Loss of Cr results in decreased dendritic complexity of L5a pyramidalneurons
P0–P15 is a key time window for the experience-dependent development of the barrel cortex[22, 43-46]. Interestingly, we previously observed dynamic expression of Cr in L5a pyramidal neurons during this period[30]. We then employed Cr KO mice to explore the role of CR in the development of L5a pyramidal neurons. We first confirmed the disruption of CR in the developing barrel cortex at P8 by immunohistochemical staining (Fig. 1A) and Western blotting (Fig. 1B). Since an inducible Cre recombinase (CreER) was targeted to the Cr locus, the AI9-RFP line was next introduced to trace CR+ L5a pyramidal neurons (Fig. 1C). We analyzed the dendritic morphology of RFP+ neurons at P20 and found that control mice exhibited a typical morphology of L5a pyramidal neurons with an apical dendrite extending to L1 and several basal dendrites stretching to the sides and deeper[20]; however, the complexity of basal dendrites was significantly reduced in Cr KO mice (Fig. 1D). We measured the dendritic length of RFP+ neurons and detected significant reductions in the apical, basal and total dendritic lengths in Cr KO mice compared with those in control mice (Fig. 1E). The apical dendritic length decreased by approximately 25%, and the basal and total dendritic lengths decreased by approximately 35%. We also observed a marked decrease in dendritic complexity in Cr KO mice (Fig. 1F). These results suggest that ablation of Cr led to abnormal dendritic development of L5a pyramidal neurons.
Impaired organization of barrels and abnormal formation of the barrel wall in Cr KO mice
To investigate the contribution of L5a to the development of the barrel cortex, we carefully profiled the morphological changes of L5a pyramidal neurons during the time window of P0–P30 by double immunostaining of CR and vGlut2, a marker commonly used to label cortical barrels[47]. At P4, the CR+ L5a pyramidal neurons displayed a serrated pattern of alignment underneath the barrels, with their dendrites starting to extend towards the intervals between barrels (Fig. 2A). From P8 to P15, the serrated alignment pattern of the CR+ L5a cell bodies became more distinct, and more dendrites were observed in the intervals and formed septa-like structures (Fig. 2B, C)[48]. From P15–P30, as the maturation of the barrel cortex proceeded, the expression level of Cr in L5a gradually decreased. Until P30, the barrels in L4 were well developed (Fig. 2D). The expression of Cr was decreased to a very low level both in L5a cell bodies and in septa-like structures (Fig. 2D).
L4 is the main recipient layer of the whisker-barrel cortex system, whereas L5a is the main output layer, and the two layers are monosynaptically connected[2, 6, 49]. The connections of L4 to L5a form a “short circuit” between afferent signals to the cortex and efferent signals that leave the barrel cortex from L5a[8]. To assess the effects of abnormal L5a dendrite on the development of barrel/septum microcircuitry, we examined the barrel cortex at P8. As shown in Figure 2E, in contrast to the regularly organized barrels at L4 in control mice, we found barrels with disrupted organization in Cr KO mice, with some barrels deviating from their intrinsic level.
During the development of the barrel cortex, thalamocortical axons (TCAs) reach L4 at around P1, and clusters of axons can be detected in the barrel cortex at P3, reflecting an increase in the complexity of axonal endings[22, 50]. Subsequently, L4 spiny stellate cells reorganize around TCA axonal clusters to form barrel walls[45]. At P7, barrel walls are clearly visible. L5a pyramidal neurons are reported to preferentially connect with barrel walls, but whether L5a is required for the formation of barrel walls remains unclear. In our control mice, consistent with previous reports, at P8, L4 spiny stellate cells gathered to form a clear barrel wall between barrels, as shown by Nissl staining. Interestingly, we found that barrel wall-like structures were completely missing in the entire Cr KO barrel cortex (Fig. 2F). These data indicate that in addition to the TCAs endings, the L5a CR+ dendritic tree is also very important for the organization of barrels and the formation of barrel walls.
