Distribution of brain iron accrual in adolescence: Evidence from cross-sectional and longitudinal analysis.

To track iron accumulation and location in the brain across adolescence, we repurposed diffusion tensor imaging (DTI) and functional magnetic resonance imaging (fMRI) data acquired in 513 adolescents and validated iron estimates with quantitative susceptibility mapping (QSM) in 104 of these subjects. DTI and fMRI data were acquired longitudinally over 1 year in 245 male and 268 female, no-to-low alcohol-consuming adolescents (12-21 years at baseline) from the National Consortium on Alcohol and NeuroDevelopment in Adolescence (NCANDA) study. Brain region average signal values were calculated for susceptibility to nonheme iron deposition: pallidum, putamen, dentate nucleus, red nucleus, and substantia nigra. To estimate nonheme iron, the corpus callosum signal (robust to iron effects) was divided by regional signals to generate estimated R2 (edwR2 for DTI) and R2 * (eR2 * for fMRI). Longitudinal iron deposition was measured using the normalized signal change across time for each subject. Validation using baseline QSM, derived from susceptibility-weighted imaging, was performed on 46 male and 58 female participants. Normalized iron deposition estimates from DTI and fMRI correlated with age in most regions; both estimates indicated less iron in boys than girls. QSM results correlated highly with DTI and fMRI results (adjusted R2 = 0.643 for DTI, 0.578 for fMRI). Cross-sectional and longitudinal analyses indicated an initial rapid increase in iron, notably in the putamen and red nucleus, that slowed with age. DTI and fMRI data can be repurposed for identifying regional brain iron deposition in developing adolescents as validated with high correspondence with QSM.

Focal Stroke in the Developing Rat Motor Cortex Induces Age- and Experience-Dependent Maladaptive Plasticity of Corticospinal System.

Motor system development is characterized by an activity-dependent competition between ipsilateral and contralateral corticospinal tracts (CST). Clinical evidence suggests that age is crucial for developmental stroke outcome, with early lesions inducing a "maladaptive" strengthening of ipsilateral projections from the healthy hemisphere and worse motor impairment. Here, we investigated in developing rats the relation between lesion timing, motor outcome and CST remodeling pattern. We induced a focal ischemia into forelimb motor cortex (fM1) at two distinct pre-weaning ages: P14 and P21. We compared long-term motor outcome with changes in axonal sprouting of contralesional CST at red nucleus and spinal cord level using anterograde tracing. We found that P14 stroke caused a more severe long-term motor impairment than at P21, and induced a strong and aberrant contralesional CST sprouting onto denervated spinal cord and red nucleus. The mistargeted sprouting of CST, and the worse motor outcome of the P14 stroke rats were reversed by an early skilled motor training, underscoring the potential of early activity-dependent plasticity in modulating lesion outcome. Thus, changes in the mechanisms controlling CST plasticity occurring during the third postnatal week are associated with age-dependent regulation of the motor outcome after stroke.

Spontaneous activity and functional connectivity in the developing cerebellorubral system.

The development of the cerebellar system depends in part on the emergence of functional connectivity in its input and output pathways. Characterization of spontaneous activity within these pathways provides insight into their functional status in early development. In the present study we recorded extracellular activity from the interpositus nucleus (IP) and its primary downstream target, the red nucleus (RN), in unanesthetized rats at postnatal days 8 (P8) and P12, a period of dramatic change in cerebellar circuitry. The two structures exhibited state-dependent activity patterns and age-related changes in rhythmicity and overall firing rate. Importantly, sensory feedback (i.e., reafference) from myoclonic twitches (spontaneous, self-generated movements that are produced exclusively during active sleep) drove neural activity in the IP and RN at both ages. Additionally, anatomic tracing confirmed the presence of cerebellorubral connections as early as P8. Finally, inactivation of the IP and adjacent nuclei using the GABAA receptor agonist muscimol caused a substantial decrease in neural activity in the contralateral RN at both ages, as well as the disappearance of rhythmicity; twitch-related activity in the RN, however, was preserved after IP inactivation, indicating that twitch-related reafference activates the two structures in parallel. Overall, the present findings point to the contributions of sleep-related spontaneous activity to the development of cerebellar networks.

