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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. The majority of cases are sporadic (sALS), while the most common inherited form is due to C9orf72 mutation (C9ALS). A high burden of inclusion pathology is seen in glia (including oligodendrocytes) in ALS, especially in C9ALS. Myelin basic protein (MBP) messenger RNA (mRNA) must be transported to oligodendrocyte processes for myelination, a possible vulnerability for normal function. TDP43 is found in pathological inclusions in ALS and is a component of mRNA transport granules. Thus, TDP43 aggregation could lead to MBP loss. Additionally, the hexanucleotide expansion of mutant C9ALS binds hnRNPA2/B1, a protein essential for mRNA transport, causing potential further impairment of hnRNPA2/B1 function, and thus myelination. Using immunohistochemistry for p62 and TDP43 in human post-mortem tissue, we found a high burden of glial inclusions in the prefrontal cortex, precentral gyrus, and spinal cord in ALS, which was greater in C9ALS than in sALS cases. Double staining demonstrated that the majority of these inclusions were in oligodendrocytes. Using immunoblotting, we demonstrated reduced MBP protein levels relative to PLP (a myelin component that relies on protein not mRNA transport) and neurofilament protein (an axonal marker) in the spinal cord. This MBP loss was disproportionate to the level of PLP and axonal loss, suggesting that impaired mRNA transport may be partly responsible. Finally, we show that in C9ALS cases, the level of oligodendroglial inclusions correlates inversely with levels of hnRNPA2/B1 and the number of oligodendrocyte precursor cells. We conclude that there is considerable oligodendrocyte pathology in ALS, which at least partially reflects impairment of mRNA transport. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

The corticospinal tract (CST) is the major descending pathway controlling voluntary hand function in primates, and though less dominant, it mediates voluntary paw movements in rats. As with primates, the CST in rats originates from multiple (albeit fewer) cortical sites, and functionally different motor and somatosensory subcomponents terminate in different regions of the spinal gray matter. We recently reported in monkeys that following a combined cervical dorsal root/dorsal column lesion (DRL/DCL), both motor and S1 CSTs sprout well beyond their normal terminal range. The S1 CST sprouting response is particularly dramatic, indicating an important, if poorly understood, somatosensory role in the recovery process. As rats are used extensively to model spinal cord injury, we asked if the S1 CST response is conserved in rodents. Rats were divided into sham controls, and two groups surviving post-lesion for ~6 and 10 weeks. A DRL/DCL was made to partially deafferent one paw. Behavioral testing showed a post-lesion deficit and recovery over several weeks. Three weeks prior to ending the experiment, S1 cortex was mapped electrophysiologically, for tracer injection placement to determine S1 CST termination patterns within the cord. Synaptogenesis was also assessed for labeled S1 CST terminals within the dorsal horn. Our findings show that the affected S1 CST sprouts well beyond its normal range in response to a DRL/DCL, much as it does in macaque monkeys. This, along with evidence for increased synaptogenesis post-lesion, indicates that CST terminal sprouting following a central sensory lesion, is a robust and conserved response., (© 2019 Wiley Periodicals, Inc.)

