remains intact, developed normally. In the SI cortex, however, thala-mocortical afferents (TCAs) formed only small, rudimentary patterns (as visualized with histochemical and immunohistochemical markers) in regions corresponding to the representation of larger whiskers. Fur-thermore, layer IV granule cells failed to develop barrels even in regions where there were rudimentary patterning of TCAs (Iwasato et al., 2000).
These results demonstrated critical roles of cortical NMDAR-mediated activity in the patterning of both presynaptic component (TC axonal termini) and postsynaptic component (layer IV granule cell bodies and dendrites). Rudimentary patterning of TCAs in CxNR1KO mice might be due to NMDAR activation in cortical GABAergic cells, which escaped the NR1 deletion. This is a possibility that remains to be experimentally tested. Another interesting observation in these mice was that neonatal whisker-induced structural plasticity followed the same time course as in wild type mice, and rudimentary cortical patterns could be altered up to postnatal day 3 but not thereafter (Datwani et al., 2002a).
An important caveat to all these reports underscoring the involvement of NMDARs in patterning of developing somatosensory pathways (as well as the vast majority of other studies documenting barrel cortex phe-notypes in other lines of mutant mice) is that the morphological assays have been done at a gross microscopic level using a variety of histolog-ical and immunohistochemhistolog-ical markers for barrel patterns (Erzurumlu and Kind, 2001; L ´opez-Bendito and Moln´ar, 2003). If NMDARs serve as coincidence detectors between pre and postsynaptic elements, how is presynaptic terminal and dendritic differentiation of pattern forming el-ements in somatosensory centers affected in mice with various types of genetic alterations of the NMDAR function? What are the downstream signaling mechanisms that allow detection of patterning of presynap-tic inputs by postsynappresynap-tic cells, and consolidation of patterns at both sites? To date there is very little documentation of fine structural de-fects in mice with reported barrel pattern dede-fects. Below we review our recent results on axonal and dendritic differentiation in mice with NR1 mutations.
NMDAR-Mediated Differentiation of Trigeminal
subsequently to the barrel cortex), but rudimentary patterns can be seen in portions of the spinal trigeminal nucleus, subnucleus interpolaris (SpI) only in NR1KD mice. Peripheral trigeminal (IO) axons invade the developing whisker fields around embryonic day (E) 10 in the mouse, and their central counterparts lay down the trigeminal tract in the brain-stem by E13 (Stainier and Gilbert 1990; 1991). Once the tract extends cau-dally to the level of the cervical spinal cord, axon extension is halted, and these single axons begin emitting radial collaterals into the brainstem trigeminal nuclei, where they eventually start to form whisker-specific patchy terminals by E17.
Carbocyanine dye labeling of single trigeminal axons from individ-ual whisker follicles during development of the central trigeminal path-way in NMDAR mutant and control animals revealed striking differ-ences (Lee et al., 2005a; Figure 3). In control, NR1 knockout and NR1KD mice initial arborization patterns in the PrV are simple and similar at E15, but by E17 terminal fields show notable differences. Trigeminal arbors in control cases show patchiness and elaboration of small termi-nal branches, while much larger and highly branched termitermi-nal arbor field is emergent in NR1KD and more so in NR1 knockout cases. At the time of birth, the whisker afferent arbors are the largest and most complex in NR1 knockout mice and conspicuously larger in NR1KD animals in comparison to controls. By P5, after the end of the critical period for whisker-lesion induced morphological plasticity (Woolsey 1990), trigeminal terminal arbors in the NR1KD PrV occupy five-fold larger area than those in control cases. Clearly wide spread terminal arbors, increased branch tips and overlapping distribution of whisker afferents within the PrV of NR1 knockout and NR1KD mice are major de-fects that contribute to the absence of barrelette patterns. Most whisker afferents bifurcate upon entry into the brainstem, one branch extends rostrally to form the ascending component of the central trigeminal tract and the other elongates caudally to contribute to the descending trigemi-nal tract (Jacquin et al., 1993). In NR1KD mice, there are differential levels of expression of NR1 between the PrV and SpI, the latter having more expression (Iwasato et al., 1997). Consequently in the middle portion of the SpI in NR1KD mice, there is rudimentary whisker-specific pat-terning. Comparison of single axons terminating in the PrV and SpI in NR1 knockout and NR1KD mice revealed that the same axon can form restricted terminal patches in the SpI of the NR1KD mice in compari-son to wide terminal fields in the PrV, whereas both branches formed extensive terminals in the SpI and PrV of the NR1 knockout mice (Lee and Erzurumlu, unpublished observations). These findings, along with those from CxNR1KO mice (see below), provide a strong argument for the involvement of postsynaptic NMDARs in restricting terminal arbor fields of whisker afferents and formation of whisker-specific patches.
