The α5 Subunit Drives the Developmental Peak in Layer VI Nicotinic Currents
To examine the role of nAChR α5 accessory subunits on electrophysiological responses in mPFC layer VI neurons during postnatal development, we prepared acute brain slices from male mice that were either wild-type (α5+/+
) or constitutively deleted for the α5 subunit (α5−/−
). We elicited nicotinic currents in response to 1 mmol/L ACh (in the presence of 200 nmol/L atropine) at four postnatal ages (). Presence of the nAChR α5 subunit had striking consequences on the magnitude of the nicotinic currents elicited at each age and also on the developmental peak in nicotinic currents in mPFC layer VI neurons [; F
(1,166) = 80.72, p
< .0001 for the genotype effect]. While neurons from wild-type mice showed a developmental peak during week 3 that was significantly greater than each of the other ages examined for wild-type mice (p
< .05 for each comparison), neurons from α5−/−
mice showed consistently lower nicotinic currents with negligible developmental changes. Exemplary nicotinic inward current traces for each experimental group are shown in .
Figure 1 Changes in the excitation of medial prefrontal layer VI pyramidal neurons by acetylcholine during postnatal development are dependent on the nicotinic receptor α5 subunit. (A) The peak inward current response to 10-second bath application of 1 (more ...)
The α5 Subunit Underlies Normal Developmental Changes in Layer VI Neuron Morphology
The timing of the developmental peak in mPFC layer VI neuron nicotinic currents is of great interest because nicotinic stimulation can modulate the growth and retraction of neurites for cultured neurons (14
) and because the observed peak coincides temporally with a critical period of circuit refinement for the developing rodent cerebral cortex (12
). We next tested whether nicotinic signaling influences the in vivo development of mPFC layer VI neurons by examining dendritic morphology in wild-type and α5−/−
mice at postnatal week 3 (near the beginning of the critical developmental period) and in young adulthood. For these experiments, we included Neurobiotin in the patch pipettes, which diffused throughout the neurons while we performed electrophysiological recordings, and then used multiphoton imaging to visualize the filled neurons in situ within fixed brain slices. As demonstrated in , in week 3, almost all layer VI neurons from mice of both genotypes had long apical dendrites that reached into layer I of the mPFC (9 of 11 neurons for wild-type mice and 11 of 12 neurons for α5−/−
= .6). This finding in the mPFC of young mice (mean age of P17 ± 1 day) contrasts greatly with previous morphological studies in sensory and motor cortices across development, where layer VI neuron apical dendrites predominantly terminate within the midlayers of the cortex (reviewed recently in [20
]). More consistent with this morphological work in other cortical areas, we found in our young adult wild-type mice (mean age of P77 ± 4 days) that the majority of mPFC layer VI neurons also terminate before layer I (11 of 20 neurons, p
= .07 compared with week 3 wild-type neurons), suggesting that a retraction of apical dendrites normally occurs for a subset of mPFC layer VI neurons during postnatal brain maturation. Importantly, we did not observe this maturational change in layer VI neurons of young adult α5−/−
mice (mean age of P87 ± 5 days); almost all neurons (12 of 13) retained the immature phenotype with apical dendrites reaching into layer I (; p
= 1.0 compared with week 3 α5−/−
= .009 compared with adult wild-type neurons). These results suggest that ontogenic changes in the morphology of mPFC layer VI neuron apical dendrites during postnatal development depend on the presence of the nicotinic α5 subunit. Exemplary z-projection photomicrographs of a typical adult wild-type neuron and a typical adult α5−/−
neuron are shown in .
Figure 2 Qualitative analysis of medial prefrontal layer VI pyramidal neuron apical dendrites. Pie charts in (A) show the proportion of neurons from wild-type (WT) mice and nicotinic receptor α5 subunit knockout (α5−/−) mice at (more ...)
To investigate the developmental changes occurring in wild-type neurons and the striking genotype effect in adulthood, we performed a detailed quantitative analysis of neuronal morphology by tracing and analyzing each neuron in three dimensions using Neurolucida software. Representative z-projections of traced neurons are shown in . Results from three-dimensional Sholl analyses are shown in , where we found significant effects of genotype on neuronal complexity not only in adulthood (; p < .0001) but also at week 3 (; p = .01), suggesting that the influence of α5-containing nicotinic receptors on apical dendrite morphology is already in progress at this age. Analysis of wild-type neurons only found a significant effect of age on apical dendrite complexity, with the main difference appearing within approximately the distal 300 μm (500–800 μm) from the soma (; p = .01), whereas comparison of Sholl plots for α5−/− neurons only found no effect of age (; p = .6), providing further evidence that developmental changes in apical dendrite morphology are dependent on the α5 nAChR subunit. Further analysis of traced neurons has also found genotype effects on total apical dendrite length (; p = .02), the distance from the soma to the most distal apical dendrite terminal (; p = .03), and the total number of apical dendrite terminals (; p = .03).
Representative z-projections of traced neurons are shown for wild-type and nicotinic receptor α5 subunit knockout (α5−/−) mice at postnatal week 3 and in adulthood.