Corticospinal neurons (CSNs) undertake direct cortical outputs to the spinal cord and innervate the upper limb through the brachial plexus. Our previous study has shown that the contralateral middle trunk transfer to the paralyzed upper extremity due to cerebral injury can reconstruct the functional cerebral cortex and improve the function of the paralyzed upper extremity. To interpret the cortical reconstruction and the motor improvement after the middle trunk transfer, we explored the distribution of CSNs connecting to the middle, upper, and lower trunk of the brachial plexus by retrograde trans-neuronal tracing using pseudorabies virus (PRV-EGFP or PRV-mRFP). We show that, rather than an individual specific area, these CSNs labelled by each trunk of the brachial plexus were widespread and mainly assembled within the primary motor cortex (M1), secondary motor cortex (M2), primary somatosensory cortex (S1), and slightly within the secondary somatosensory cortex (S2). The three trunk-labelled CSNs were intermingled in these cortices, and mostly connected to more than two trunks, especially the middle trunk-labelled CSNs with higher proportion of co-labelled neurons. Our findings revealed the distribution features of CSNs connecting to the adjacent spinal nerves that innervate the upper limb, which can improve our understanding of the corticospinal circuits associated with motor improvement and the functional cortical reconstruction after the middle trunk transfer., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Reorganization of residual descending motor circuits underlies poststroke recovery. We previously clarified a causal relationship between the cortico-rubral tract and intensive limb use-induced functional recovery after internal capsule hemorrhage (ICH). However, other descending tracts, such as the cortico-reticular tract, might also be involved in rehabilitation-induced compensation. To investigate whether rehabilitation-induced recovery after ICH involves a shift in the compensatory circuit from the cortico-rubral tract to the cortico-reticular tract, we established loss of function of the cortico-rubral tract or/and cortico-reticular tract using two sets of viral vectors comprising the Tet-on system and designer receptors exclusively activated by the designer drug system. We used an ICH model that destroyed almost 60% of the corticofugal fibers. Anterograde tracing in rehabilitated rats revealed abundant sprouting of axons from the motor cortex in the red nucleus but not in the medullary reticular formation during the early phase of recovery. This primary contribution of the cortico-rubral tract was demonstrated by its selective blockade, whereas selective cortico-reticular tract silencing had little effect. Interestingly, cortico-rubral tract blockade from the start of rehabilitation induced an obvious increase of axon sprouting in the reticular formation with substantial functional recovery. Additional cortico-reticular tract silencing under the cortico-rubral tract blockade significantly worsened the recovered forelimb function. Furthermore, the alternative recruitment of the cortico-reticular tract was gradually induced by intensive limb use under cortico-rubral tract blockade, in which cortico-reticular tract silencing caused an apparent motor deficit. These findings indicate that individual cortico-brainstem pathways have dynamic compensatory potency to support rehabilitative functional recovery after ICH. SIGNIFICANCE STATEMENT This study aimed to clarify the interaction between the cortico-rubral and the cortico-reticular tract during intensive rehabilitation and functional recovery after capsular stroke. Pathway-selective disturbance by two sets of viral vectors revealed that the cortico-rubral tract was involved in rehabilitation-induced recovery of forelimb function from an early phase after internal capsule hemorrhage, but that the cortico-reticular tract was not. The sequential disturbance of both tracts revealed that the cortico-reticular tract was recruited and involved in rehabilitation-induced recovery when the cortico-rubral tract failed to function. Our data demonstrate a dynamic compensatory action of individual cortico-brainstem pathways for recovery through poststroke rehabilitation., (Copyright © 2019 the authors.)

The axonal composition of cortical projections originating in premotor, supplementary motor (SMA), primary motor (a4), somatosensory and parietal areas and descending towards the brain stem and spinal cord was characterized in the monkey with histological tract tracing, electron microscopy (EM) and diffusion MRI (dMRI). These 3 approaches provided complementary information. Histology provided accurate assessment of axonal diameters and size of synaptic boutons. dMRI revealed the topography of the projections (tractography), notably in the internal capsule. From measurements of axon diameters axonal conduction velocities were computed. Each area communicates with different diameter axons and this generates a hierarchy of conduction delays in this order: a4 (the shortest), SMA, premotor (F7), parietal, somatosensory, premotor F4 (the longest). We provide new interpretations for i) the well-known different anatomical and electrophysiological estimates of conduction velocity; ii) why conduction delays are probably an essential component of the cortical motor command; and iii) how histological and dMRI tractography can be integrated.

Uncontrollable itch-scratching cycles lead to serious skin damage in patients with chronic itch. However, the neural mechanism promoting the itch-scratching cycle remains elusive. Here, we report that tachykinin 1 (Tac1)-expressing glutamatergic neurons in the lateral and ventrolateral periaqueductal gray (l/vlPAG) facilitate the itch-scratching cycle. We found that l/vlPAG neurons exhibited scratching-behavior-related neural activity and that itch-evoked scratching behavior was impaired after suppressing the activity of l/vlPAG neurons. Furthermore, we showed that the activity of Tac1-expressing glutamatergic neurons in the l/vlPAG was elevated during itch-induced scratching behavior and that ablating or suppressing the activity of these neurons decreased itch-induced scratching behavior. Importantly, activation of Tac1-expressing neurons induced robust spontaneous scratching and grooming behaviors. The scratching behavior evoked by Tac1-expressing neuron activation was suppressed by ablation of spinal neurons expressing gastrin-releasing peptide receptor (GRPR), the key relay neurons for itch. These results suggest that Tac1-expressing neurons in the l/vlPAG promote itch-scratching cycles., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. This complexity presents a major challenge to interpreting functional outcomes from therapies that target supraspinal connectivity after injury or disease, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate that injection of AAV2-Retro to the cervical spinal cord of adult female mice results in highly efficient transduction of supraspinal populations throughout the brainstem, midbrain, and cortex. Some supraspinal populations, including corticospinal and rubrospinal neurons, were transduced with >90% efficiency, with robust transgene expression within 3 d of injection. In contrast, propriospinal and raphe spinal neurons showed much lower rates of retrograde transduction. Using tissue clearing and light-sheet microscopy we present detailed visualizations of descending axons tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors resulted in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde vectors were also highly efficient when injected after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs. SIGNIFICANCE STATEMENT The complexity of descending inputs to the spinal cord presents a major challenge in efforts deliver therapeutics to widespread supraspinal systems, and to interpret their functional effects. Here we demonstrate highly effective gene delivery to diverse supraspinal nuclei using a retrograde viral approach and combine it with tissue clearing and 3D microscopy to map the descending projectome from brain to spinal cord. These data highlight newly developed retrograde viruses as therapeutic and research tools, while offering new insights into supraspinal connectivity., (Copyright © 2018 the authors 0270-6474/18/3810566-16$15.00/0.)

