Coordinated forms of noradrenergic plasticity in the locus coeruleus and primary auditory cortex

Abstract

The cerebral cortex is plastic and represents the world according to the significance of sensory stimuli. However, cortical networks are embodied in complex circuits, including neuromodulatory systems such as the noradrenergic locus coeruleus, providing information about internal state and behavioral relevance. Although norepinephrine is important for cortical plasticity, it is unknown how modulatory neurons themselves respond to changes of sensory input. We examined how locus coeruleus neurons are modified by experience and the consequences of locus coeruleus plasticity for cortical representations and sensory perception. We made whole-cell recordings from rat locus coeruleus and primary auditory cortex (A1), pairing sounds with locus coeruleus activation. Although initially unresponsive, locus coeruleus neurons developed and maintained auditory responses afterwards. Locus coeruleus plasticity induced changes in A1 responses lasting at least hours and improved auditory perception for days to weeks. Our results demonstrate that locus coeruleus is highly plastic, leading to substantial changes in regulation of brain state by norepinephrine.

Figure 1

Locus coeruleus responses are plastic. a, In vivo whole-cell or cell-attached recording from locus coeruleus (“LC”) neurons. b, Locus coeruleus pairing procedure. Scale: 0.3 mV, 25 msec. c, Current-clamp recording from locus coeruleus neuron. Dotted line, baseline tone-evoked EPSP (0.0±0.1 mV). Red line, tone-evoked EPSP after pairing (0.7±0.1 mV, P=10⁻⁸, Student’s unpaired two-tailed t-test; z-score: 3.0). d, Recording with AP5 infusion (1 mM); no response to tones before (0.0±0.1 mV) or after pairing (−0.1±0.1 mV, P=0.3; z-score: −0.3). e, Three locus coeruleus neurons for 7+ hours before and after pairing. Left, first recording ten minutes before (gray; tone-evoked EPSPs: −0.4±0.1 mV) and ten minutes post-pairing (red; tone-evoked EPSPs: 0.3±0.3 mV, P=0.004, z-score: 1.1); third recording 420 minutes post-pairing (black; tone-evoked EPSPs: 0.6±0.2 mV, z-score: 1.8). Right, second cell-attached recording 360 minutes post-pairing (0.2±0.02 spikes/tone; z-score: 3.4). f, Summary of new tonal responses in locus coeruleus neurons after pairing. Left, synaptic responses (33 measurements, n=14 neurons, N=9 animals; z-score 5–15 minutes post-pairing: 3.1±0.8, P=0.0009; z-score 3–10 hours post-pairing: 2.0±0.5, P=0.02). Filled diamonds, experiments pairing with single-cell depolarization instead of extracellular stimulation (‘ES’). Open symbols, AP5 in locus coeruleus (n=7, N=3; z-score 5–15 minutes post-pairing: −0.2±0.1, P=0.3; z-score 3–10 hours post-pairing: 0.04±0.05, P=0.4). Right, spiking (35 measurements, 20 neurons, 10 animals; z-score 5–15 minutes post-pairing: 1.6±0.6, P=0.02; z-score 3–10 hours post-pairing: 2.9±0.4, P=10⁻⁴; AP5, n=13, N=5; z-score 5–15 minutes post-pairing: −0.7±0.2, P=0.2; z-score 3–10 hours post-pairing: −1.0±0.7, P=0.2). Error bars indicate s.e.m.

