A critical period for nicotine-induced disruption of synaptic development in rat auditory cortex

Abstract

Cholinergic markers in the middle layers of rat auditory cortex are transiently upregulated during the second postnatal week, at which time alpha 7 nicotinic acetylcholine receptors (nAChRs) selectively regulate NMDA receptor (NMDAR)-mediated EPSPs. To investigate the developmental role of this regulation, we determined whether manipulating nAChR function at specific times during the first 4 weeks after birth could alter subsequent neuronal function. Rat pups were injected twice daily with nicotine (1 or 2 mg/kg) or saline during approximately the first, second, or fourth postnatal week (i. e., before, during, or after the peak upregulation of nAChRs). Glutamate EPSPs and intrinsic membrane properties were measured during whole-cell recordings from visually identified pyramidal neurons in layers II-IV of brain slices prepared at least 15 hr after the last injection. Chronic nicotine exposure (CNE) had little effect on intrinsic membrane properties and during week 1 or 4 did not affect synaptic function. However, CNE during week 2 resulted in EPSPs with long durations, multiple peaks, and enhanced NMDAR components. These changes remained significant even 10 d after CNE. Rapid application of nicotine, which in control neurons selectively enhances NMDAR EPSPs during week 2, produced only weak effects after CNE. Receptor binding studies showed that CNE-induced EPSP alterations occurred in the absence of altered alpha 7 nAChR numbers or agonist binding affinity. Thus, altered stimulation of nAChRs by CNE during week 2, but not before or after, disrupts the development of glutamate synapses in rat auditory cortex.

Fig. 1.

Normal development of EPSPs in pyramidal neurons of layers II–IV of auditory cortex. A,Maximal subthreshold EPSPs recorded in neurons from control P8–26 rats. B, Data on maximal subthreshold EPSPs grouped into four age ranges (values in Table 1). Bi, Latency to peak decreased over development. Bii, EPSP amplitude increased with age. Biii, EPSP duration, measured at1/3, 1/2, and 2/3 peak (PK) (A, arrows), decreased with age.

Fig. 2.

CNE group I: nicotine treatment for approximately the first postnatal week does not affect glutamate EPSPs.Ai, Representative EPSP in a control P10 neuron;V_m −66 mV. Aii, Nicotine injections from P1–9 resulted in an EPSP that was similar to controls; P10 neuron, V_m −61 mV. B, CNE group I EPSP durations (measured at 1/3,1/2, and 2/3 Pk) did not differ from age-matched controls (Table 1; p > 0.10).

Fig. 3.

CNE group II: nicotine treatment during postnatal week 2 alters EPSPs. Ai, Representative EPSP from a control P13 neuron; V_m −73 mV.Aii, Nicotine injections (1 or 2 mg/kg) from P8 through P11–14 produced EPSPs that were larger in duration and magnitude and had multiple peaks on their descending phase (asterisks). Top trace,V_m −66 mV; middle trace,V_m −66 mV; bottom trace,V_m −77 mV. B, The time of occurrence of miniature fluctuations is plotted for 50 msec before and 260 msec after the stimulus (10 msec bins; comparisons using Fisher's PLSD). Bi, In 22 control neurons (P9–15), the number of events occurring before or 60 msec after the stimulus did not differ (p > 0.10). Bii, For 22 CNE group II neurons (1 and 2 mg/kg doses combined), the number of events within 60 msec after the stimulus was greater than during either the prestimulus period or the same 60 msec poststimulus period in control neurons (p > 0.10). C, EPSP durations recorded from CNE group II neurons (measured at $\frac{1}{3}$, ½, and $\frac{2}{3}$ peak; histogram shading as in Fig. 2) were longer than age-matched controls (Table 1; p < 0.01). The increase in duration was similar for the two nicotine doses (p > 0.10).

Fig. 4.

