Nicotinic neuromodulation in auditory cortex requires MAPK activation in thalamocortical and intracortical circuits

Abstract

Activation of nicotinic acetylcholine receptors (nAChRs) by systemic nicotine enhances sensory-cognitive function and sensory-evoked cortical responses. Although nAChRs mediate fast neurotransmission at many synapses in the nervous system, nicotinic regulation of cortical processing is neuromodulatory. To explore potential mechanisms of nicotinic neuromodulation, we examined whether intracellular signal transduction involving mitogen-activated protein kinase (MAPK) contributes to regulation of tone-evoked responses in primary auditory cortex (A1) in the mouse. Systemic nicotine enhanced characteristic frequency (CF) tone-evoked current-source density (CSD) profiles in A1, including the shortest-latency (presumed thalamocortical) current sink in layer 4 and longer-latency (presumed intracortical) sinks in layers 2-4, by increasing response amplitudes and decreasing response latencies. Microinjection of the MAPK kinase (MEK) inhibitor U0126 into the thalamus, targeting the auditory thalamocortical pathway, blocked the effect of nicotine on the initial (thalamocortical) CSD component but did not block enhancement of longer-latency (intracortical) responses. Conversely, microinjection of U0126 into supragranular layers of A1 blocked nicotine's effect on intracortical, but not thalamocortical, CSD components. Simultaneously with enhancement of CF-evoked responses, responses to spectrally distant (nonCF) stimuli were reduced, implying nicotinic "sharpening" of frequency receptive fields, an effect also blocked by MEK inhibition. Consistent with these physiological results, acoustic stimulation with nicotine produced immunolabel for activated MAPK in A1, primarily in layer 2/3 cell bodies. Immunolabel was blocked by intracortical microinjection of the nAChR antagonist dihydro-β-erythroidine, but not methyllycaconitine, implicating α4β2*, but not α7, nAChRs. Thus activation of MAPK in functionally distinct forebrain circuits--thalamocortical, local intracortical, and long-range intracortical--underlies nicotinic neuromodulation of A1.

Fig. 1.

Laminar profile of characteristic frequency (CF) tone-evoked responses in A1. Left: representative example of local field potentials (LFPs) recorded with a 16-channel silicon multiprobe (12 channels shown, with the trace from the recording site visible at the cortical surface labeled 0 μm). The derived, 1-dimensional current-source density (CSD) traces (middle) and interpolated color plot (right) show the CSD response to the stimulus (20 kHz, 70 dB SPL, 100-ms duration). Green CSD trace and white arrow indicate the layer 4 sink that includes the presumed thalamocortical response. Red trace and black arrow indicate the layer 2/3 (intracortical) current sink. Color scale indicates response amplitudes normalized to the largest current sink (reds/black) and largest current source (blues/white) and also applies to all subsequent CSD figures.

Fig. 2.

Effects of systemic nicotine on group CSD profiles in A1 for control animals (A) and after thalamic (C) or intracortical (D) microinjection of MEK inhibitor. A: nicotine enhanced CSD profiles evoked by CF stimuli (70 dB, n = 7) after control injections of U0124 targeting the initial thalamocortical pathway. Vertical dashed lines indicate 20-ms time window expanded in panels at bottom. B, left: example of nicotine's effects on current sinks in layers 4 and 2/3 (inset shows points of measurement); arrowheads indicate onset of CF stimulus, 70 dB SPL. Right: example of thalamic injection site. Auditory thalamocortical pathway [superior thalamic radiation (STR)] is indicated by white arrow in image of a near-horizontal (“thalamocortical” plane) brain section (top). Fluorescence image of same section (bottom) reveals injection site in STR. A similar procedure was carried out in each animal to identify thalamic or cortical injections sites. AC, auditory cortex; MG, medial geniculate; LG, lateral geniculate; hip, hippocampus. C: group CSD profiles after thalamic injection of the MEK inhibitor U0126 reveal strong nicotine effects in upper layers but lesser effects on initial responses in layer 4. D: group CSD profiles after intracortical injection of U0126 reveal little effect of nicotine on supragranular responses but preserved enhancement of initial layer 4 responses.

Fig. 3.

