NMDA receptor-dependent refinement of somatotopic maps.

We have examined the role of NMDA receptor-mediated neural activity in the formation of periphery-related somatosensory patterns, using genetically engineered mice. We demonstrate that ectopic expression of a transgene of an NMDAR1 splice variant rescues neonatally fatal NMDAR1 knockout (KO) mice, although the average life span varies depending on the level of the transgene expression. In NMDAR1 KO mice with "high" levels of the transgene expression, sensory periphery-related patterns were normal along both the trigeminal and dorsal column pathways. In the KO mice with "low" levels of the transgene expression, the patterns were absent in the trigeminal pathway. Our results indicate that NMDA receptor-mediated neural activity plays a critical role in pattern formation along the ascending somatosensory pathways.

Neural activity: sculptor of 'barrels' in the neocortex.

A major portion of the primary somatosensory cortex of rodents is characterized by the discrete and patterned distribution of thalamocortical axons and layer IV granule cells ('barrels'), which correspond to the spatial distribution of whiskers and sinus hairs on the snout. In recent years several mutant mouse models began unveiling the cellular and molecular mechanisms by which these patterns emerge presynaptically and are reflected postsynaptically. Neural activity plays a crucial role in conferring presynaptic patterns to postsynaptic cells via neurotransmitter receptor-mediated intracellular signals. Here we review recent evidence that is finally opening the doors to understanding the cellular and molecular mechanisms of pattern formation in the neocortex.

Thalamic adenylyl cyclase 1 is required for barrel formation in the somatosensory cortex.

Cyclic AMP signaling is critical for activity-dependent refinement of neuronal circuits. Global disruption of adenylyl cyclase 1 (AC1), the major calcium/calmodulin-stimulated adenylyl cyclase in the brain, impairs formation of whisker-related discrete neural modules (the barrels) in cortical layer 4 in mice. Since AC1 is expressed both in the thalamus and the neocortex, the question of whether pre- or postsynaptic (or both) AC1 plays a role in barrel formation has emerged. Previously, we generated cortex-specific AC1 knockout (Cx-AC1KO) mice and found that these animals develop histologically normal barrels, suggesting a potentially more prominent role for thalamic AC1 in barrel formation. To determine this, we generated three new lines of mice: one in which AC1 is disrupted in nearly half of the thalamic ventrobasal nucleus cells in addition to the cortical excitatory neurons (Cx/pTh-AC1KO mouse), and another in which AC1 is disrupted in the thalamus but not in the cortex or brainstem nuclei of the somatosensory system (Th-AC1KO mouse). Cx/pTh-AC1KO mice show severe deficits in barrel formation. Th-AC1KO mice show even more severe disruption in barrel patterning. In these two lines, single thalamocortical (TC) axon labeling revealed a larger lateral extent of TC axons in layer 4 compared to controls. In the third line, all calcium-stimulated adenylyl cyclases (both AC1 and AC8) are deleted in cortical excitatory neurons. These mice have normal barrels. Taken together, these results indicate that thalamic AC1 plays a major role in patterning and refinement of the mouse TC circuitry.

Efferent connections of the brainstem trigeminal complex with the facial nucleus of the rat.

The sensory surface of the face of the rat is topographically represented in the brainstem trigeminal complex (Nord, '67), and in parallel with this the underlying facial musculature is also represented in a topographic fashion in the facial nucleus (Papez, '27; Martin and Lodge, '77; Watson and Sakae, '78). It has been recently reported that in the young rat three distinct representations of the vibrissae are present in the sensory portion of the brainstem trigeminal complex (Belford and Killackey, '79). Within this perspective, the specific connectivity between the brainstem trigeminal complex and the facial nucleus was investigated in adult rats by Fink-Heimer technique. The two major sensory nuclei of the brainstem trigeminal complex, the spinal trigeminal nucleus and the principal sensory nucleus, differ in their projection patterns to the facial nucleus. While the principal sensory nucleus sends sparse projections to the ipsilateral lateral and dorsal subdivisions of the facial nucleus, the spinal trigeminal nucleus send differential projections to various subdivisions of the facial nucleus depending on their origin with respect to three cytoarchitectonically different subnuclei that compose the spinal trigeminal nucleus. It is concluded that the magnocellular portion of subnucleus caudalis projects rather heavily to the ipsilateral lateral subdivision of the facial nucleus, while the projections from the subnucleus interpolaris are sparser and distributed more widely to parts of the lateral, dorsal and intermediate subdivisions of the facial nucleus ipsilaterally. In contrast to ipsilateral facial projections from the rest of the brainstem trigeminal complex, the projections from the subnucleus oralis of the spinal trigeminal nucleus are bilateral and confined to the intermediate subdivision of the facial nucleus. However, ipsilateral projections of the subnucleus oralis are denser than the the very sparse contralateral projections. In addition to the facial projections from the brainstem trigeminal complex, projections from the upper portions of the cervical cord to the medial subdivision of the facial nucleus were observed. These projections ar bilateral, and those fibers destined for the contralateral medial subdivision cross over below the level of the pyramidal decussation.

