalamocortical activity before eye-opening is driven by central transmission of retinal waves . Neither VC alone , nor an intact LGN-VC loop , is capable of producing more than brief bursts of activity . In contrast , without VC , firing in LGN is dramatically reduced but sustained activity is still present . Only the longest events—caused by sustained firing between waves occurring in a cluster ( Blankenship and Feller , 2010 ) —are suppressed by VC silencing , demonstrating that corticothalamic feedback maintains activity during waves but does not initiate activity . The fact that VC and retina are both necessary for over 50% of LGN spiking shows their drive is multiplicative , not additive . Together our findings suggest that VC makes a feed-forward excitatory loop with thalamus that amplifies the drive from retina . In ferrets corticothalamic feedback synchronizes activity between eye-specific lamina ( Weliky and Katz , 1999 ) , but its role in amplification and oscillation generation is unknown , as is whether this function is unique to rodents . In addition to amplifying retinal activity , central circuits transform ganglion cell firing during waves into spindle-burst oscillations ( Colonnese and Khazipov , 2010 ) . Almost all activity in early VC , as well as every other cortical region examined , occurs as rapid oscillations necessary for synaptic stabilization in the developing circuit ( Khazipov et al . , 2013a , 2013b ) . Determining the similarities and differences between the cortical areas in how these rhythms are generated will inform our understanding of how regional specialization develops . In all areas , rapid oscillations occur in response to spontaneous and evoked firing of the dominant input ( eg . whisker in barrel cortex ) , with a frequency inversely proportional to its temporal and spatial specificity ( Khazipov et al . , 2013b ) . Here we identify LGN as the locus of generation of VC spindle-bursts , similar to somatosensory ( VPN ) thalamus ( Yang et al . , 2013a ) . Unlike VPN , corticothalamic feedback plays a critical role for setting the frequency and amplitude of LGN firing . During the first post-natal week , corticothalamic connections are capable of directly driving LGN , but only at long latency and with poor reliability , consistent with their low density in LGN at this age ( Seabrook et al . , 2013; Grant et al . , 2016 ) , and they are not necessary for rhythmogenesis . How spindle-bursts are generated in LGN is unknown , but likely involves a combination of functional connectivity with the thalamic reticular nucleus ( Evrard and Ropert , 2009 ) and intrinsic excitability of relay neurons ( Lo et al . , 2002 ) . Between the first and second post-natal weeks , roughly synchronized to the switch between cholinergic and glutamatergic waves ( Huberman et al . , 2008 ) , the timing and reliability of corticothalamic connectivity improves , changing its role from simple amplification to synchronization and rhythmogenesis . LGN can still generate its own weak ( 15 Hz ) oscillations in the first week , but it requires VC to generate normal ( 25 Hz ) activity in the second week . Because corticothalamic projections are only capable of synchronizing LGN at these frequencies at this age ( Figure 4 ) , and L5/6 neurons are synchronized by the spindle-oscillations ( Colonnese and Khazipov , 2010 ) , we suggest that the faster spindle-burst oscillations are generated in the feedback circuit between the two structures . The role of corticothalamic feedback appears specific to spindle-bursts , as it is not important for the generation of the other major early oscillation , Early-Gamma Oscillations ( EGOs ) . In contrast to spindle-bursts , EGOs activate only the input and superficial layers of a single column , and occur only in response to temporally and spatially limited input ( Khazipov et al . , 2013b ) , for example in response to a single deflection of the principle whisker in barrel cortex . VC EGOs do not occur in response to retinal waves , but can be induced by visual stimulation ( Colonnese et al . , 2010 ) , allowing us to examine their cortical dependence . While LGN spiking during the period of EGOs was reduced by VC silencing , the normalized oscillation power was unaffected , suggesting that VC provides amplification but not rhythmogenesis for EGOs . Poor modulation of EGOs by corticothalamic projections is consistent with