Figure 2

AI plasticity induced by locus coeruleus pairing with electrical stimulation. a, Setup: stimulation electrode (“Stim”) in locus coeruleus (“LC”) and recordings (“Rec”) from AI neurons. b, Current-clamp recording of responses to paired 16 kHz and unpaired 4 kHz tones. c, Synaptic (top) and spiking (bottom) tuning curves from five neurons before and 0–11 hours post-pairing from current-clamp (filled) or cell-attached recordings (open). Each recording from same AI location. Upper left, first recording ten minutes before (gray) and fifteen minutes after (black) pairing with 16 kHz. After pairing, best frequency shifted to 16 kHz (100% shift) and tuning width increased from 2.4 octaves to 5.3 octaves (221% width). EPSPs increased across frequencies (paired 16 kHz EPSPs: 2.0±0.4 mV pre-pairing, 18.3±2.3 mV post-pairing, P=10⁻⁸; unpaired EPSPs across other frequencies: 1.7±0.3 mV pre-pairing, 13.4±0.8 mV post-pairing, P=10⁻⁵). Same cell as b. Inset, 16 kHz EPSPs before (gray) and after (black) pairing; scale: 6 mV, 25 msec. Upper right, cell-attached recording 55 minutes post-pairing (best frequency shift: 100%, tuning curve width: 1.8 octaves). Middle left, second recording 320 minutes post-pairing (shift: 100%, width: 3.8 octaves). Middle right, third recording 485 minutes post-pairing (shift: 100%, width: 0.9 octaves). Lower left, fourth recording 600 minutes post-pairing (shift: 100%, width: 3.3 octaves). Lower right, fifth recording 660 minutes post-pairing (shift: 100%, width: 0.8 octaves). Error bars indicate s.e.m.

Figure 3

AI plasticity induced by locus coeruleus pairing with optical stimulation in TH-Cre rats. a, Optogenetic control of locus coeruleus. Left, schematic of viral injection. Animals had an AAV expressing the ChETA variant of channelrhodopsin-2 and YFP (AAV5Ef1a-DIO ChETA-EYFP) stereotaxically injected into right locus coeruleus. Middle, TH and YFP immunostaining in locus coeruleus imaged at 10X; red, TH; green, YFP; blue, DAPI. Tissue was examined this way in one animal. Arrow, injection site. Scale, 500 μm. Right, zoom-in of white boxed region in middle panel showing co-labeling of TH and YFP expression. Scale, 40 μm. b, Synaptic and spiking tuning curves from four neurons before and 0–7 hours post-pairing from current-clamp (filled) or cell-attached recordings (open). Each recording from same AI location. Upper left, first recording ten minutes before (gray) and ten minutes after (black) pairing optical stimulation with 16 kHz (arrow). EPSPs increased across frequencies (paired 16 kHz EPSPs: 0.1±0.05 mV pre-pairing, 0.8±0.2 mV post-pairing, P=0.001; unpaired EPSPs across other frequencies: 0.1±0.02 mV pre-pairing, 0.9±0.06 mV post-pairing, P=10⁻¹⁵). Upper right, second cell-attached recording 115 minutes post-pairing (best frequency shift: 75%). Middle left, third recording 225 minutes post-pairing (shift: 100%). Middle right, third recording 240 minutes post-pairing (shift: 75%). Lower left, fourth recording 420 minutes post-pairing (shift: 100%). Lower right, fourth recording 435 minutes post-pairing (shift: 100%). Error bars indicate s.e.m.

Figure 4

Changes to synaptic and spiking tuning curves after locus coeruleus pairing. a, Changes to paired inputs from individual recordings. Circles, all recordings (5–10 minutes: 335.9±74.8%, n=37 neurons, P=0.0003; 45–60 minutes: 261.2±62.8%, n=17, P=0.01); squares, current-clamp (n=21); triangles, voltage-clamp (n=16). b, Changes to unpaired inputs from individual recordings for all recordings (5–10 minutes: 231.1±38.3%, P=0.03; 45–60 minutes: 151.0±49.4%, P=0.2). c, Best frequency shift of synaptic tuning over multiple recordings (circles, n=87 neurons, N=37 animals; 5–30 minutes: 90.6±10.7%, P=10⁻⁹; 7–12 hours: 65.6±12.5%, n=27, N=13, p<P=0.0002; squares, current-clamp, n=47; triangles, voltage-clamp, n=40). d, Synaptic tuning curve width over multiple recordings (5–30 minutes: 146.6±14.8%, n=37, N=37, P=10⁻⁴0.; 7–12 hours: 103.3±7.8%, n=27, N=13, P=0.1). Same recordings as c. e, Best frequency shifts of spiking tuning curves over multiple recordings (circles, n=72, N=34; 5–30 minutes: 89.7±11.4%, n=29, N=29, P=10⁻⁵; 7–12 hours: 66.1±11.5%, n=21, N=11, P=0.002; filled squares, current-clamp, n=22; open squares, cell-attached, n=50). f, Widening of spiking tuning curves over multiple recordings (5–30 minutes: 188.5±15.1%, n=29, N=29, P=0.001; 7–12 hours: 93.6±11.5%, n=21, N=11, P=0.7). Same recordings as e. All comparisons with Student’s paired two-tailed t-tests. Error bars indicate s.e.m.