Motor Cortex Activity Organizes the Developing Rubrospinal System.

The corticospinal and rubrospinal systems function in skilled movement control. A key question is how do these systems develop the capacity to coordinate their motor functions and, in turn, if the red nucleus/rubrospinal tract (RN/RST) compensates for developmental corticospinal injury? We used the cat to investigate whether the developing rubrospinal system is shaped by activity-dependent interactions with the developing corticospinal system. We unilaterally inactivated M1 by muscimol microinfusion between postnatal weeks 5 and 7 to examine activity-dependent interactions and whether the RN/RST compensates for corticospinal tract (CST) developmental motor impairments and CST misprojections after M1 inactivation. We examined the RN motor map and RST cervical projections at 7 weeks of age, while the corticospinal system was inactivated, and at 14 weeks, after activity returned. During M1 inactivation, the RN on the same side showed normal RST projections and reduced motor thresholds, suggestive of precocious development. By contrast, the RN on the untreated/active M1 side showed sparse RST projections and an immature motor map. After M1 activity returned later in adolescent cat development, RN on the active M1/CST side continued to show a substantial loss of spinal terminations and an impaired motor map. RN/RST on the inactivated side regressed to a smaller map and fewer axons. Our findings suggest that the developing rubrospinal system is under activity-dependent regulation by the corticospinal system for establishing mature RST connections and RN motor map. The lack of RS compensation on the non-inactivated side can be explained by development of ipsilateral misprojections from the active M1 that outcompete the RST. Significance statement: Skilled movements reflect the activity of multiple descending motor systems and their interactions with spinal motor circuits. Currently, there is little insight into whether motor systems interact during development to coordinate their emerging functions and, if so, the mechanisms underlying this process. This study examined activity-dependent interactions between the developing corticospinal and rubrospinal systems, two key systems for skilled limb movements. We show that the developing rubrospinal system competes with the corticospinal system in establishing the red nucleus motor map and rubrospinal tract connections. This is the first demonstration of one motor system steering development, and ultimately function, of another. Knowledge of activity-dependent competition between these two systems helps predict the response of the rubrospinal system following corticospinal system developmental injury.

Postnatal maturation of the red nucleus motor map depends on rubrospinal connections with forelimb motor pools.

The red nucleus (RN) and rubrospinal tract (RST) are important for forelimb motor control. Although the RST is present postnatally in cats, nothing is known about when rubrospinal projections could support motor functions or the relation between the development of the motor functions of the rubrospinal system and the corticospinal system, the other major system for limb control. Our hypothesis is that the RN motor map is present earlier in development than the motor cortex (M1) map, to support early forelimb control. We investigated RN motor map maturation with microstimulation and RST cervical enlargement projections using anterograde tracers between postnatal week 3 (PW3) and PW16. Microstimulation and tracer injection sites were verified histologically to be located within the RN. Microstimulation at PW4 evoked contralateral wrist, elbow, and shoulder movements. The number of sites producing limb movement increased and response thresholds decreased progressively through PW16. From the outset, all forelimb joints were represented. At PW3, RST projections were present within the cervical intermediate zone, with a mature density of putative synapses. In contrast, beginning at PW5 there was delayed and age-dependent development of forelimb motor pool projections and putative rubromotoneuronal synapses. The RN has a more complete forelimb map early in development than previous studies showed for M1, supporting our hypothesis of preferential rubrospinal rather than corticospinal control for early movements. Remarkably, development of the motor pool, not intermediate zone, RST projections paralleled RN motor map development. The RST may be critical for establishing the rudiments of motor skills that subsequently become refined with further CST development.

Sequence of synaptogenesis in the fetal and neonatal cerebellar system - part 1: Guillain-Mollaret triangle (dentato-rubro-olivo-cerebellar circuit).