the conduction delays ( >20 ms ) and lack of L5/6 firing during EGOs ( Colonnese et al . , 2010 ) which should prevent corticothalamic projections from contributing to such rapid synchronization of LGN . In barrel cortex , cortical silencing reduces frequency power for EGOs but not spindle-bursts ( Yang et al . , 2013a ) . Thus corticothalamic feedback may be differentially tuned by region , as EGOs occur spontaneously in barrel-cortex but not in VC ( Yang et al . , 2009; Cichon et al . , 2014 ) . Finding the circuit specializations that allow for transmission of activity during early development will help determine genetic and environmental causes of neurological disorders ( Kanold and Luhmann , 2010 ) . Defective transient circuits can disrupt circuit formation and then disappear , leaving no trace in the adult . Failure to reorganize early circuits into their mature configurations is another likely cause of adult dysfunction ( Ben-Ari , 2008 ) . Important transitional synaptic properties include elevated input resistance , long duration NMDA currents with poor Mg++ block , glutamate spillover , and plateau potentials ( Lo et al . , 2002; Hauser et al . , 2014 ) . Transitional circuits include early-born subplate neurons and L5 somatostatin neurons , both of which receive early thalamocortical synapses ( Kanold and Luhmann , 2010; Marques-Smith et al . , 2016; Tuncdemir et al . , 2016 ) . Here we show clear evidence in vivo that a transient circuit outside the retina ( Blankenship and Feller , 2010 ) participates in the transmission of early activity . Amplification involves functional strength that seems larger than that predicted given VC terminal density ( Seabrook et al . , 2013; Grant et al . , 2016 ) , but also the late integration of inhibition into the circuit . The source of corticothalamic feedback could include contributions from the normal L6 projection as well as L5 , whose axons traverse LGN without extensive arborization due to competition from retinal axons ( Grant et al . , 2016 ) . The capacity of these axons to sprout following retinal blockade suggests that axon terminals are present . Our results identify corticothalamic feedback as a potential contributor to circuit refinements that are dependent on retinal waves in LGN and cortex ( Huberman et al . , 2008 ) . The amplification identified here likely contributes to the defects in retinal ganglion cell targeting caused by genetic ablation of cortex ( Shanks et al . , 2016 ) . We predict that less dramatic manipulations such as silencing corticothalamic feedback should also disrupt refinement of retinotopy and other receptive field properties at eye-opening in LGN and VC . However , the inability to specifically block corticothalamic feedback , without disrupting the intra-cortical connections of L5/6 circuits , make definitive experiments unfeasible . The prominent role of corticothalamic feedback in patterning LGN responses to glutamatergic waves suggests it is particularly critical for synaptic refinement driven by these waves . The role of glutamatergic waves remains controversial ( Kerschensteiner , 2016 ) , but their perturbation reduces segregation of C-lamina and maintenance of A-lamina segregation in LGN ( Davis et al . , 2015a , 2015b ) as well as orientation selectivity in VC ( Huberman et al . , 2008 ) . One of the most surprising roles for corticothalamic feedback is the acceleration of spindle-oscillations during late pre-visual development . A similar near doubling of occipital delta-brush frequencies occurs in human infants between 27 and 35 gestational weeks ( André et al . , 2010 ) . Thus , acceleration of early oscillations is a conserved feature of cortical development , for which we have identified a mechanism . Why does frequency increase ? Driving cortical inputs at 10 Hz causes synaptic depression in immature somatosensory cortex at age equivalents ( Minlebaev et al . , 2011 ) , while higher frequencies cause potentiation . Thus the acceleration in oscillation frequency may be a function of the change from ‘burst-based’ plasticity rules used to grossly align topography ( Butts et al . , 2007 ) , to more precise spike-timing dependent plasticity that can take advantage of the increasing specificity of firing patterns within retinal waves ( Kerschensteiner and Wong , 2008 ) to reduce poly-innervation of relay cells ( Jaubert-Miazza et al . , 2005 ) and refine