The ratio of barrel/septum size is decreased after Cr deletion
To further investigate the effect of Cr deletion in L5a on the maturation of the barrel/septum microcircuit, we explored the morphology of the barrel field by immunostaining of vGlut2 at P30 when barrel cortex development was completed[43]. Control mice exhibited well-arranged barrels (Fig. 3A). Cr KO mice still displayed a disrupted arrangement pattern; furthermore, the individual barrel was more dispersed and indistinct (Fig. 3A). Nissl staining showed that at P30 in control mice, barrel walls clearly formed a “thin” wall. In Cr KO mice, barrel walls generally formed and were positioned in the expected area; however, it seemed that L4 spiny stellate neurons were distributed more broadly in Cr KO mice than in control mice (Fig. 3B), consistent with the widened intervals viewed by vGlut2 staining (Fig. 3A). This phenotype was further confirmed by DAPI staining (Fig. 3C).
We prepared flattened tangential cortical slices to examine the holonomic barrel field by double immunostaining of vGlut2 with DAPI[47, 51], and the entire barrel field was reconstructed and analyzed using ImageJ software (Fig. 3E, F). We calculated and compared the size of the entire barrel field and found that there was no remarkable difference in the size of the entire barrel field in Cr KO mice compared with control mice (Fig. 3G). We then explored the size of individual barrels and septa within the major mystacial whisker barrels (from A2 to E4). Our results showed that the barrel/septum ratio was significantly increased (Fig. 3H), indicating that the individual barrel columns were contractible and that the septa columns were correspondingly outstretched. This change was further confirmed by the decreased barrel/ (barrel + septum) ratio (Fig. 3I) and increased septum/ (barrel + septum) ratio (Fig. 3J). Taken together, these results indicate that CR is required for the normal formation of barrels and septum in the barrel cortex due to its regulation of the development of L5a pyramidal neuron dendrites.
Both membrane excitability and excitatory synaptic transmission are increased in Cr KO L5a pyramidal neurons
As an intrinsic Ca2+ buffer, CR plays an important role in the regulation of neuronal excitability and neurotransmitter release[25, 52]. We next performed whole-cell patch-clamp recording on RFP+ neurons using acute brain slices to investigate the effect of Cr deletion on the maturation and excitability of L5a pyramidal neurons. We investigated intrinsic cell electroresponsiveness through current-clamp recordings. The resting membrane potential was recorded immediately after perforating the cell membrane and was found to be comparable between control and Cr KO neurons (Fig. 4A)[37]. However, a significant decrease in the action potential current threshold was detected after Cr deletion (Fig. 4B). Moreover, the mean input resistance and amplitude of the AHP were significantly higher in Cr KO neurons than in control neurons (Fig. 4C, D). Although the action potential half-width showed a slight tendency to decrease, there was no statistical significance between control and Cr KO neurons (Fig. 4E). Unsurprisingly, in response to a series of suprathreshold depolarizing current injections with amplitudes ranging from −50 to 300 pA (with an increment of 50 pA), the number of action potentials recorded from Cr KO neurons was significantly higher than that of the control neurons (Fig. 4F, G).
To assess the functional consequences of Cr deletion on synaptic transmission, we next tested the basic synaptic transmission of L5a pyramidal neurons and compared them with the amplitude and frequency of spontaneous miniature EPSCs (mEPSCs) (Fig. 4H)[53]. BMI and TTX were applied to block GABA receptor-mediated inhibitory currents and action potential-dependent synaptic transmission, respectively. We found that the mean amplitude of mEPSCs was unaffected (Fig. 4I), while the mean frequency was strongly increased in Cr KO neurons compared with control neurons (Fig. 4J). Collectively, these results suggest that loss of Cr leads to increased membrane excitability and excitatory synaptic transmission of L5a pyramidal neurons.
