In order to determine how nicotinic regulation of primary auditory cortex (A1) varies across development, we measured tone-evoked current-source density (CSD) profiles at regular intervals before and after administration of nicotine in 44 mice ranging in age from postnatal day (P) 21 (~1 week after hearing onset) to adult. In each animal, we inserted a 16-channel silicon multiprobe into A1 to record local field potentials (LFPs) evoked by characteristic frequency (CF) and nonCF (two octaves below CF) stimuli, and constructed CSD profiles off line. To examine peri-adolescent trends, separately for male and female mice, we grouped data into five age ranges that, for convenience, we refer to as pre-adolescence (P21–25, 3 males, 4 females), early-adolescence (P26–30, 5 males, 5 females), mid-adolescence (P31–35, 3 males, 4 females), late-adolescence (P36–40, 4 males, 3 females) and adult (P70–100, 7 males, 6 females). The age range for adolescence is based on that previously established for the rat (P28–45; Spear, 2000
). We also examined sex differences within each of these age groups.
Tone-evoked cortical responses in A1 of adolescent mice
CSD profiles indicate the location of current sinks, i.e., putative sites of synaptic inputs. In young mice (P21–40), as in adults (Kawai et al., 2007
), CF tones generated one or two large current sinks in the upper layers (). The earliest-onset current sink in the middle layers occurred near the border of layers 3 and 4 (referred to as the ‘layer 3/4 sink’) (, green CSD trace). This current sink or the one immediately above it (, red CSD trace), was the largest amplitude current sink in each animal (the ‘CF-main sink’). We also observed a small amplitude sink with a short duration in the lower layers (the ‘layer 5/6 sink’; , blue CSD trace). Onset latencies of the layer 5/6 sink were shorter than those of the layer 3/4 sink in most animals (43 out of 44 animals, p
< 0.05, paired t
-test), suggesting that thalamocortical synaptic inputs first contact the lower layers of A1 before reaching layers 3/4. This CSD profile in mouse A1 is in general agreement with previous findings in other species, where the lemniscal thalamocortical input from the ventral division of medial geniculate nucleus (MGv) to layers 3/4 is rapidly amplified intracortically to generate large current sinks, while thalamocortical collateral inputs to layer 5/6 generate smaller current sinks (monkey: Müller-Preuss and Mitzdorf, 1984
; Steinschneider et al., 1992
; rat: Barth and Di, 1990
; Kaur et al., 2005
; Sakata and Harris, 2009
; gerbil: Happel et al., 2010
Fig. 1 Tone-evoked CSD profiles in adolescent mouse A1. LFPs and derived CSD profiles evoked in response to CF (A; 22 kHz) and nonCF (B; 5.5 kHz, both 60 dB SPL) tones in a P32 female mouse. In CF-evoked CSD traces (top, middle column), the layer 3/4 sink (green (more ...)
We also recorded responses to a spectrally-distant (nonCF) tone two octaves below CF at the same intensity. NonCF stimuli evoked the largest amplitude current sink (the ‘nonCF-main sink’) at the same cortical depth as CF tones did in most animals (26 of 30 pre-adolescent and adolescent animals; 10 of 13 adult animals; see below), or at an adjacent recording site 100 µm above or below the CF-main sink (). Compared to the CF-evoked sinks, the nonCF-evoked sinks exhibited significantly longer onset latencies with low magnitude (see below for quantification), remained elevated longer and spread more widely across the upper cortical layers (i.e
., multiple recording sites responding with similar sink magnitude) (). Direct thalamocortical inputs likely do not contribute to the nonCF-main sink, which more likely reflects intracortical synaptic activity (Metherate and Cruikshank, 1999
; Kaur et al., 2005
, Happel et al., 2010
Cortical processing matures early in adolescence
To determine whether CSD profiles change across peri-adolescent development, we analyzed the magnitude of CF-evoked current sinks. CSD traces in the upper layers were divided into three phases―Input, Early Intracortical, and Late Intracortical (labeled 1–3 in , right)―and areas under the traces for each phase were quantified. The Input phase is the first 3 ms of the layer 3/4 sink, capturing the linearly rising component of the sink prior to its peak. This phase reflects presumed thalamocortical inputs. The Early Intracortical phase (phase 2 in ) reflects the sink magnitude 3~20 ms after onset of the CF-main sink, which includes its peak response. In addition, we often observed a late component of current sinks with a secondary peak several tens of milliseconds later; this Late Intracortical phase (phase 3 in ) reflects sink magnitude 30~80 ms after onset of the CF-main sink. Comparison of the three phases showed no difference among the five age groups (, see figure legends for statistics), suggesting that the magnitude of tone-evoked thalamocortical and intracortical synaptic activities does not change beyond the third week in mouse A1.