orientation selectivity ( Huberman et al . , 2008 ) . Overall our results show that thalamocortical circuits function as an integrated circuit even during early development , and suggest that spontaneous retinal activity influences the development of corticothalamic feedback to LGN and reticular thalamus ( RT ) . This is a systematic investigation of the functional development of a ‘modulator’ ( Sherman and Guillery , 2009 ) feedback pathway in vivo . Previous investigations have focused on driver inputs , such as retinogeniculate and thalamocortical synapses ( Ackman and Crair , 2014 ) . For drivers , feedforward inhibition develops later than excitation ( Ziburkus et al . , 2003; Colonnese , 2014 ) , but their functional effect remains excitatory . Here we show that for a modulator its role during development is different from its role in the adult , switching from an excitatory and amplifying role early , to a modulatory , net-inhibitory role later . This principle may apply to other modulatory feedback projections , such as intra-cortical feedback , which largely synapse on interneurons and develop later than feedforward connections ( Berezovskii et al . , 2011; Yang et al . , 2013b ) . It should be noted we used coarse assays of corticothalamic function which , while sufficient to reveal developmental differences in this pathway , reveal little about its adult function , which appears to have excitatory or inhibitory effects that are strongly dependent on stimulus and behavioral state ( Denman and Contreras , 2015; Crandall et al . , 2015 ) . The lack of thalamocortical feedback inhibition has implications for seizure susceptibility during infancy , because thalamocortical synchronization through RT is an important mechanism of seizure spread ( Paz et al . , 2013 ) . Preterm infants and neonates are highly susceptible to seizures ( Rakhade and Jensen , 2009 ) , and excitatory feedback amplification through thalamus may amplify paroxysmal events . Conversely , lack of inhibitory recruitment by cortex likely contributes to the differences in frequency and propagation of neonatal seizures . Our results suggest that manipulating RT function would be a poor candidate for seizure prevention in preterm populations . The mechanism by which functional corticothalamic inhibition develops between P11 and P13 likely involves the recruitment of RT , and not LGN , interneurons , as they are poorly driven by VC ( Jurgens et al . , 2012 ) . It is also unlikely to be due to a change in the polarity of GABAA currents , as GABA is inhibitory in LGN during the first post-natal week . Regardless of the localization of the relevant synaptic potentiation to the cortico-reticular or reticular-LGN synapse , it is clear that the development of effective feedforward inhibition brings to a close the net excitatory feedback loop that amplifies retinal-wave transmission , as well as the ability of cortex to synchronize rapid oscillations within LGN . Feedforward inhibition in both VC and LGN is potentiated between P11 and P13 ( Lo et al . , 2002; Colonnese , 2014 ) , suggesting a common factor is responsible . One prominent candidate for future work includes an increase in continuous activity driven by midbrain neuromodulatory inputs ( Colonnese et al . , 2010 ) and/or spontaneous non-wave retinal activity . We have identified a clear developmental role for the late development of inhibition , relative to excitation . A high excitatory to inhibitory ratio during development has been suggested to compensate for low synaptic density ( Le Magueresse and Monyer , 2013 ) , however a clear correlation with specific developmental events is lacking . By showing a strong contribution to corticothalamic gating of retinal waves , we establish a specific role for late inhibitory development , and provide potential experimental predictions to test its role in circuit formation .