Figure 5

Cortical circuit mechanisms of enhanced AI responses after locus coeruleus pairing. a, Voltage-clamp recording showing new tone-evoked responses during pairing. Inset, responses before (gray), during pairing (black); scale:150 pA, 10 msec. b, Current-clamp recording showing sub- to suprathreshold tone-evoked responses. Dashed line, spike threshold. Scale:5 mV, 10 msec. c, Synaptic changes. Left, voltage-clamp recordings (pre: −15.7±2.8 pA, post: −31.3±6.2 pA; n=16, P=0.006). 10/16 recordings had significant tone-evoked responses before pairing, 14/16 post-pairing (P<0.05 for each cell). Right, current-clamp recordings (pre: 2.0±0.4 mV, post: 5.1±1.1 mV; n=21, P=0.002). 18/21 recordings had significant tone-evoked responses before pairing, 20/21 post-pairing. d, Spiking changes. Left, current-clamp recordings (pre: 0.05±0.03 spikes/tone, post: 0.25±0.07 spikes/tone; n=21, P=0.004). 4/21 recordings had 1+ tone-evoked spikes before pairing, 13/21 post-pairing. Right, cell-attached recordings (pre: 0.5±0.2 spikes/tone, post: 0.9±0.3 spikes/tone; n=12, P=0.04). 9/12 recordings had 1+ tone-evoked spikes before pairing, 12/12 post-pairing. e, Spontaneous IPSCs. Top, pairing decreased spontaneous IPSC rate (pre:9.2 Hz, post:5.0 Hz) but not amplitude (pre: 70.8 pA, post: 61.3 pA). Left, IPSC rate (pre: 4.3±0.8 Hz, post: 3.1±0.6 Hz; n=16, P=0.01). Right, amplitude (pre: 24.6±4.7 pA, post: 24.7±4.9 pA; n=16, P=0.9). ‘n.s.’, non-significant. f, Spontaneous EPSCs. Top, pairing did not affect spontaneous EPSC rate (pre: 12.4 Hz, post: 15.4 Hz) or amplitude (pre: −44.3 pA, post: −56.7 pA). Same recording as e. Left, EPSC rate (pre: 5.7±1.0 Hz, post: 6.3±1.1 Hz; n=16, P=0.3). Right, amplitude (pre: −21.1±3.0 pA, post: −35.7±13.7 pA; n=16, P=0.2). All comparisons with Student’s paired two-tailed t-tests. Error bars indicate s.e.m.

Figure 6

Noradrenergic receptor activation is required for expression of AI plasticity. a, Norepinephrine pairing (‘NE pairing’) leads to shorter-term but not sustained AI changes. Top, experimental design; pure tones were paired with NE iontophoresis (0.1 mM) in AI. Middle, cell-attached recording before and after NE pairing with 2 kHz (arrow). Initially, best frequency (open arrowhead) shifted from 8 kHz to 2 kHz, but returned to 8 kHz one hour later. Bottom, NE pairing summary (best frequency shift 10 minutes post-pairing: 95.8±5.1%, n=4, N=4, P=10⁻⁵; shift 45+ minutes post-pairing: 8.3±10.2%, n=4, N=4 animals, P=0.3). b, Cortical phentolamine (0.1–1 mM) only during LC pairing prevents shorter-term changes, but longer-term changes emerge when phentolamine is removed. Top, experimental design. Middle, whole-cell recording before and after pairing, and cell-attached recording one hour post-pairing. Original best frequency was 4 kHz, tuning was unchanged 10 minutes post-pairing in presence of phentolamine (0.1 mM), but shifted to paired 16 kHz tone one hour later when phentolamine was removed. Bottom, phentolamine during pairing summary (shift 10 minutes post-pairing: 1.5±62%, n=6, N=6, P=0.7; shift 45+ minutes post-pairing: 67.8±17.7%, n=8, N=7, P=0.0006). c, Phentolamine applied after pairing for hours shortens AI shift duration. Top, experimental design-phentolamine (0.1 mM) was applied to AI for subsequent recordings after pairing. Middle, two cell-attached recordings before and after LC pairing. Bottom, phentolamine after pairing summary (shift 10 minutes post-pairing: 85.0±10.7%, n=6, N=6, P=10⁻⁴; shift 3–8 hours post-pairing: 6.3±6.3%, n=8, N=5, P=0.3). All comparisons with unpaired two-tailed t-tests. Error bars indicate s.e.m.