Fig. 2 Peri-adolescent development of tone-evoked current sink magnitudes (A) and onset latencies (B). (A) Magnitudes of CF tone-evoked current sinks for Input (black), Early Intracortical (white), and Late Intracortical (gray) phases for five age groups, normalized (more ...)
We also analyzed the onset timing of tone-evoked responses. The onset latencies of the layer 3/4 sink, the layer 5/6 sink and the nonCF main sink in pre-adolescence were longer than those in the adult (). However, by early adolescence these same latencies had shortened to adult-like values. Consistent with this, the latency difference between the response onset in layer 3/4 and that in layer 5/6, which likely reflects conduction times in thalamocortical afferents, significantly decreased from preadolescence (4.7 ± 0.9 ms, n = 7) to adult-like values in early-adolescence (2.3 ± 0.5 ms, n = 10; preadolescence vs. early-adolescence p < 0.05, t-tests) (cf., 2.8 ± 0.5 ms, n = 7, in mid-adolescence, 2.4 ± 0.3 ms, n = 7, in late-adolescence, and 2.0 ± 0.4 ms, n = 13, in adults; all values significantly different from preadolescence). Finally, the difference between the onset latencies of the CF-evoked layer 3/4 sink and the nonCF-main sink showed a tendency to decrease from preadolescence (11.2 ± 1.2 ms) to adult-like values in early-adolescence (8.4 ± 2.1 ms; cf., 8.4 ± 2.8 ms in mid-adolescence, 8.7 ± 2.0 ms in late-adolescence), but this trend did not reach significance (p > 0.05, t-tests). The latency difference in preadolescence was, however, significantly different from that in adults (6.9 ± 1.4 ms, n = 13; p < 0.05, t-tests), suggesting again that intracortical processing is immature in preadolescence. Overall, these data indicate that some basic aspects of synaptic connectivity in the auditory pathway mature by early-adolescence.
Nicotine regulation of tone-evoked CSD profiles in A1 varies with age and sex
We next examined the effects of acute, systemic nicotine (0.7 mg/kg, i.p.) on tone-evoked CSD profiles. This nicotine dose had been determined in preliminary experiments to be low but effective, since lower doses (0.17 mg/kg and 0.35 mg/kg) did not alter tone-evoked responses. shows examples of CSD profiles for CF-evoked responses in young mice. Qualitatively, nicotine had little effect on a pre-adolescent female () but strongly enhanced responses in an early-adolescent female (; response amplitude color scale normalized to peak value in nicotine). To quantify nicotine effects, we analyzed current sink areas for the three phases of CF-evoked responses (labeled 1–3 in ), measured at regular intervals (~4 min) before and after systemic nicotine. shows the time course for the Input phase of the layer 3/4 sink (presumed thalamocortical input), separately for male and female animals in each of the five age groups. No nicotine enhancement was seen in pre-adolescents of either sex. In all other age groups, nicotine enhanced the Input phase, an effect that for most groups peaked within ~12 min and declined over ~30–60 min. In early-adolescent females, the nicotine enhancement was especially strong, peaked after the initial 12 min period and lasted ~50 min. The effect of nicotine on Input responses for each sex and age group is summarized in (bottom), which depicts current sink magnitude averaged over 12 min time windows (vertical lines in , top) up to ~48 min post nicotine. Nicotine enhanced Input phase beginning in early adolescence for both sexes (asterisks in ; pre- vs. post-nicotine, p < 0.05; paired t-tests). Enhancement in females peaked during early-adolescence (for 12–24 min: F(2,11) = 4.27, p = 0.042, 3-age (preadolescence, early-adolescence, adult) ANOVA; for 24–36 min, F(4,17) = 4.01, p = 0.018, ANOVA). Post-hoc t-tests show enhancement for early-adolescence and adults compared to preadolescence. The peak enhancement in females differed from the nicotine effect in males of the same age for post-nicotine intervals of 12–48 min (p < 0.05, t-tests). Nicotine enhancement in males first became apparent in early-adolescence, and increased in magnitude during late-adolescence (P36–40; 0–12 min: F(4,17) = 3.64, p = 0.026, ANOVA). Post-hoc t-tests show significant enhancement for all adolescent age groups and adults over preadolescence.