All experiments were conducted with approval from The George Washington University School of Medicine and Health Sciences Institutional Animal Care and Use Committee , in accordance with the Guide for the Care and Use of Laboratory Animals ( eighth Edition , National Academies Press ) . Long–Evans female rats with litters at postnatal day 4 ( P4 , birth P0 ) , or pregnant female rats at embryonic day 11–19 , were acquired from Hilltop Lab Animals ( Scottdale , PA ) and housed at one litter per cage on a 12 hr light 12 hr dark cycle . Both males and females were used . No formal sorting or randomization of subjects was applied , and experiments and analysis was not blind to age or treatment . Eyelid opening occurred between P13 and P14 . In vivo recording methods are extensively described ( Colonnese and Khazipov , 2010; Berzhanskaya et al . , 2016 ) . Topical lidocaine ( 2 . 5% ) and systemic Carprofen ( 5 mg/kg ) were used as analgesic pre-operatively . To place the headplate , under isoflurane anesthesia ( 3% induction , 1 . 5–2% maintenance , verified by toe pinch ) , the scalp was resected , the skull was cleaned , and a stainless plate with a hole was placed so that the region over occipital cortex was accessible , and the plate was fixed with dental cement . The pups were monitored for signs of stress after recovery from anesthesia . For recording , the animal was head-fixed , and the body was supported within a padded enclosure and warmed with an electric blanket . Temperature was monitored with a thermocouple placed under the abdomen and maintained at 34–36°C . Movement was detected using a piezo-based detector placed under the enclosure . For VC recording , the skull above the monocular primary visual cortex was thinned , and the monocular primary visual cortex was targeted with the following coordinates: 0 . 5–1 . 0 mm anterior from the lambda suture , and 2 . 5–3 . 0 ( P5-7 ) , 2 . 8–3 . 3 ( P9-11 ) , or 3 . 0–3 . 5 ( P13-14 ) mm lateral from lambda . Co-ordinates for LGN were 2 . 0–2 . 5 mm anterior and 3 . 0–3 . 5 mm lateral ( p5-7 ) , 2 . 3–2 . 8 mm anterior and 3 . 3–3 . 8 mm lateral ( p9-11 ) , or 2 . 5–3 . 0 mm anterior and 3 . 5–4 . 0 mm lateral from the lambda ( p13-14 ) . Extracellular activity was recorded using single shank linear multi-electrode arrays ( NeuroNexus , MI ) . A combination of linear ‘edge’ arrays with 100 or 20 µm separation or ‘Poly2’ design of two parallel rows with 50 um separation were used . Electrodes were coated with DiI ( Life Technologies , CA ) before insertion for histological verification of electrode location . In animals older than P8 , recording was initiated only after verifying that contralateral visual evoked firing on presumptive LGN electrodes preceded that of V1 electrodes ( three animals were not recorded for this reason ) . For P5-7 animals ( prior to visual responsiveness ) , recordings were initiated if both presumptive LGN or V1 locations displayed prominent 1–5 s firing periods that were interrupted by 10–60 s periods of network silence , indicative of retinal waves at this age ( Hanganu et al . , 2006 ) . Post-mortem reconstruction of the electrode tract was used to verify placement within central dLGN or monocular V1 ( Figure 1B ) by reference to a developmental atlas ( Khazipov et al . , 2015 ) . In total , three P5-7 animals were excluded post-mortem because the electrode was not located in the dLGN . An Ag/AgCl wire was placed over right frontal cortex ( ~1 mm posterior and 3 mm lateral to bregma ) as ground . Electrical signals were digitized using the Neuralynx ( Bozeman , MT ) Digital Lynx S hardware with Cheetah ( v5 . 5 ) software . dEEG signals were band-pass filtered between 0 . 1 Hz to 9 kHz , and digitized at 32 kHz . Cortical recordings were referenced to a contact site located in underlying white matter . LGN recordings were referenced to a contact just dorsal to LGN . MUA was extracted by threshold crossing of −50 µV following 300 Hz–9 kHz band-pass . Visual stimuli were provided by a 100 ms whole-field flash ( 100 lux ) every 30 s to the contralateral or ipsilateral eye on a background of low light ( <1 lux ) . For LGN silencing , the LGN electrode was removed after control recording , and 1 μl of 1 mM Muscimol and 1% Chicago Sky Blue in saline was injected using a Nanoject II ( Drummond ) at the same stereotaxic coordinates used for the LGN recording . Recordings began 10 min after application of the solution . The injection site was confirmed to be located in LGN in all animals by post-recording histology . Retinal silencing was also performed acutely after obtaining baseline visual responses . The surface of the eye was prepared with 2 . 