Nicotinic excitability in layer VI is reduced in α5−/− mice and eliminated in β2−/− mice
First, we examined the nicotinic depolarization of layer VI pyramidal neurons in WT, α5−/−, and β2−/− mice since these subunits are known to be the principal constituents of the nicotinic receptors expressed in this layer along with the α4 subunit. In the presence of atropine (200 nM) to block muscarinic receptors, stimulation with ACh (1 mM, 15 s) resulted in significantly different depolarization across the genotypes (F(2,49) = 47.89, p = 0.0001). As illustrated in , ACh depolarized layer VI neurons in WT mice to the greatest extent (n = 24), depolarized α5−/− neurons to a lesser degree (n = 21), and did not alter the membrane potential in β2−/− neurons (n = 7). Nicotinic excitation was sufficient to elicit action potentials in the majority of WT neurons, fewer α5−/− neurons (p < 0.05), and none of the β2−/− neurons.
Figure 1 Nicotinic excitability of layer VI pyramidal neurons is reduced in α5−/− and eliminated in β2−/−mice. A, Stimulation of only nicotinic receptors following blockade of muscarinic receptors by atropine (200 (more ...)
To examine nicotinic effects on the excitability of already-depolarized neurons, we injected neurons with positive current to elicit action potentials (~1 Hz) at baseline. The current required did not differ significantly across genotypes, consistent with the lack of significant difference in input resistance. As illustrated in , ACh increased the frequency of action potential firing in a genotype-dependent manner (F(2,31) = 11.78, p = 0.0002). This treatment resulted in a large increase in the firing frequency of WT (n =15) and α5 −/− neurons (n =14) but not β2 −/− neurons (n = 5).
Cholinergic excitation of layer VI cortical neurons primarily involves nicotinic receptors unless nicotinic drive is impaired
The striking differences across genotypes in the response to nicotinic stimulation raised the question of how layer VI neurons normally respond to ACh in the absence of atropine, when it can stimulate both nicotinic and muscarinic receptors together. This experiment is shown in (we use “cholinergic” to identify conditions where both nicotinic and muscarinic receptors are stimulated). In layer VI neurons from WT mice, we find that cholinergic depolarization does not differ greatly from nicotinic depolarization alone (cholinergic: 20.3 ± 1.3 mV, n = 23; nicotinic: 19.2 ± 1.0 mV, n = 24; t = 0.7, p = 0.5). By contrast, layer VI neurons from α5 −/− mice show greater depolarization when both nicotinic and muscarinic receptors are stimulated (cholinergic: 15.3 ± 1.1 mV, n = 26; nicotinic: 11.9 ± 1.1 mV, n = 21; t =2.2, p = 0.03). More strikingly in β2 −/− mice, the muscarinic component appears dominant since the response to nicotinic stimulation is negligible in comparison with the response to cholinergic stimulation (cholinergic: 4.3 ±0.6 mV, n =23; nicotinic: 0.1 ± 0.2 mV, n = 7; t = 3.8, p = 0.0005). Yet, there are significant differences in the magnitude of depolarization elicited by cholinergic stimulation across the genotypes (F(2,69) = 63.12, p = 0.0001). Compared to nicotinic stimulation alone, cholinergic stimulation had a significantly greater effect on the ability to elicit action potentials from rest in neurons from α5 −/− mice, and was able to elicit spiking in some neurons from β2 −/− mice.
Figure 2 Cholinergic (nicotinic and muscarinic receptor) stimulation depolarizes WT neurons most, but it increases spike frequency similarly across genotypes. A, Cholinergic stimulation depolarizes neurons to a different degree across genotypes (p <0.0001), (more ...)
In contrast to the differences in nicotinic effects in layer VI neurons across genotypes, cholinergic stimulation increased the frequency of action potential firing similarly across all three groups. As illustrated in , we observed no significant differences in the percentage increase in action potential frequency at the peak of the cholinergic response between layer VI neurons from WT (n =22), α5 −/−(n =17), and β2 −/−(n =24) mice (F(2,60) = 0.67, p = 0.5). The similar effects of ACh across genotypes on spike frequency cannot be attributed to a ceiling effect, since injected depolarizing current was consistently able to elicit faster spiking across the genotypes (paired t test, n =59, t = 20.7, p < 0.0001).
Notably, the time course of cholinergic effects was genotype dependent, with slower onset and substantially longer currents in layer VI neurons from β2 −/− mice. The 10–90% rise times of cholinergic responses in β2 −/− mice were slower for depolarization (WT: 13.3 ±1.1 s, α5 −/− : 14.7 ±0.6 s, β2 −/−: 25.8 ±2.9 s; F(2,45) =10.1, p =0.0002; p <0.01 for WT vs β2 −/−) and action potential frequency (WT: 14.6 ± 0.8 s, α5 −/−: 13.4 ± 1.3 s, β2 −/−: 20.7 ± 2.6 s; F(2,42) = 4.1, p = 0.02). Yet, the peak cholinergic effects lasted much longer in β2 −/− neurons, as shown by the τ (63% decay time) of depolarization (WT: 53.0 ± 8.3 s, α5 −/−: 53.4 ± 9.2 s, β2 −/−: 257.3 ± 22.8 s; F(2,45) = 48.3, p <0.0001; p <0.001 for WT vs β2 −/−) and action potential frequency (WT: 42.7 ± 4.4 s, α5 −/−: 56.1 ± 10.8 s, β2 −/−: 111.6 ± 25.5 s; F(2,38) = 5.5, p <0.01; p <0.01 for WT vs β2 −/−).