Fig. 3 Effects of systemic nicotine on tone-evoked CSD profiles become apparent during adolescence. (A) Nicotine had little effect on CSD profile in a pre-adolescent female (P22; CF stimulus, 21 kHz, 65 dB SPL), but enhanced CSD profile (B) in an early-adolescent (more ...)
Fig. 4 Nicotinic effects on CF-evoked CSD profiles peak during adolescence, earlier for females than males. (A) Time course of nicotine’s effect on the Input phase of layer 3/4 sink. Current sink areas are normalized to pre-nicotine baseline in males (more ...)
For those age groups and time windows with significant enhancement of the Input phase, we also analyzed nicotine effects on the layer 5/6 current sink (i.e., presumed thalamocortical collateral; sink area measured over 3 ms from sink onset). Unexpectedly, nicotine did not enhance the layer 5/6 sink during the 0–12 min post-nicotine interval for any age or either sex, and for later post-nicotine intervals (up to 48 min), enhancement occurred in only 25% of cases (7 of 28 12-min intervals: 12–48 min in P26–30 males; 24–48 min in P36–40 males and 24–48 min in >P70 females). These data indicate that there is little correspondence between nicotine effects in layer 3/4 and in layer 5/6.
The effects of nicotine on the Intracortical phases of CF-evoked responses also peaked during adolescence and showed striking sex differences. For the Early Intracortical phase (), enhancement in females peaked in early adolescence and disappeared in older adolescents and adults (P26–30, 12–24 min, F(4,17) = 3.93, p = 0.019, ANOVA; 12–24 min and 36–48 min, p < 0.05, post-hoc t-tests). In males, after a slight nicotinic suppression in pre-adolescence, enhancement occurred only in late adolescents and adults (pre- vs. post-nicotine, paired t-tests). Late adolescence was the only age group that differed from preadolescent males (post-hoc t-tests). The late-adolescent enhancement also differed significantly from that in females of the same age (for 12–24 min and 24–36 min ranges, p < 0.05, t-tests). For the Late Intracortical phase (), females again showed heightened nicotinic enhancement in early adolescence, which declined at later ages (for 12–24 min, F(4,17) = 6.84, p = 0.002, all-age ANOVA), whereas males showed nicotinic enhancement in mid-adolescence and thereafter (pre vs. post-nicotine, paired t-tests). The early-adolescent enhancement in females differed significantly from that in males of the same age (for 12–24 min and 36–48 min ranges, p < 0.05, t-tests).
Thus, nicotinic enhancement of CF-evoked current sinks begins, and peaks, during adolescence. Notable sex differences include the peak in nicotinic enhancement that occurs ~10 days earlier in females than in males, and the diminished intracortical effects in females at any age after early adolescence.
Nicotine effect on thalamocortical latency
Nicotine generally decreased the onset latency of the CF-evoked current sink in layer 3/4, and this effect occurred earlier in development than effects on sink magnitude (above). Pre-adolescent females, but not males, exhibited nicotine-induced reduction of onset latency (), even though current sink magnitude was not affected (). Peak effects of ~10% reduction in onset latency occurred during early-adolescence for females and mid-adolescence for males, with lesser but significant effects persisting into adulthood. The nicotine-induced decrease in onset latency persisted for ~30 min or more, post injection (data not shown). Thus, again, nicotine effects peaked during adolescence and occurred earlier for females than for males. Nicotinic effects on sink onset latency, but not magnitude, during pre-adolescence implies regulatory mechanisms with different development time courses and, possibly, different locations (e.g., subcortical and cortical, respectively).
Nicotinic regulation of nonCF tone-evoked current sinks
Nicotinic regulation of nonCF-evoked CSD profiles differed from that of CF-evoked responses. shows an example: in contrast to the uniform enhancement of CF-evoked response phases (as described above), nicotine reduced the early component of the nonCF-main sink and enhanced the late component. Reduction of the early component was observed in ~70% of animals across ages and in both sexes (males: 10/15 peri-adolescents, 5/7 adults; females: 11/15 peri-adolescents, 5/6 adults). For quantitative analysis, we divided the nonCF-main sink into an Early phase (0–20 ms after sink onset; labeled 4 in and ) and a Late phase (from 30 ms post onset to 80–100 ms; labeled 5 in and ). The time course of nicotine’s effect on the Early phase is shown in ; quantification of the first three time points after nicotine (shaded areas in ) indicated that nicotine either suppressed or did not affect the Early phase in all age groups and both sexes (). No enhancement of the Early phase was seen at any age. The most consistent, and long-lasting suppressive effects of nicotine were observed in adults of both sexes.