Regulation of neurotrophin-induced axonal responses via Rho GTPases.

Nerve growth factor (NGF) and related neurotrophins induce differential axon growth patterns from embryonic sensory neurons (Lentz et al. [1999] J. Neurosci. 19:1038-1048; Ulupinar et al. [2000a] J. Comp. Neurol 425:622-630). In wholemount explant cultures of embryonic rat trigeminal ganglion and brainstem or in dissociated cell cultures of the trigeminal ganglion, exogenous supply of NGF leads to axonal elongation, whereas neurotrophin-3 (NT-3) treatment leads to short branching and arborization (Ulupinar et al. [2000a] J. Comp. Neurol. 425:622-630). Axonal responses to neurotrophins might be mediated via the Rho GTPases. To investigate this possibility, we prepared wholemount trigeminal pathway cultures from E15 rats. We infected the ganglia with recombinant vaccinia viruses that express GFP-tagged dominant negative Rac, Rho, or constitutively active Rac or treated the cultures with lysophosphatitic acid (LPA) to activate Rho. We then examined axonal responses to NGF by use of the lipophilic tracer DiI. Rac activity induced longer axonal growth from the central trigeminal tract, whereas the dominant negative construct of Rac eliminated NGF-induced axon outgrowth. Rho activity also significantly reduced, and the Rho dominant negative construct increased, axon growth from the trigeminal tract. Similar alterations in axonal responses to NT-3 and brain-derived neurotrophic factor were also noted. Our results demonstrate that Rho GTPases play a major role in neurotrophin-induced axonal differentiation of embryonic trigeminal axons.

Trigeminal projections to the superior colliculus of the rat.

The deep layers of the rodent superior colliculus contain a vibrissae-related organization that is in "spatial register" with the overlying visuotopic organization (Dräger and Hubel, '76). The distribution of vibrissae-related afferents and their cells of origin were determined with a number of anatomical techniques. The brainstem trigeminal complex afferents to the superior colliculus terminate in the lateral portions of the strata album intermediate and griseum profundum and, to a lesser degree, in deep portions of the stratum griseum intermediate. The cells giving rise to these afferents are located mainly in the ventral portions of the contralateral principal sensory nucleus, subnucleus oralis, and subnucleus interpolaris. The majority of tectal projection cells are found in subnucleus interpolaris, and the fewest in the principal sensory nucleus. Further, the density of projection cells in the three components of the brainstem trigeminal complex can be correlated with the density of their projections to the superior colliculus. The afferents from the somatosensory cortex terminate in a continuous band in the strata album intermediate and griseum intermediate. The cells of origin of this projection are located in layer Vb of the agranular zones of the ipsilateral somatosensory cortex. The present results suggest that the organization of trigeminal afferents to the deep portion of the superior colliculus is similar to that of the visual afferents to the superficial laminae. Further, the results suggest that observations on the nature of afferent termination pattern should be made with care, considering both the techniques employed and the idiosyncrasies of the local neuropil.

Development of order in the rat trigeminal system.