Time course and peak magnitude of systemic nicotine's effects on CF-evoked current sinks in layers 4 and 2/3 after thalamic (A) or intracortical (B) microinjection of control drug (U0124) or MEK inhibitor (U0126). Measurements reflect thalamocortical input (“Layer 4” indicates sink onset latency and amplitude at 5 ms, cf. Fig. 2B) or intracortical activity (“Layer 2/3” indicates sink peak latency and peak amplitude). A: nicotine effects on latency (top) and amplitude (bottom) measures after thalamic injections of control drug (U0124, n = 7) or MEK inhibitor (U0126, n = 6). Arrows indicate time of thalamic microinjection (white arrows) and systemic nicotine (black arrows). Data normalized to average values over 30-min prenicotine period. Thalamic MEK inhibition blocked nicotine's effect on the layer 4, but not layer 2/3, current sink. Statistics: for time course data, asterisks (U0124) and crosses (U0126) indicate significant difference from prenicotine response (post hoc tests with Bonferroni correction after MANOVA; postnicotine data grouped in 14-min bins); for histograms, which show average significant nicotine effect in each condition, or 30-min average if no effect, asterisks above each bar indicate level of significance (*P < 0.05, **P < 0.01, ***P < 0.001), and asterisks between bars indicate difference between groups (U0124 vs. U0126, *P < 0.05, **P < 0.01). B: nicotine's effects on latency and amplitude measures after intracortical control injections (U0124, n = 6) or MEK inhibition (U0126, n = 6). Intracortical MEK inhibition blocked nicotine's effects on the layer 2/3 sink but not on the initial layer 4 sink. Conventions as in A.

Fig. 4.

Effects of nicotine and MEK inhibition are similar at all stimulus intensities tested. Graphs show effects of nicotine on current sinks in layer 4 (initial amplitude) and layer 2/3 (peak amplitude) evoked by CF stimuli at 3 intensities: acoustic threshold (10–20 dB), 40 dB, and 70 dB SPL. Top: thalamic injections: in control experiments (U0124), nicotine enhanced all sink amplitudes evoked by stimuli at each intensity (pre- vs. postnicotine, paired t-tests, P < 0.001–0.05, n = 7). Thalamic inhibition of MEK (U0126) blocked nicotinic enhancement of the initial layer 4 current sink at each intensity (pre- vs. postnicotine, paired t-tests, P > 0.05, n = 6) but did not prevent nicotine's enhancement of layer 2/3 current sink (P < 0.05). Bottom: intracortical injections: in control experiments (U0124) nicotine enhanced all tone-evoked sink amplitudes at each intensity (pre- vs. postnicotine, paired t-tests, P < 0.01–0.05, n = 5 or 6). Intracortical inhibition of MEK (U0126) blocked nicotinic enhancement of the peak layer 2/3 sink (pre- vs. postnicotine, paired t-tests, P > 0.05, n = 6) but did not prevent enhancement of the initial layer 4 sink (P < 0.01–0.05, n = 6).

Fig. 5.

Effects of nicotine and MEK inhibition on CSD profiles evoked by spectrally distant (nonCF) stimuli. A: in this example, systemic nicotine simultaneously enhanced CF-evoked current sinks (top) and reduced most activity evoked in response to nonCF stimuli (2 octaves below CF; bottom). CSD profiles show nicotine effect after control (U0124) intracortical injections; “Difference” profiles on right obtained by subtracting prenicotine from postnicotine CSDs (red colors indicate enhancement, blue colors indicate reduction). B: group data showing nicotine's effect on the largest-amplitude current sink evoked by nonCF stimuli in each animal, after microinjection of control drug (U0124) or MEK inhibitor (U0126) into the thalamus or cortex. In controls, nicotine significantly reduced the current sink peak amplitude (pre- vs. postnicotine, paired t-test, thalamic: P < 0.05, n = 6; cortical: P < 0.05, n = 6), while after MEK inhibition (U0126) nicotine had no effect (thalamic: P > 0.05, n = 5; cortical: P > 0.05, n = 6). *P < 0.05.

Fig. 6.

Distribution of cells with activated MAPK in A1 and dependence of activation on intracortical nicotinic acetylcholine receptors (nAChRs). A: horizontal tissue section with brightfield view (left) and fluorescence (right) showing location of drug injection site in A1. Scale bar, 1 mm. B: representative tissue sections showing immunolabeled cells in upper layers of A1 in response to acoustic (white noise) stimulation and systemic nicotine after intracortical microinjection of control solution [artificial cerebrospinal fluid (ACSF), left], dihydro-β-erythroidine (DHβE, 1 μM, middle) and methyllycaconitine (MLA, 10 nM, right). Scale bar, 50 μm. Control and DHβE examples are from opposite hemispheres in the same animal. DHβE prevented labeling of cell bodies and reduced somewhat the level of “background” immunofluorescence. C: depth distribution of immunolabeled cell bodies (mean number of labeled cell bodies per 100-μm depth) after control (ACSF) intracortical injections in A1 and after intracortical injection of either DHβE (n = 3 animals) or MLA (n = 3) in the opposite hemisphere of the same animals. *P < 0.05.