Figure 7

Locus coeruleus plasticity controls the duration of cortical plasticity. a, Three AI recordings for 7+ hours pre/post-pairing with AP5 in locus coeruleus. Left, first current-clamp recording ten minutes before (gray) pairing; third current-clamp recording 460 minutes post-pairing (black). 4 kHz was original best frequency (arrowhead); 1 kHz was paired frequency (arrow). Right, second cell-attached recording 200 minutes post-pairing. b, Summary of AI best frequency shift with AP5 in locus coeruleus. Left, synaptic AI best frequency shift. After 2–10 hours post-pairing, best frequency returned to baseline (shift: 33.3±21.1%, P=0.1, n=6 neurons, N=3 animals). Shaded area, mean±s.e.m. of shifts from Figure 4c. Right, spiking best frequency shift (shift: 20.0±20.0%, P=0.3, n=5, N=4). Shaded area, mean±s.e.m. from Figure 4e. Error bars indicate s.e.m.

Figure 8

Locus coeruleus pairing improves sensory perception. a, Enhanced detection after pairing 30 dB SPL, 4 kHz tones with electrical stimulation (‘ES’) of locus coeruleus. Hits (circles) at 20–40 dB SPL increased after 12 hours (pre-pairing, black: 8.3±5.3%, post-pairing, red: 43.7±14.0%, P=0.04); foil responses (triangles) were unchanged (pre-pairing: 3.6±1.7%, post-pairing: 6.8±1.8%, P=0.2), increasing d′ (0.5 to 1.4). b, Enhanced detection after pairing 30 dB SPL, 4 kHz tones with optogenetic locus coeruleus stimulation (‘opto’). Hits at 20–40 dB SPL increased after 12 hours (pre-pairing, black: 5.0±5.0%, post-pairing, blue: 38.7±11.8%, P=0.04); foils were unchanged (pre-pairing: 2.7±1.6%, post-pairing: 3.8±2.5%, P=0.7), increasing d′ (0.3 to 1.1). c, d′ values (before: 0.49±0.06, 12 hours after: 1.12±0.12, N=21, P=10⁻⁵, Student’s paired two-tailed t-test). AP5 in locus coeruleus prevented improvement (open circles; d′ before: 0.65±0.07, after: 0.57±0.10, N=6, P=0.5). d, Detection was enhanced four days (circles; d′ before: 0.49±0.09, after: 0.98±0.12, N=12, P=0.004) and 20 days after pairing (stars; before: 0.44±0.09, after: 0.89±0.14, N=10, P=0.01). e, Reversal learning without pairing; rewarded frequency was changed to 16 kHz. f, One pairing episode (at 16 kHz) accelerated reversal learning. g, Accelerated reversal learning after electrical LC pairing. Sliding t-tests (width: two days) used to determine when performance recovered to baseline (black, control: 22 days, N=11; red, paired animals: 13 days, N=6). h, Accelerated reversal learning after optogenetic LC pairing. Performance recovered faster in paired animals (black, control: 17 days, N=4; blue, paired animals: 13 days, N=4). Error bars indicate s.e.m.