Precise temporal and spatial sequences of synaptogenesis occur in the cerebellar system, as in other synaptic circuits of the brain. In postmortem brain sections of 172 human fetuses and neonates, synaptophysin immunoreactivity was studied in nuclei of the Guillain-Mollaret triangle: dentato-olivo-rubro-cerebellar circuit. Synaptophysin demonstrates not only progressive increase in synaptic vesicles in each structure, but also shows the development of shape from amorphous globular neuronal aggregates to undulated nuclei. Intensity of synaptophysin reactivity is strong before the mature shape of these nuclei is achieved. Accessory olivary and deep cerebellar nuclei are intensely stained earlier than the principal olivary and dentate nuclei. The dorsal blades of both form earlier than the ventral, with reactivity initially peripheral. Initiation of synaptophysin reactivity is at 13 weeks in the inferior olive (r6, r7) and at 16 weeks in the dentate (r2). Initial synaptic vesicles are noted at 13 weeks in the red nucleus (r0); synapses form initially on the small neurons at 13 weeks but thereafter simultaneously on small and large neurons. Form and reactivity follow caudorostral, dorsoventral and mediolateral gradients in the axes of the rhombencephalon. This study provides control data to serve as a basis for interpreting aberrations in synaptogenesis in malformations of the cerebellar system, genetic disorders and acquired insults to the cerebellum and brainstem during fetal life, applicable to tissue sections and complementing biochemical and molecular techniques.

Development of the human parvocellular red nucleus. A morphological study.

Morphology of the human parvocellular red nucleus (RNp) was investigated in 14 fetuses aged from 12 to 39 weeks of gestation (WG). The brains were processed into celloidin-embedded serial sections. At 12 WG, the anlage of RNp was observed as an ovoid mass of immature neurons clustering into some groups. Lobular appearance in cross-sectional images was conspicuous during the early stages (12-23 WG), particularly at rostral levels. The fasciculus retroflexus of Meynert was seen as a prominent bundle of fibers surrounded by the most rostral part of RNp. Two types of neurons were identified: large and small neurons. Large neurons were earlier observed at 16 WG, and had a polygonal or multipolar perikaryon with abundant Nissl bodies from 28 WG onwards. Small neurons later appeared among large neurons at 21 WG, and had a triangular or ovoid perikaryon with scanty Nissl bodies. The volume of RNp showed an exponential increase with age during 20-39 WG. The mean of neuronal perikaryonal areas showed a linear increase with age in both types during 16-39 WG, although the degree of change was much greater in large neurons than small neurons. The current study has clearly demonstrated the presence of two neuronal populations and their differential growth in developing human RNp.

Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury.

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.

The growth of the feline brain from fetal into adult life. II. A morphometric study of subcortical nuclei.

As a continuation of the morphometric studies on the preceding paper, here we report on the rate of growth of the caudate nucleus (n.), thalamus, red n., and the substantia (s.) nigra using, with few exceptions, the same cohort of cats. The same previously used brains (n=64 cats) were allocated to the following age groups: fetal (E) 59 days, postnatal (P) days 1, 7, 15, 30, 45, 60, 90, 120, and 180. Sixteen additional cats, interspersed within the groups, were substituted for the red n. and s. nigra studies. There were six subjects per group (except for E59, n=4). Using a projection microscope and cytochrome oxidase-stained coronal sections, a combined (left plus right sides) total of 4693, 3822, 1636, and 1180 sections were drawn for the caudate, thalamus, s. nigra, and red n., respectively. With computer assistance, the drawings were digitized to calculate mean cross-sectional areas and then the mean volume of each structure per group. The growth time tables for the caudate n., thalamus and s. nigra were fairly synchronous. In terms of percentage of the adult volume, for the left side (both sides grew at a similar rate), the three structures grew at a fast pace between E59 and P30. Thus, at E59 their respective percentages relative to adult volume were 23.7, 29.8 and 22.6% and by P30 the percentages were within adult range (85.2, 115.1 and 87.5%, respectively). Starting at P30, for the thalamus and at P45 for the caudate n., there was a consistent tendency to an overgrow which ranged between 4.3 and 30.9% (at P180, P<0.5) for the caudate and between 0.3 and 15.1% for the thalamus. In addition, starting at P30, the right thalamus tended to be consistently larger than the left by a margin ranging between 0.5 and 11.2% (P120, P<0.05). The red n. grew at a different, slower pace. Starting from a fetal volume equivalent to an 18.6% of adult size, its volume was only a 61.0% of the adult value at P30 and came within range of adulthood size only by P60 (81. 3%). Neither the s. nigra nor the red n. showed any consistent tendency to overgrow or to asymmetry. These findings are discussed in the context of the literature. Furthermore, we discuss general conclusions and considerations pertaining to both papers as well as draw comparisons with the maturational time tables of other developmental landmarks in cats. Finally, in a comparison with growth of human brain structures, we point at the limitations and complexities involved in studying human material and, noting interspecies similarities, we propose that the present data from an advanced gyrencephalic mammal may form the bases for a model of structures maturation in humans.