The trigeminal system of the rat is characterized by a high degree of order. The pattern of the distribution of vibrissae follicles on the face is replicated at each synaptic station between face and somatosensory cortex (Belford and Killackey, '80). The present study details the development of the trigeminal nerve, its intrinsic organization, and its relationship with its peripheral and central targets. We have observed that at early embryonic ages (E12 and E13) the trigeminal ganglion neurons grow out in straight lines without crossing, and the distance between these neurons and their peripheral and central targets is very short. We have found that fibers reach the periphery before follicle formation is first detectable (E14). At all ages, the trigeminal fibers show a marked tendency to fasciculate. After the development of the pattern of vibrissae follicles on the face, the pattern of fasciculation within the nerve can be clearly related to the rows of vibrissae and the buccal pad. This peripherally related order in the nerve was experimentally verified by injecting horseradish peroxidase into the follicles of individual rows and selectively sectioning portions of the nerve. Further, we provide evidence that the discrete brainstem pattern reflecting vibrissae distribution develops after organization is detectable in the nerve and in a temporal sequence from lateral to medial, which replicates the developmental sequence of vibrissae follicles from ocular to nasal on the face. This sequence is detectable in both the distribution of afferent terminals as measured with succinic dehydrogenase histochemistry and of horseradish peroxidase back-labeled trigeminothalamic relay cells. We interpret our results as suggesting that a number of factors may play a role in the establishment of specific neuronal topographies in the rodent trigeminal system.

Peripheral nerve transection induces innervation of embryonic neocortical transplants by specific thalamic fibers in adult mice.

Embryonic neocortical tissue survives and differentiates when grafted to injured adult neocortex. While these transplants are readily innervated by the host cholinergic fibers, specific thalamic fibers fail to innervate them. The present study was designed to test whether changing the activity levels of the thalamic ventrobasal projection neurons would promote sprouting of their axons into the embryonic cortical implants placed in the barrel field cortex. To achieve this the main input to these thalamic neurons was eliminated two synapses away, by blocking the peripheral sensory input to the barrel field cortex. Adult hosts underwent unilateral transection of the infraorbital nerve and two days later the contralateral barrel field cortex was lesioned enough to insert an embryonic neocortical graft. Following a one month post-transplantation period we examined the amount of specific thalamic axon ingrowth into the transplants by injecting the ventrobasal nucleus with horseradish peroxidase. The control cases without prior nerve damage confirmed previous observations that ventrobasal nucleus neurons fail to innervate the implanted neocortex. Transection of the infraorbital nerve prior to transplantation resulted in an unprecedented ingrowth of specific thalamic axons into the transplants. There was no significant difference in the amount of thalamic fiber ingrowth into the transplants when the peripheral nerve was (transection) or was not (cautery) allowed to regenerate. However, transection of the infraorbital nerve permits the nerve to regenerate and at least partially reconnect the sensory periphery, thus leading to the possibility of functional integration of the neocortical transplants into the host trigeminal system. The morphology and distribution of host acetylcholinesterase-positive fibers that grow into the transplants under both experimental and control conditions were distinctly different from those of thalamic axons. These results provide the first demonstration of peripheral sensory nerve induction of regenerative propensity in specific thalamocortical projection neurons. The thalamic fiber ingrowth should lead to enhanced functional innervation of the neocortical implants and better incorporation of the graft into the adult host brain circuitry.

Peripheral target-specific influences on embryonic neurite growth vigor and patterns.

We examined axon-target interactions in cocultures of embryonic rat trigeminal, dorsal root, nodose, superior cervical ganglia or retina with a variety of native or foreign peripheral targets such as the whisker pad, forepaw, and heart explants. Axon growth into these peripheral target tissues was analyzed by the use of lipophilic tracer DiI. Embryonic day 15 dorsal root and trigeminal axons grew into isochronic normal and foreign cutaneous targets. Both axon populations avoided the same age heart tissue, but grew profusely into younger (embryonic day 13) or older (postnatal) heart explants. In contrast, embryonic day 15 superior cervical or nodose ganglion axons grew heavily into the same age heart and forepaw explants and to a lesser extent into the whisker pad explants. Embryonic day 15 retinal axons grew into all three peripheral targets used in this study. Primary sensory and sympathetic axons, but not retinal axons, formed target-specific patterns in the whisker pad and forepaw explants. DiI-labeling and immunostaining of primary sensory neurons in coculture revealed that these neurons retain their bipolar characteristics, and express class-specific markers such as parvalbumin, calcitonin gene-related peptide and TrkA receptors. In the whisker pad explants, axons positive for all three markers were seen to form patterns around the follicles. Our results indicate that developing peripheral targets can attract and support axon growth from a variety of sources. Whereas neurotrophins play a major role in attracting and supporting survival of subpopulations of sensory neurons, other substrate-bound or locally released molecules must regulate sensory neurite growth into specific peripheral and central targets.