Fig. 5 Nicotine has a different effect on nonCF-evoked CSD profiles than on CF-evoked profiles. (A) An example of nicotine’s differential effects on CSD profiles evoked by CF vs. nonCF stimuli (P30, female). Nicotine enhanced all three phases of the (more ...)
Analysis of nicotine effects on the Late phase of the nonCF-main sink () shows a developmental pattern that more closely resembles regulation of CF-evoked responses (cf. ). Nicotinic enhancement of the Late phase peaked in early-adolescent females and late-adolescent males. Thus, CF and nonCF tones may recruit partially overlapping intracortical neural circuits, and nicotine may affect late-intracortical processing for each in a similar manner.
Intracortical injection of nAChR antagonists, DHβE or MLA
To determine whether the effects of systemic nicotine were due to nicotinic acetylcholine receptors (nAChRs) located in A1—and if so, which nAChR subtypes—we injected nicotinic antagonists directly into the cortex via a fluid port assembly fused to the 16-channel multiprobe. The fluid port opening was positioned between recording channels, approximately in layer 4. Experiments were done at ages of peak nicotine responsiveness for each sex, i.e., in late-adolescent males and early-adolescent females. Following baseline recordings of CF-evoked responses, 100 nl of artificial cerebrospinal fluid (ACSF) or drug was infused slowly (10 nl/min over 10 min; vertical shading in ) and recordings resumed for ~20 min before delivery of systemic nicotine (solid vertical lines in ). Cortical infusion of dihydro-β-erythroidine (DHβE, 1 µM at port), an antagonist at α4β2* nAChRs (Mulle and Changeux, 1990
; Whiting et al., 1991
; Alkondon and Albuquerque, 1993
; Wong et al., 1995
; Buisson et al., 1996
), blocked nicotine enhancement of the CF-evoked Early () and Late () Intracortical phases, but did not block enhancement of the Input phase (), in both sexes. Notably, in females only, DHβE alone (prior to nicotine administration) reduced the magnitude of the Intracortical sinks (), but not the Input sink, implying sex-specific actions of endogenous transmitter on intracortical processes. DHβE also blocked nicotinic enhancement of the nonCF-evoked Late phase (data not shown; normalized nicotine effect after ACSF: 1.56 ± 0.54 in males (n = 6), 1.27 ± 0.18 in females (n = 7), paired t-tests comparing pre vs. post-nicotine, p < 0.05; nicotine effect after DHβE, 1.14 ± 0.13 in males (n = 5), 0.90 ± 0.11 in females (n = 6); paired t-tests, p >0.05). Thus, cortical α4β2* nAChRs mediate enhancement of CF- and nonCF-evoked intracortical, but not subcortical, processes in both sexes, with females exhibiting evidence for similar regulation by endogenous ACh.
Fig. 6 Effects of intracortical DHβE injection on nicotinic regulation of CF tone-evoked current sinks. Time course of effects on Input (A), Early Intracortical (B), and Late Intracortical (C) current sinks are shown for males (P36–40; left column) (more ...)
Cortical infusion of methyllycaconitine (MLA, 10 nM at port), a specific antagonist at α7 nAChRs (Alkondon et al., 1992
; Drasdo et al., 1992
), had no effect on nicotinic enhancement of any CF-evoked current sink: Input, Early or Late Intracortical (). However, MLA did prevent nicotinic enhancement of the nonCF-evoked Late phase (data not shown; normalized nicotine effect after MLA, 0.83 ± 0.07 in males (n = 7), 1.00 ± 0.13 in females (n = 5); paired t
-tests comparing pre vs. post-nicotine, p > 0.05). Thus, α7 nAChRs and α4β2* nAChRs both regulate the nonCF-evoked Late current sink.
Fig. 7 Effects of intracortical MLA injection on nicotinic regulation of CF tone-evoked current sinks. Time course of effects on Input (A), Early Intracortical (B) and Late Intracortical (C) current sinks are shown for males (P36–40; left column) and (more ...)
These antagonist data suggest that α4β2* nAChRs located in A1 mediate enhancement of CF-evoked intracortical processing, while both α4β2* and α7 nAChRs mediate enhancement of nonCF-evoked intracortical processing. Administration of antagonists to A1 did not block enhancement of the CF-evoked Input current sink, likely due to dependence on subcortical nAChRs.