Cr KO mice exhibit pronounced exploratory behavior deficits
Rodents use their whiskers as multipurpose organs for behaviors ranging from object detection, including object localization, judgment of shape and texture, and discrimination, to movement coordination, such as detecting distance and motor coordination[1-3, 6]. Since L5a plays a crucial role in integrating information resources and coordinating the movement of the whiskers[9, 30], we performed a series of behavioral tests related to the barrel cortex. First, we conducted an open-field test to evaluate spontaneous motor ability and found that the mean velocity and the total distance traveled within a 30-min duration were comparable between Cr KO and control mice (Fig. 5A, B), indicating that locomotor activity was unaffected. However, within the first five minutes, the time spent in the center zone and the frequency of entering the center zone were obviously decreased in Cr KO mice (Fig. 5C, D). This result suggested two possibilities: increased anxiety or decreased desire to explore.
To further examine the level of anxiety, we conducted elevated O-maze and elevated plus maze tests[34, 54]. As shown in Figure 5E and F, there were no significant differences in the time spent in the open arms between the control and Cr KO mice, demonstrating that loss of Cr had no influence on anxiety levels. To test the exploratory behaviors, a novel object was introduced to the center of the open field after the mice became fully familiar with the environment[40]. As shown in Figure 5G, the trajectories of control mice were more concentrated in the central area around the novel object than those of Cr KO mice. Although the frequency of entering the center zone was comparable (Fig. 5H), Cr KO mice spent significantly less time investigating the object than control mice (Fig. 5I). Taken together, these data indicate that deletion of Cr in L5a has no effects on spontaneous motor ability but impairs exploratory behaviors.
Cr KO mice display defects in whisker-associated tactile sensation behavior
To further investigate exploratory behavior deficits, we performed an “S” curve test. This test is designed to simulate a narrow nocturnal environment. When the mice were introduced into an unfamiliar curve, they explored the curve with their whiskers while moving forward[6]. Cr KO mice spent more time going through the “S” curve to reach their destination (incubation) than the control mice (Fig. 6A); moreover, Cr KO mice also spent more time exploring the opening area near the exit before leaving the curve (Fig. 6A).
To measure tactile responses after Cr deletion in L5a, we performed a sticky paper test[39, 41]. This test is designed to measure tactile responses to adhesive paper stuck on the palmar surface of mouse hind paws. Mice were required to detect the tape with the help of whiskers and then to remove it. The results showed that Cr KO mice spent more time finding the tape (incubation); accordingly, they spent more time removing the tape from their hind paws (Fig. 6B).
Next, we examined accurate recognition ability through a texture discrimination test[1, 55, 56]. Mice were first allowed to move freely to explore different textures in the open field box. Ten minutes later, two glass bottles, one of which was wrapped in sandpaper, were placed in the opposite corners of the box. The time that mice stayed in each corner in the next ten minutes was assessed. We found that the control mice spent more time in the corner where the unwrapped glass bottle was placed, while the Cr KO mice did not exhibit any preference for the two corners (Fig. 6C), suggesting that the texture discrimination of Cr KO mice was impaired. We also assessed the time that Cr KO mice stayed with two identical bottles and found that they had no position preference (Fig. 6D, E).
Finally, we performed the gap crossing test[39, 42], a specific test to detect the distance perception ability of cortical whiskers. It consists of a series of trials requiring the mice to accurately measure gaps of variable distances and to cross the gaps to reach a safe platform (Fig. 6I). The average gap distance crossed in Cr KO mice was significantly shorter than that in control mice (Fig. 6F). Weight data confirmed that the disparity was not related to body size (Fig. 6G). As the gap distances increased, the percentage of mice that were able to cross the gap gradually decreased (Fig. 6H). Collectively, our data suggest that tactile sensation was impaired in Cr KO mice.
Deletion of Cr impairs social novelty preference
In addition to discriminating simple tactile properties, whiskers of rodents also play an important role in social interaction[3, 57]. The three-chamber test showed that neither control nor Cr KO mice displayed a preference for either of the two empty chambers during the habituation phase (Fig. 7A), and they all spent more time with the first stimulating mouse (Fig. 7B), indicating that the loss of Cr had no effect on social ability. In the social novelty test, control mice displayed a preference for the novel mouse, while Cr KO mice did not show any preference (Fig. 7E), suggesting that loss of Cr impairs social novelty preference. In summary, loss of Cr affects the normal formation of the barrel and septa columns in the barrel cortex so that tactile information cannot be processed properly, leading to related behavioral deficits.