Transient patterns of GAP-43 expression during the formation of barrels in the rat somatosensory cortex.

The development of the rat barrel field cortex was investigated with an antibody to the axonal membrane-specific phosphoprotein GAP-43 in order to examine the developmental pattern of afferent projections, and with cytochrome oxidase histochemistry and Nissl stains to reveal the morphogenesis of cortical barrels. On the first two days after birth, GAP-43 immunostaining in the cortical plate was light and diffuse, then became intense in the presumptive layer IV of the parietal cortex on PND3 (day of birth = PND0). Immunoreactive densities were visible as small, focal patches within the centers of prospective barrels. These densities increased in size and intensity over the next few days and then diminished abruptly. On PND7, the distribution of GAP-43 was coextensive with barrels, as defined by cytochrome oxidase histochemistry and Nissl staining. GAP-43 virtually disappeared from the barrels after PND7. From the second postnatal week, GAP-43 immunostaining was evident in the septa between barrels and in the dysgranular regions of SI cortex. This pattern of GAP-43 distribution was complementary to the pattern of cytochrome oxidase activity, and persisted into maturity. In an attempt to identify possible source(s) of GAP-43 positive afferents in the developing barrels, we examined the effects of altering the sensory periphery on the distribution of GAP-43 immunostaining in the cortex. Rat pups had row C whiskers cauterized on PND0 and were sacrificed on PND3 or PND5. Whereas immunopositive densities corresponding to intact whiskers developed in a normal, punctate pattern, cortical representation of the lesioned whiskers formed a continuous band of labeling that was evident as early as PND3. We argue that the disjunctive expression of GAP-43 in the barrel field reflects the pattern of distribution of afferents (most likely from the ventro-basal thalamic nucleus) to the barrel field cortex, and that this pattern may be instructive in the formation of barrels as cytoarchitectonic units. The rapid alteration in patterns of immunostaining following whisker lesions lends further support to the conclusion that the "barrel template" is conveyed to the neocortex by incoming afferents. The possible significance of the transient expression of GAP-43 in the maturing barrel field is discussed.

Differential effects of NGF and NT-3 on embryonic trigeminal axon growth patterns.

We examined the effects of neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT-3) on trigeminal axon growth patterns. Embryonic (E13-15) wholemount explants of the rat trigeminal pathway including the whisker pads, trigeminal ganglia, and brainstem were cultured in serum-free medium (SFM) or SFM supplemented with NGF or NT-3 for 3 days. Trigeminal axon growth patterns were analyzed with the use of lipophilic tracer DiI. In wholemount cultures grown in SFM, trigeminal axon projections, growth patterns, and differentiation of peripheral and central targets are similar to in vivo conditions. We show that in the presence of NGF, central trigeminal axons leave the tract and grow into the surrounding brainstem regions in the elongation phase without any branching. On the other hand, NT-3 promotes precocious development of short axon collaterals endowed with focal arbors along the sides of the central trigeminal tract. These neurotrophins also affect trigeminal axon growth within the whisker pad. Additionally, we cultured dissociated trigeminal ganglion cells in the presence of NGF, NT-3, or NGF+NT-3. The number of trigeminal ganglion cells, their size distribution under each condition were charted, and axon growth was analyzed following immunohistochemical labeling with TrkA and parvalbumin antibodies. In these cultures too, NGF led to axon elongation and NT-3 to axon arborization. Our in vitro analyses suggest that aside from their survival promoting effects, NGF and NT-3 can differentially influence axon growth patterns of embryonic trigeminal neurons.