CxNR1KO mice develop normal barrelette and barreloid patterns in the brainstem and thalamus, respectively, but in the barrel cortex, cellu-lar aggregates (barrels) fail to form (Figure 1). A rudimentary patterning corresponding to the large whiskers can be visualized with serotonin
Figure 3Illustration of trigeminal afferent terminals in the PrV of control, NR1KD, and global NR1 knockout, mice and TCA terminals in control, and CxNR1KO mice at different developmental time points. Top two rows show examples of single whisker afferent terminals in the PrV at E15, E17, and P0 for control (C), NR1KD (KD), and NR1 global knockout (KO) cases (from left to right) and control and NR1KD cases at P5. Bottom two rows show examples of single TCA terminals labeled from the VPM for control (C) and CxNR1KO (CxKO) cases at P0, P3, P5, and P7 (from left to right). Cortical laminae are indi-cated to the left of each pair. Note that both in the PrV and SI cortex these afferent terminals start branching in a similar fashion at early stages of development, but when NR1 gene is disrupted afferent terminals grow extensive branches.
Bottom panel shows comparison of single TCA terminals with respect to CO-dense patches seen in the control and CxNR1 cortex. Figure adapted from Lee et al., 2005a and 2005b).
transporter (5-HTT) immunohistochemistry and cytochrome oxidase (CO) histochemistry (Iwasato et al., 2000; Datwani et al., 2002b). Because developing TCAs transiently express 5-HTT (Lebrand et al., 1996; 1998), it has been used as a reliable marker for developing somatosensory,
visual and auditory TCAs. CO histochemistry is also a common bar-rel pattern marker for pre and postsynaptic zones rich in mitochon-dria (Wong-Riley and Welt, 1980; Wong-Riley, 1989). These individual patches in the CxNR1KO mice are much smaller and inter-patch dis-tances are wider than those in the wild-type mice. The emergence of whisker specific patterning in the wild-type mice barrel cortex was vi-sualized with 5-HTT immunohistochemistry as early as P3. This marker for TCAs and a CO histochemistry show the emergence and con-solidation of whisker-specific TCA patterns in the wild-type mouse barrel cortex between P3-7. In CxNR1KO mice, these patterns con-solidate during the same period as in wild-type mice (Lee et al., 2005b).
Detailed analyses of single TCA development in the barrel cortex of CxNR1KO mice between P1-7 revealed that while whisker-specific TCAs target proper cortical layers at first and begin arborization similar to that seen in control cases, their growth is not confined to layer IV (Lee et al., 2005b). At P1, TCAs invade the cortical plate as simple axons with few small branches, and their morphological appearance is similar in both control and CxNR1KO mice (Figure 3). By P3, TCAs of the control animals display focalized branches in layer IV and to a lesser extent in layer VI. In contrast, in CxNR1KO cortex, TCAs display a wider termi-nal territory and more branches in other layers. At P5 and later on, as the normal TCAs consolidate their focal terminal arbors in layer IV and fewer terminal arbors in layer VI, TCAs of CxNR1KO mice continue their expansion and branching in other layers. In this study (Lee et al., 2005b) it was calculated that in control animals, bifurcation points and terminal tips were mostly distributed in layer IV (about 75-80% of the total number), with some in layer VI (10–15%). In CxNR1KO cases from P5 and on, significantly reduced numbers of both bifurcation points and terminal tips were counted in layer IV. Greatly increased bifurcations of TCAs, as well as their terminals, were found in layers II/III and V in CxNR1KO cortices at P3 and older ages, also suggesting that when corti-cal excitatory neurons lack functional NMDARs, TCAs fail to recognize any putative layer-specific “stop’’ signals (Moln´ar and Blakemore, 1995;
see Yamamoto et al., 2005, this volume). Interestingly, the total num-bers of bifurcation points and terminal tips in all cortical layers for each age did not show any significant differences, however, the total axonal branch length within the terminal field of all reconstructed single ax-ons for each age was significantly higher in CxNR1KO cases beginning on P5. This increase indicates that as the terminal arbors begin shap-ing, terminal branch segments get longer, thereby contributing to the wider span of terminal arbors seen in CxNR1KO animals. Despite these large arbors in the CxNR1KO cortex, zones of TCA terminal condensa-tions were seen in layer IV. These terminal condensacondensa-tions correspond to the rudimentary patterning seen with histochemical and immunohisto-chemical markers.
In addition to terminal spreading in several cortical layers, the medi-olateral span of CxNR1KO TCAs was also double that of control TCAs by P7 (Lee et al., 2005b). Since in control and knockout phenotypes
TCA arbor mediolateral extent is similar during initial phases of cortical target invasion, exuberant growth of TCA terminals in CxNR1KO SI cortex indicate that postsynaptic NMDARs might act as “stop and elab-orate’’ signals for their presynaptic partners. Both studies (Lee et al., 2005a, b) at the level of the first (brainstem) and third (SI cortex layer IV) relay stations of the trigeminal pathway clearly show that NMDAR deficiency leads to exuberant presynaptic axon terminal branching, sug-gesting the presence of retrograde signals released through NMDAR activation of cortical cells that control pruning and patterning of presy-naptic terminals.