CNE group III: EPSP alterations produced by CNE treatment during week 2 last for at least 1 week. Ai, Representative EPSP from a control P24 neuron;V_m −80 mV. Aii, EPSPs recorded 7 d after nicotine injections from P8–16. Top trace, V_m −68 mV; bottom trace, V_m −79 mV. B, CNE group III EPSP durations (measured at1/3, 1/2, and 2/3 Pk) were longer than age-matched controls (Table 1; p < 0.01).

Fig. 5.

CNE group IV: nicotine treatment during postnatal week 4 does not affect EPSPs. Ai, Representative EPSP from a control P26 neuron; V_m −78 mV.Aii, CNE from P20 through P23–25 resulted in EPSPs that in general were similar to age-matched controls. Top trace, V_m −79 mV. However, in two of nine neurons, maximal subthreshold EPSPs had enhanced durations, whereas lower intensity stimuli produced control-like durations (bottom traces, V_m −75 mV).B, CNE group IV EPSP durations did not differ from age-matched controls (Table 1; p > 0.10).

Fig. 6.

NMDARs mediate the enhanced EPSPs and stimulus-evoked miniature events produced by CNE during week 2.Ai, CNE group II: APV (50 μm) reduced the long-duration and multiple-peaked EPSP in a P13 neuron;V_m −70 mV. Aii, The area of the non-NMDAR EPSP (the EPSP in APV) in CNE group II neurons did not differ from control (control, 619 ± 104 mV · msec,n = 7; CNE II, 557 ± 99 mV · msec,n = 15; p > 0.10). In contrast, the area of the NMDAR EPSP (pre-APV area minus area in APV) was twice as large in CNE group II neurons as in control neurons (control, 493 ± 102 mV · msec; CNE II, 994 ± 117 mV · msec; p < 0.01). Aiii, APV reduced the stimulus-evoked increase in miniature events (p < 0.01, n = 11 neurons; for each neuron, miniature events were counted in three consecutive EPSPs, 10 sec interstimulus interval). Bi, CNE group III: APV decreased EPSP area in a P23 neuron;V_m −68 mV. Bii, The non-NMDAR EPSP area was similar in control and CNE group III neurons (control, 344 ± 44 mV · msec, n = 7; CNE III, 258 ± 69 mV · msec, n = 5;p > 0.10). However, the NMDAR EPSP area was more than twice as large in CNE group III neurons as in control neurons (control, 180 ± 48 mV · msec; CNE III, 476 ± 131 mV · msec; p < 0.05). Ci, CNE group IV: APV produced a small decrease in the late EPSP; P25 neuron,V_m −79 mV. Cii, The non-NMDAR EPSP area was similar in control and CNE group IV neurons (control, 344 ± 44 mV · msec, n = 7; CNE IV, 360 ± 49 mV · msec, n = 9;p > 0.10). Similarly, the NMDAR EPSP area did not differ between control and CNE group IV neurons (control, 180 ± 48 mV · msec; CNE IV, 306 ± 40 mV · msec;p > 0.10).

Fig. 7.

Effect on EPSPs of acute, rapid nicotine application to control and CNE group II neurons. All data are from P13 neurons. A, In a control neuron, pressure-ejected nicotine enhanced the late portion of the EPSP;V_m −70 mV. In a CNE group II neuron, nicotine increased the EPSP magnitude only slightly;V_m −62 mV. Each trace is an average of five responses at 10 sec interstimulus intervals.B, Rapid application of nicotine (25 μm, 10–40 msec, 20 psi) significantly enhanced EPSPs in control neurons (n = 17; paired t test,p < 0.02), but not CNE group II neurons (n = 8; p > 0.2).C, EPSP enhancement for the 25 neurons whose responses contribute to the histograms in B is shown for neurons that had either an increase (●) or no effect (○) in response to rapid nicotine application. For control neurons, nicotine increased EPSP areas 34–289% (mean 114%). In contrast, increases for CNE group II neurons were only 30–34% (mean 32%). Dashed linesindicate statistical significance at the 0.05 level (see Materials and Methods). Arrows indicate measurements for responses shown in A.