Excess nerve growth factor in the periphery does not obscure development of whisker-related patterns in the rodent brain.

We have addressed the issue of whether or not peripherally expressed nerve growth factor (NGF) influences the formation of whisker-specific patterns in the brain by regulating the survival of sensory neurons. Transgenic mice that overexpress an NGF cDNA in the skin were examined. In these animals, excess NGF expression is controlled by promoter and enhancer sequences of a keratin gene, thus restricting the higher levels of NGF expression to basal keratinocytes of the epidermis. Twice the number of trigeminal sensory neurons survive in transgenic mice as in normal animals, and a corresponding hyperinnervation of the whisker pad is noted, both around the vibrissa follicles and along the intervibrissal epidermis. However, the increased survival of sensory neurons and the enhanced peripheral projections do not interfere with the development of whisker-specific patterns in the trigeminal brainstem, in the ventrobasal thalamic complex or in the face-representation region of the primary somatosensory (SI) cortex. These results demonstrate that vibrissa-related central patterns are able to form in the virtual absence of trigeminal ganglion cell death and suggest that mechanisms other than a selective elimination of sensory neurons control the development of whisker-specific neural patterns in the brain.

Distribution of morphologically different retinal axon terminals in the hamster dorsal lateral geniculate nucleus.

Afferent terminal arbors in the hamster LGBd were labelled with horseradish peroxidase (HRP) implanted into the optic tract. Three morphologically distinct terminal types, each with a different regional distribution, were observed. Type R1 terminals are large, ovoid swellings and are predominantly distributed medially within the nucleus. Type R2 terminals are very small, clustered varicosities and are distributed laterally and ventrally. Type R3 terminals are medium in size and their distribution overlaps with that of Type R1 and R2 terminals.

Central correlates of peripheral pattern alterations in the trigeminal system of the rat. III. Neurons of the principal sensory nucleus.

We provide evidence that the discrete clustered distribution of the trigeminothalamic projection neurons in the principal sensory nucleus of the trigeminal is dependent on the integrity of the trigeminal nerve during development. Nerve section at birth abolishes all evidence of cellular clustering in the principal sensory nucleus, while more discrete damage to specific rows of follicles results in changes in the cellular clustering pattern only in the portion of the nucleus related to the afflicted rows of vibrissae.

Differential organization of thalamic projection cells in the brain stem trigeminal complex of the rat.

Following large injections of horseradish peroxidase into the thalamus of the adult rat, labeled neurons can be detected in the contralateral principal sensory nucleus and the subnucleus interpolaris of the spinal trigeminal nucleus. Thalamic projection neurons in the principal sensory nucleus are arranged in discrete aggregates which replicate the organization of the vibrissae and other parts of the face. This cellular organization corresponds very well with the pattern of vibrissae related afferent termination in this region of the nucleus as seen in SDH preparations. There is no such cellular organization in the subnucleus interpolaris.

Peripheral nerve regeneration induces elevated expression of GAP-43 in the brainstem trigeminal complex of adult hamsters.

A polyclonal antibody was used to delineate the pattern of GAP-43 expression in central processes of trigeminal ganglion cells while their peripheral processes were undergoing regeneration. Two weeks after transection of the infraorbital nerve, levels of GAP-43 ipsilateral to the transection were greatly increased along the trajectory of infraorbital axons in the central trigeminal tract and also within the target neuropil. We conclude that elevated levels of GAP-43 in central processes of injured trigeminal ganglion cells occur in direct response to the regenerative response of the cell body and may have important implications for 'plasticity'-related changes seen in the adult trigeminal system.

Target specific differentiation of peripheral trigeminal axons in rat-chick chimeric explant cocultures.