Regeneration of supraspinal axons after transection of the thoracic spinal cord in the developing opossum, Didelphis virginiana.

When the thoracic spinal cord of the North American opossum is transected early in development, supraspinal axons grow through the lesion. In the experiments reported here, we asked whether regeneration of cut axons contributes to such growth. Fast Blue (FB) was injected into the lumbar cord on postnatal day (PD)5, 8, 15, or 20. Five days later, FB was removed by gentle suction, and the spinal cord was transected at thoracic levels. Fourteen days later, rhodamine B dextran was injected between the site of the FB injection and the lesion. The pups were maintained for an additional 7-10 days before killing and perfusion. We assumed that supraspinal neurons that contained FB survived axotomy and those that contained both FB and rhodamine B dextran supported regenerating axons. In the PD5 group (lesioned at PD10), regenerative growth was documented for axons originating in all of the supraspinal nuclei that innervate the lumbar cord by PD10. When the injections were made at the later ages, however, neurons that supported regenerative growth were fewer in number and regionally restricted. In some cases, they were limited primarily to the red nucleus, the medullary raphe, and the adjacent reticular formation. Our results show that regeneration of cut axons contributes to growth of supraspinal axons through the lesion after transection of the thoracic cord in developing opossums and that the critical period for regenerative growth is not the same for all axons.

Preferential termination of corticorubral axons on spine-like dendritic protrusions in developing cat.

The formation of synaptic contacts is a crucial event during neural development and is thought to be achieved by complex interactions between incoming axons and the neurons in the target. We have focused on spine-like dendritic protrusions (SLDPs), which are transient pleomorphic protrusive structures seen in developing brains. Although the functional significance of SLDPs remains unknown, accumulating in vitro evidence suggests that the SLDP plays an important role in synaptogenetic interactions with axons. As a test of this idea, the present study was performed to examine whether the SLDPs are the preferential sites of synapse formation in vivo. The ultrastructure of biocytin-labeled corticorubral (CR) terminals was examined in serial thin sections during the period of synaptogenesis in newborn cats. We found that a major proportion (86%) of the CR synapses was formed on SLDPs. The presynaptic terminals were often invaginated by fine processes extending from the tips of SLDPs. Synaptic structures presumably of cortical origin were also found on SLDPs of HRP-labeled rubrospinal cells, suggesting that SLDPs postsynaptic to labeled CR terminals originate at least in part from rubrospinal cells. Taken together, these results indicate that SLDPs may represent preferred sites of synapse formation and support the notion that SLDPs play a role in synaptogenic interactions during brain development.

Requirement for Brn-3.0 in differentiation and survival of sensory and motor neurons.

Specific families of transcription factors mediate events in the sequential maturation of distinct neuronal phenotypes. Members of one such family, the class IV POU domain transcription factor Brn-3.0, and two highly related factors Brn-3.1 and Brn-3.2, are differentially expressed in the developing and mature mammalian nervous system. The expression pattern of Brn-3.0 suggested that it has an important role in the development of sensory ganglia, as well as red nucleus, inferior olive, and nucleus ambiguus. Analysis of mice null for the Brn-3.0 locus shows that Brn-3.0 is required for the survival of subpopulations of proprioceptive, mechanoreceptive and nociceptive sensory neurons, where deletion of the gene affects neurotrophin and neurotrophin-receptor gene expression. Deletion of Brn-3.0 also alters either differentiation, migration or survival of specific central neuronal populations.