Figure 4 Quantitative analysis of medial prefrontal layer VI pyramidal neuron apical dendrites. (A–D) Three-dimensional Sholl analysis measuring the number of dendrite intersections at concentric spheres of varying distance from the soma for neurons from (more ...)
The proposed α5 subunit-dependent retraction appears to be specific for the apical dendrite tree. In these same neurons, the distance between the soma and the most distal basal dendrite terminal was longer in wild-type neurons compared with α5−/− neurons: at week 3, 285 ± 34 μm (n = 8) for wild-type neurons and 206 ± 10 μm (n = 9) for α5−/− neurons; at adulthood, 289 ± 27 μm (n = 18) for wild-type neurons and 241 ± 21 (n = 12) for α5−/− neurons; F(1,43) = 5.27, p = .03 for effect of genotype. Yet, the mean number of basal dendrite trees per neuron was significantly lower in wild-type neurons compared with α5−/− neurons: at week 3, 4.9 ± .4 (n = 8) for wild-type neurons and 5.6 ± .4 (n = 9) for α5−/− neurons; at adulthood, 4.2 ± .2 (n = 18) for wild-type neurons and 5.3 ± .3 (n = 12) for α5−/− neurons; F(1,43) = 8.89, p = .005 for effect of genotype.
We thoroughly compared electrophysiological and morphological properties of traced neurons in this study and found that neuronal input resistance correlated negatively with a number of measures for neuronal size. For all traced neurons, there were significant correlations between input resistance and: soma volume (r = −.45, p = .002), total dendrite volume (r = −.41, p = .006), overall neuron volume (soma + dendrites, r = −.35, p = .02), and total dendrite length (r = −.35, p = .02). Spike amplitude in these neurons correlated positively with soma volume (r = .37, p = .01); however, there were no other significant correlations between spike amplitude or resting membrane potential and any measure of neuronal morphology.
Loss of the α5 Subunit Does Not Alter the Morphology of the Medial Prefrontal Cortex
We next performed a control experiment to test whether genetic deletion of the nAChR α5 subunit in our mouse model alters the morphology of the mPFC or the distribution pattern for neurons expressing α4* nAChRs (presumably α4β2* nAChRs) within layer VI of the mPFC. We performed this experiment by crossbreeding α5−/−
mice with a knockin mouse line in which all nAChR α4 subunits are tagged with the YFP motif (18
) to create experimental animals having YFP-labeled α4 subunits that were either homozygous wild-type for the α5 subunit or null for the α5 nAChR subunit. Immunohistochemistry for the exogenous YFP motif and immunostaining analysis were performed in 400 μm thick coronal sections as described in Supplement 1
and in a previous study (5
As shown in , there was an intense band of immunostaining for α4-YFP within layer VI of the mPFC that was more prominent at week 3 compared with adulthood. However, there were no effects of α5 subunit genotype on mPFC morphology or α4-YFP immunostaining pattern at either age. The mean width of the mPFC, as measured from the layer VI/white matter boundary and the pial surface medial to layer I, was not affected by age [F(1,29) = .00; p = 1.00] or by α5 genotype [F(1,29) = .03, p = .87]. Values at week 3 were 871 ± 20 μm (n = 11) for wild-type and 841 ± 16 μm (n = 11) for α5−/− mice and at adulthood were 837 ± 35 μm (n = 5) for wild-type and 874 ± 18 (n = 6) for α5−/− mice. The width of the layer VI immunoreactive band was lower at week 3 compared with adulthood: at week 3, 209 ± 11 μm (n = 11) for wild-type and 188±10 μm (n=11) for α5−/− mice; at adulthood, 237±11 μm (n=5) for wild-type and 239 ± 5 μm (n = 6) for α5−/− mice; F(1,29) = 12.66, p = .001, but was not affected by genotype [F(1,29) = .68, p = .42]. The percentage of neurons within layer VI expressing α4-YFP was slightly greater at week 3 compared with adulthood: at week 3,75.9 ± 1.3% (n = 11) for wild-type and 74.3±2.3% (n = 11) for α5−/− mice; at adulthood, 70.3±1.8% (n = 5) for wild-type and 69.2±3.0% (n = 6) for α5−/− mice; F(1,29) = 5.52, p = .03), but again was not affected by α5 genotype [F(1,29) = .34, p = .57]. We also examined the expression of α4-YFP positive neurons throughout the remainder of the mPFC (layers I–V) and found no effect of age or genotype (data not shown). These results demonstrate that the observed α5 subunit-dependent developmental changes in mPFC layer VI neuron apical dendrite morphology occur in the absence of overt changes to the morphology of the mPFC itself or to the distribution pattern of this α4* nAChR-expressing neuronal population.
Figure 5 Immunohistochemistry for α4* nicotinic acetylcholine receptors within the medial prefrontal cortex. (A) Low magnification photomicrographs within medial prefrontal slices showing immunohistochemical staining for nicotinic receptor α4 subunits (more ...)