Avian and rodent trigeminal ganglion (TG) neurons share common features in their neurotrophin requirements and axonal projections between the sensory periphery and the brainstem. In rodents, the whisker pad (WP) is a major peripheral target of the infraorbital (IO) nerve component of the TG. The chick IO nerve is much smaller and innervates the maxillary process (MP). In the embryonic WP, IO axons course in fascicles from a caudal to rostral direction and form terminal plexuses around follicles. In the chick, IO axons travel as a thin bundle to the MP and branch out with no specific patterning. We cocultured E15 rat TG with E5-6 chick MP or chick TG with rat WP explants to examine target influences on trigeminal axon growth patterns as visualized with DiI labeling or neurofilament immunohistochemistry. Chick TG axons showed robust growth into WP explants, and the ganglion increased in size. Thick bundles of axons traveled between rows of follicles and formed a distinct pattern as they developed terminal arbors around individual follicles. In contrast, rat TG axon growth was sparse in chick MP explants and the ganglion size reduced over time. Furthermore, rat TG axons did not show any patterning in the chick MP. Similar target-specific growth patterns were observed when TG explants were given a choice between chick MP and rat WP explants. Collectively these results indicate that both the chick and rat TG cells respond to similar target-specific peripheral cues in the establishment of innervation density and patterning in peripheral orofacial targets.

Maintenance of discrete somatosensory maps in subcortical relay nuclei is dependent on an intact sensory cortex.

Lesions of the rat barrelfield cortex drastically alter the discrete representations of the somatosensory periphery in the central nervous system. We have found that lesions placed in the parietal cortex, after the formation of barrels (postnatal day 5), can irreversibly abolish vibrissae- and extremity-related patterns of cytochrome oxidase activity in the principal sensory nucleus of the trigeminal nerve and in the dorsal column nuclei. Furthermore, abnormal patterns of enzymatic activity occur in the remaining primary somatosensory cortex and the ventrobasal nucleus of the thalamus. We conclude that cortical barrels are essential in maintenance of periphery-related discrete morphological organization in the rodent somatosensory system.

Thalamic axons confer a blueprint of the sensory periphery onto the developing rat somatosensory cortex.

In order to study the role of afferents in the maturation of cortical axons projecting from the ventrobasal thalamic complex (VB) to the barrel field (SI) cortex were labeled with the carbocyanine dye DiI, in aldehyde-fixed embryonic and newborn rat brains. Our results reveal that the first few thalamic axons are in the cortical plate by embryonic day (E) 19. Between E19 and the day of birth (E21 = PND 0), layers V and VI differentiate from the lower part of the cortical plate. On PND 0, a plexus of growth-cone tipped thalamic axons is present within the cortical plate and a few VB fibers have reached the marginal zone. Increasing numbers of thalamic afferents invade and ramify within the cortical plate on PND 1 and, over the course of the next 24 h, form a vibrissa-specific pattern in the lower part of this zone, prior to the differentiation of layer IV into a distinct lamina. This periphery-related organization is exhibited by VB afferents earlier than reported for other afferents to the cortex, by glia- or neuron-associated extracellular elements or by the cytoarchitectonic specializations (barrels) of stellate cells. Our observations, in conjunction with the previous studies, demonstrate that thalamic afferents may have a pivotal role in determining the morphological specification of the primary somatosensory cortex.

Morphological specification of trigeminal neurites depends on target fields.

Primary sensory neurons bridge the sensory periphery to the central nervous system (CNS) via their two axonal processes. The morphological patterning of the terminals of each process in its respective target is unique. Whether the differences between peripheral and central axons result from an intrinsic developmental program of the ganglion cell body, or from target-derived signals is not known. To explore this issue, we have used an explant coculture system in which embryonic (E15) trigeminal ganglion explants were placed between a vibrissa pad and a brainstem explant, but the explants were rotated 180 degrees relative to their normal orientation. In other experiments, individual ganglia were placed between two vibrissa pad explants or between two slices taken through the brainstem. The cultures were fixed after several days and ganglion cell processes were labeled with the lipophilic tracer DiI. Results of the ganglion rotation experiments suggest that trigeminal axons which would be directed centrally in vivo can regenerate into peripheral targets, and peripheral axons can grow into CNS tissue. Similarly, in cocultures with two peripheral or two central targets, both processes of trigeminal ganglion cells can simultaneously invade vibrissa pad explants or project into brainstem slices. Moreover, in all cocultures the differentiation of each set of processes is specific to the target innervated by it. These results show that the axons of embryonic sensory neurons are not selective in their choice of targets, and that their morphological patterning is dictated by target-derived signals.