5% lidocaine . Eyes were stabilized with blunt forceps and 2–5 μl of APV ( 5 mM ) /CNQX ( 2 mM ) in saline was injected intravitreally under the microscope . Recordings resumed at least 15 min after recovery from anesthesia . Retinal silencing was verified by loss of visual response ( n = 2 removed for remaining visual response ) . For VC silencing , 1–3 μl of APV ( 5 mM ) /CNQX ( 2 mM ) in saline was applied over the small hole above the visual cortex . Recordings started 10 min after application of the solution . VC silencing was confirmed by verifying the complete secession of firing in all layers of VC for the electrodes under the drug placement . Because LGN and VC recordings were not aligned , we cannot be certain that the aligned portion of VC was completely inhibited by the treatment . However , the drug eliminated activity at all depths ( ~1 . 2 mm ) of VC . Assuming a similar horizontal spread ( 2 mm diameter ) , inhibition will , at a minimum , affect all of monocular VC . Recording began 10 min after application and effects were stable for at least 30 min after application , when recording was terminated . AAV9-Synapsin-promoter-Chronos-GFP ( tier 3 . 5e13 GC/ml ) was obtained from Penn Vector Core . P0-1 rat pups were cold anesthetized and 50–100 nl of viral solution was injected into the visual cortex at multiple depths ( Nanoject II ) . At least 5 days post injection yielded strong expression of Chronos-GFP in VC and in cortical axons in LGN , and expression was verified in all animals recorded . To activate Chronos in vivo , a 400 µm diameter optic fiber ( Thorlabs , Newton , NJ ) coupled with 470 nm LED ( Thorlabs ) was placed over VC , and 2 . 5 mW flashes of 470 nm LED illumination were used for activation . Neural signals were imported into Matlab ( Mathworks , MA ) . Spike-times and dEEG were down sampled to 1 kHz . Before analysis , animals were eliminated for unstable baseline LFPs ( fluctuation larger than the maximum amplitude of visual response ) or variable spike activity ( > 20% change between the start and end of recording ) ( n = 5 ) . Cortical L4 was identified in each recording as the channel with the earliest negative deflection and the fastest spike response in the mean visual evoked response as previously described ( Berzhanskaya et al . , 2016 ) . For LGN , contralateral- and ipsilateral- responsive regions were identified by 100 ms flashes to either eye . The channel for analysis in LGN was selected by the earliest spike response to contralateral visual stimuli and used for detailed analysis . For P5-7 , because of the lack of visual response at this age , cortical L4 was identified as that with the largest spike response during spindle bursts , and the recording site in LGN with the strongest spike activity during spindle bursts was used for detailed analysis . For all analyses , activity for each animal was calculated from the entirety of a continuous 20 min period prior and 20 min period after manipulation ( following 15 min recovery from anesthesia ) . For spectral analysis in VC , LFP spectra were obtained over a fixed window ( 2 s for spontaneous and 0 . 3 s for visually evoked response ) by the multitaper method using the freely available Chronux package ( Mitra and Bokil , 2007 ) with taper parameters [3 5] . Animal mean spectra were calculated by averaging spectral windows . To reduce the effect of the 1/f relationship , mean multi-taper spectra were multiplied by frequency . For LGN , animal mean MUA spectra were obtained by calculating the fourier transform of the auto-correlation of total MUA . Time-series spectra for LGN MUA used for display ( representative traces in Figures 1 , 3 , 5 ) were calculated by filtering the MUA time series with a Gaussian window ( 5 ms alpha ) and applying the multi-taper method as above . For both signals , the frequency axis was resampled on a log scale to equalize the representation of high and low frequencies and reduce the multiple comparisons problem . For normalization , frequency power at each band was divided by the mean 2–55 Hz power . Events were defined as at least two spikes occurring with intervals of less than 500 ms . Spike continuity was calculated as the proportion of total recording time that contained an event . Brains were perfused with 4% paraformaldehyde in PBS , sectioned by vibratome , and mounted with Fluoromount-G ( Electron Microscopy Sciences ) . Images were acquired using a Zeiss 710 confocal microscope with a 10x or 60x objective , or Zeiss DM6000 B microscope with a 10x objective . All hypothesis testing was conducted using non-parametric because n was less than ten . Wilcoxon signed-rank test was used for