Muscarinic responses are enhanced in layer VI of α5−/− and β 2−/− compared to WT mice
The difference between responses to nicotinic-only and total cholinergic stimulation in layer VI neurons of α5 −/− and β2 −/− mice suggests potential plasticity in muscarinic-only ACh effects. To address this question, we tested the effects of muscarinic-only stimulation using ACh in the presence of nicotinic blockers (DHβE 3 μM, MLA 10 nM), as well as antagonists for the AMPA and NMDA glutamate receptors (CNQX 10 μM, APV 50 μM) to assess whether functional upregulation of muscarinic currents occurred in layer VI neurons lacking specific nicotinic receptor subtypes. Changes in membrane potential following muscarinic stimulation were significantly different across the genotypes (F(2,47) = 4.20, p = 0.02), as illustrated in . The elicited depolarization in layer VI neurons from WT mice were small (n = 17) compared to the larger depolarization in those from α5 −/−(n =16; t =2.6, p =0.01) and β2 −/−(n =17) mice. The muscarinic antagonist atropine (200 nM, 10 min) suppressed these responses in all genotypes (n = 11, t = 3.4, p = 0.006). Furthermore, selective antagonists for the excitatory M1 and M3 muscarinic receptors (pirenzepine, 500 nM; J-104129 fumarate, 50 nM) almost completely suppressed the depolarization (22 ± 17% of that seen previously; n = 5).
Figure 3 Muscarinic responses are enhanced in α5 −/− and β2 −/− compared to WT neurons. A, Stimulation of only muscarinic receptors on layer VI neurons following blockade of nicotinic receptors (3 μM DHBE, (more ...)
There were also significant genotype differences in the increase in firing frequency elicited by muscarinic stimulation (F(2,46) = 3.86, p =0.03). Muscarinic stimulation increased peak action potential firing in layer VI neurons from WT mice (n = 14), but this increase was significantly larger in layer VI neurons from both α5 −/−(n =16; t =2.1, p =0.04) and β2 −/−(n =19; t =3.3, p =0.002) mice. Again, these muscarinic responses were suppressed by atropine across the genotypes (n = 7, t = 6.5, p = 0.0006). Similarly, M1/M3 antagonists completely eliminated the increase in action potential firing (−5 ± 6% of that seen previously, n = 7).
With muscarinic-only stimulation, all three genotypes showed the longer 10–90% rise times and T characteristic of responses mediated only by G-protein-coupled receptors. These results contrast with the kinetics of the cholinergic responses where the fast, nicotinic responses dominated in the WT and α5 −/− mice, and the slow, muscarinic response dominated in the β2 −/− mice.
Hypocretin-responsive layer VI neurons show same pattern of muscarinic responses
The differences in the magnitude of the muscarinic responses across the genotypes raises the question of whether the layer VI neurons in the mice deleted for nicotinic subunits are indeed the same type of neurons as in WT mice. To address this question, we examined a pharmacologically identified subgroup of neurons within layer VI of cortex (Bayer et al., 2004
). In these neurons excited by hypocretin (100–300 nM, 1 min), which were recorded at 32°C (based on Bayer et al., 2004
), muscarinic responses showed the same pattern across genotypes as in the previous room temperature recordings of the general population of layer VI neurons (change in membrane potential: WT, 2.5 ± 0.6 mV, n
, 8.7 ±2.3 mV, n
, 7.9 ±1.4 mV, n
= 4.2, p
= 0.02; change in spiking frequency: WT, 341 ± 94%, n
, 763 ±160%, n
, 651 ±113%, n
= 10; F(2,25)
= 2.9, p
=0.07). These findings are consistent with the interpretation that layer VI neurons, which normally display large nicotinic responses to ACh, upregulate their muscarinic responses after loss or reduction of nicotinic excitation. To ascertain the specificity of this M1/M3 muscarinic upregulation, we compared the response to hypocretin across the genotypes since it is also mediated by a Gαq
-coupled receptor. However, these responses did not differ by genotype (F(2,82)
= 0.85, p
The balance of muscarinic to nicotinic excitation differs across genotypes
Differences in the responses to nicotinic and muscarinic stimulation across genotypes are illustrated in . Analysis of changes in membrane potential shows a strong interaction between the responses to nicotinic and muscarinic stimulation (F(2,96) = 41.75, p = 0.0001) (), with the genotypes varying greatly in the ratio of the muscarinic to the nicotinic response (WT: 0.2, α5−/−: 0.6, β2−/−: 61). As illustrated in , we observed a similar interaction in the increase in action potential frequency due to nicotinic and muscarinic stimulation (F(2,77) = 15.4, p =0.0001), with the genotypes varying substantially in the ratio of the muscarinic to the nicotinic response (WT: 0.5, α5−/−: 1.3, β2−/−: 4.1). There appears to be a negligible contribution of muscarinic receptors in WT mice, a balance between nicotinic and muscarinic contributions in α5−/−, and a predominant contribution of muscarinic receptors in β2−/− mice.
Figure 4 The balance of nicotinic to muscarinic excitation is shifted in α5 −/− and β2 −/− compared to WT neurons. A, There is a significant interaction in the degree of nicotinic and muscarinic depolarization across (more ...)