Postnatal development of crossed and uncrossed corticorubral projections in kitten: a PHA-L study.

Morphological changes in individual corticorubral fibers and the pattern of crossed and uncrossed corticorubral projections were studied during the postnatal development of cats in order to understand cellular mechanisms for restriction of corticorubral projections with development. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into restricted areas of the pericruciate cortex in kittens and PHA-L-labeled axons in the red nucleus were examined at postnatal days (PND) 7-73. In accordance with our previous study (Murakami and Higashi, Brain Res. 1988; 447:98-108), a crossed corticorubral projection was observed in addition to the uncrossed one in every experimental animal. During the early period of development (PND 7-8), swellings of irregular shape were observed along the entire course of the axons and they were often interconnected with extremely fine axonal segments. These axons bifurcated only infrequently and often ended as growth cones. These features were common to both uncrossed and crossed corticorubral axons. At later stages of development (PND 28 or later), the total number of swellings decreased and axonal swellings with smooth contours became dominant. A quantitative examination of axonal branches indicated that axons on the ipsilateral side branch occurred more frequently at later stages of development. However, there was no substantial change in branching frequency for the crossed corticorubral fibers during development. In parallel with morphological changes in individual axons, the crossed projection that was initially relatively abundant was reduced during development. Since a PHA-L injection can be confined to a small region of cortex, topographic projections can easily be detected. At PND 7-8 there was no well-defined topographic order in the ipsilateral corticorubral projection. Adult-like topography was first discernible at PND 13. These observations suggest that the unilateral uncrossed corticorubral projection in the adult cat is achieved at least in part by the formation of axonal arbors in the uncrossed projection. This was accompanied by the failure of crossed fibers to form complex arbors. It is possible that a similar mechanism also operates in the formation of topographic maps.

Formation of crossed and uncrossed projections in the central nervous system.

Cellular mechanisms for decision of laterality of projections are discussed based on the observation of corticorubral and interpositorubral projections in the kitten. In both systems unilateral projections in adults appear to emerge from bilateral projections which occur early in development. Early unilateral lesions of the cerebral cortex or the interpositus nucleus cause persistent bilateral projections. Thus the neurons which eventually make a unilateral projection have the capability of forming synaptic connections with their target cells on both sides of the brain. Aberrant crossed (or uncrossed) projections in neonatal animals retract without forming complex axonal arbors, while those in lesioned animals form complex axonal arbors. Thus, success or failure in forming axonal arbors may be crucial for the maintenance of the aberrant crossed (or uncrossed) projections.

Developmental plasticity of the rubrospinal tract in the North American opossum.

We have shown previously that rubral axons can grow caudal to a lesion of their pathway at thoracic levels of the spinal cord in the developing opossum, Didelphis virginiana. In the present report we expand on that observation and present evidence which suggests that the critical period for plasticity of the rubrospinal tract ends earlier at cervical than at thoracic levels. In addition, we show that most rubrospinal neurons die as a result of axotomy during early stages of the critical period. The opossum was chosen for study because the development of its rubrospinal tract occurs after birth. In one set of experiments the area containing the rubrospinal tract was lesioned at cervical or thoracic levels and after 30 days or more, retrograde transport techniques were used to determine if rubral axons had grown caudal to the lesion. When the lesions were made at rostral cervical levels between estimated postnatal day 26 and maturity, neurons could not be labeled in the contralateral red nucleus by injections of retrograde markers ipsilateral to the lesion and caudal to it. We were not able to obtain adequate survival after cervical lesions made prior to estimated postnatal day 26. When the lesions were made at mid to caudal thoracic levels between estimated postnatal days 19 and 26, neurons could be labeled in the contralateral red nucleus. When comparable lesions were made at estimated postnatal day 40, there was usually a decrease in the number of labeled neurons, and when they were made at estimated postnatal day 54, none was labeled. In selected cases, operated at estimated postnatal day 19, cell counts provided evidence for loss of neurons in the red nucleus contralateral to the lesion. In orthograde transport experiments performed on animals with thoracic lesions of the rubrospinal tract made between estimated postnatal days 18 and 33, rubral axons could be labeled caudal to the lesion, and they seemed to take the most direct route around it. Although they sometimes assumed abnormal positions caudal to the lesion, rubral axons appeared to reach areas of the gray matter appropriate to them. When lesions were made at estimated postnatal day 54 or in older animals, labeled axons could be traced to the lesion site but not caudal to it.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for developmental plasticity of the rubrospinal tract. Studies using the North American opossum.