pairwise comparison , and mean ± standard error of the mean ( SEM ) and each data point is reported ( Figures 2A3 , B3 , C3 , 3C2 , C4 , D2 , D4 , E2 , E4 , 4G , H and 5F ) . The Kruskal–Wallis test was used for non-parametric comparison of three independent groups , and mean ± SEM and each data point is reported ( Figure 4E and F ) . Kolmogorov–Smirnov test was used for non-parametric comparison of distribution of events , and data and 95% confident interval is reported ( Figures 2A4 , B4 , C4 , 3C5 , D5 and E5 ) . Spectra were examined at each frequency for significant difference using non-parametric permutation tests corrected for multiple comparisons by the method of Cohen ( Cohen , 2014 ) ( Figures 2A5 , B5 , C5 , 3C3 , D3 , E3 and 5G ) . All tests were performed in Matlab . The number of animals is reported in all figures . Exact P-values are presented in all figures; p<0 . 001 are rounded to nearest power of ten .
Spontaneous retinal waves are critical for the development of receptive fields in visual thalamus ( LGN ) and cortex ( VC ) . Despite a detailed understanding of the circuit specializations in retina that generate waves , whether central circuit specializations also exist to control their propagation through visual pathways of the brain is unknown . Here we identify a developmentally transient , corticothalamic amplification of retinal drive to thalamus as a mechanism for retinal wave transmission in the infant rat brain . During the period of retinal waves , corticothalamic connections excite LGN , rather than driving feedforward inhibition as observed in the adult . This creates an excitatory feedback loop that gates retinal wave transmission through the LGN . This cortical multiplication of retinal wave input ends just prior to eye-opening , as cortex begins to inhibit LGN . Our results show that the early retino-thalamo-cortical circuit uses developmentally specialized feedback amplification to ensure powerful , high-fidelity transmission of retinal activity despite immature connectivity .
The brain of a developing fetus has a big job to do: it needs to create the important connections between neurons that the individual will need later in life . This is a challenge because the first connections that form between neurons are sparse , weak and unreliable . They would not be expected to be able to transmit signals in a robust or effective way , and yet they do . How the nervous system solves this problem is an important question , because many neurological disorders may be the result of bad wiring between neurons in the fetal brain . When an adult human or other mammal “sees” an object , visual information from the eye is transmitted to a part of the brain called the thalamus . From there it is sent on to another part of the brain called the cortex . The cortex also provides feedback to the thalamus to adjust the system and often acts as a brake in adults to limit the flow of information from the eyes . Murata and Colonnese investigated whether the fetal brain contains any “booster” circuits of neurons that can amplify weak signals from other neurons to help ensure that information is transferred accurately . The experiments monitored and altered visual activity in the brains of newborn rats – which have similar activity patterns to those observed in human babies born prematurely . Murata and Colonnese found that in these rats the feedback signals from the cortex to the thalamus actually multiply the visual signals from the eye , instead of restraining them . This causes a massive amplification in activity in the developing brain and explains how the fetal brain stays active despite its neurons being only weakly connected . The booster circuit stops working just before the eyes first open ( equivalent to birth in humans ) as the connections between neurons become stronger , and is replaced by the braking mechanism seen in adults . This is important , because continued amplification of signals in the adult brain might cause excessive brain activity and epilepsy . The findings of Murata and Colonnese may therefore help to explain why epileptic seizures have different causes and behave differently in children and adults . The next step following on from this work is to find out how the braking mechanism forms in young animals . Future studies will also focus on understanding the precise role the booster circuit plays in early brain development .