Using axonal tracing techniques we have shown that rubral axons are capable of growing around lesions of the rubrospinal tract during early stages of development in the North American opossum and that a critical period for such growth exists. The opossum was employed for study because it is born in a very immature state 12-13 days after conception and the entire development of its rubrospinal tract occurs postnatally.

Ultrastructural study of remodeled rubral afferents following neonatal lesions in the rat.

Following neonatal hemicerebellectomy, an aberrant ipsilateral cerebellorubral projection develops that maintains the topographic specificity of the normal contralateral projection. Similarly, neonatal lesions of the sensorimotor cortex lead to the appearance of an aberrant contralateral corticorubral projection that mirrors the topographic specificity of the normal ipsilateral input. The specificity of synaptic localization in these aberrant projections was studied by use of ultrastructural visualization of anterogradely transported HRP-WGA. Following neonatal ablations, adults received HRP-WGA injections in the unablated deep cerebellar nuclei or sensorimotor cortex. After 48 hours, animals were sacrificed and processed for ultrastructural localization of anterogradely transported HRP-WGA. In hemicerebellectomized animals, both the contralateral and ipsilateral interpositorubral projections terminated on the somatic and proximal dendritic membrane of magnocellular neurons. Some of these labeled synaptic terminals were located on somatic and dendritic spines. Following HRP-WGA injection in the unablated nucleus lateralis, anterogradely labeled synaptic terminals were located bilaterally on small- to medium-sized dendrites of parvicellular neurons. Injection of HRP-WGA in the remaining sensorimotor cortex of animals that had undergone neonatal unilateral ablation of the sensorimotor cortex resulted in labeled corticorubral synaptic terminals that contacted distal dendrites of ipsilateral and contralateral parvicellular neurons. These results demonstrate that, following neonatal deafferentation of the rat red nucleus, the topographic specificity of the aberrant rubral afferents is accompanied by a specificity of synaptic localization on discrete membrane areas of rubral neurons.

The development of rubrospinal, cerebellorubral, and corticorubral connections in the North American opossum. Evidence for asynchronism.

We have employed axonal transport and degeneration techniques to study the development of major rubral connections in the North American opossum. Opposums were chosen for study because they are born 12 d after conception and have a protracted postnatal development. Our results suggest that: The red nucleus innervates the spinal cord early in development, well before the somatic motor-sensory cortex (Cabana and Martin, 1984); the red nucleus projects to the spinal cord before it receives substantial projections from the cerebellum or cerebral cortex; and projections from the cerebellum reach the red nucleus significantly earlier than those from the cerebral cortex.

The adult organization and development of the rubrospinal tract. An experimental study using the orthograde transport of WGA-HRP in the North-American opossum.

We have employed the orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase to study the organization of rubrospinal connections in adult and pouch young opossums. Our results suggest that: in the adult opossum rubrospinal axons are distributed more widely than suggested by previous studies; rubrospinal projections are formed postnatally in the opossum, but much earlier than corticospinal connections; rubrospinal axons do not grow synchronously, as a massive bundle following a few leading axons, but by addition of axons over a protracted period of time; and the growth of rubral axons into the spinal gray matter follows a predictable rostral to caudal gradient as well as a proximal to distal one relative to the tract. Rubrospinal development is discussed in light of the growth of cerebellar and cortical axons into the red nucleus and the development of motor function.