Corticospinal tract (CST) axons from one hemisphere normally extend and terminate predominantly in the contralateral spinal cord. We previously showed that deleting the gene phosphatase and tensin homolog (PTEN) in the sensorimotor cortex enables CST axons to regenerate after spinal cord injury and that some regenerating axons extend along the "wrong" side. Here, we characterize the degree of specificity of regrowth in terms of laterality. PTEN was selectively deleted via cortical adeno-associated virus (AAV)-Cre injections in neonatal PTEN-floxed mice. As adults, mice received dorsal hemisection injuries at T12 or complete crush injuries at T9. CST axons from one hemisphere were traced by unilateral biotinylated dextran amine (BDA) injections in PTEN-deleted mice with spinal cord injury and in noninjured PTEN-floxed mice that had not received AAV-Cre. In noninjured mice, 97.9 ± 0.7% of BDA-labeled axons in white matter and 88.5 ± 1.0% of BDA-labeled axons in gray matter were contralateral to the cortex of origin. In contrast, laterality of CST axons that extended past a lesion due to PTEN deletion varied across animals. In some cases, regenerated axons extended predominantly on the ipsilateral side; in other cases, axons extended predominantly contralaterally, and in others, axons were similar in numbers on both sides. Similar results were seen in analyses of cases from previous studies using short hairpin (sh)RNA-mediated PTEN knock-down. These results indicate that CST axons that extend past a lesion due to PTEN deletion or knock-down do not maintain the contralateral rule of the noninjured CST, highlighting one aspect of how the resultant circuitry from regenerating axons may differ from that of the uninjured CST. J. Comp. Neurol. 524:2654-2676, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Evidence has suggested that cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) is correlated with the development and recurrence of pain. A recent research showed that the CSF-contacting nucleus acts as a component of the descending 5-hydroxytryptamine (5-HT) system and plays a role in descending pain inhibition. However, limited studies are conducted to investigate the relationship between the CSF-contacting nucleus and pain. In present study, we explored the effect of CSF-contacting nucleus on nociceptive behaviors in both normal and neuropathic rats via targeted ablation of the CSF-contacting nucleus in the brainstem, using cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. The CB-SAP-treated rats showed aggravated thermal hyperalgesia and mechanical allodynia. Also, results from immunohistochemical experiments showed that rostral ventromedial medulla (RVM) received fiber projection from the CSF-contacting nucleus, which disappeared after ablation of the CSF-contacting nucleus, and the CB-SAP treated rats showed downregulation of c-Fos expression in the RVM as compared with the rats receiving i.c.v. injection of phosphate buffer saline (PBS). A significant downregulation of 5-HT-labeled neurons and tryptophan hydroxylase 2 (TPH2) as the marker of 5-HT cells in the RVM, and 5-HT expression in spinal dorsal horn in both normal and chronic constriction injury (CCI) rats after i.c.v. injection of CB-SAP was observed. These results suggested that RVM may be involved in descending pain modulation originating from the CSF-contacting nucleus.

Objective: To compare the results of magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) in amyotrophic lateral sclerosis (ALS) patients., Methods: Nineteen ALS patients and thirteen age-matched healthy controls underwent MRS and DTI between October 2013 and July 2014. Fractional anisotropy (FA), apparent diffusion coefficient (ADC), N-acetylaspartate (NAA), choline (Cho), and creatine (Cr) were collected as the quantitative results of the imaging study. The ALS functional rating scale-revised (ALSFRS-R) and disease progression rate were evaluated to assess patients' disability. The imaging study results were compared between ALS patients and healthy controls. The relationship between disability assessment and imaging study results was analyzed., Results: NAA/Cr in the motor cortex and FA in the corticospinal tract (CST) of both sides were significantly lower in patients than controls. There was no significant difference between the two groups in Cho/Cr, tract length, tract volume, ADC or NAA. No relationship was found between ALSFRS-R and FA (r=0.243, p=0.316) in the right CST; NAA (r=0.095, p=0.699) or NAA/Cr (r=0.172, p=0.481) in the left motor cortex; or NAA (r=0.320, p=0.182) or NAA/Cr (r=0.193, p=0.492) in the right motor cortex. There was no relationship between the disease progression rate and FA, NAA, or NAA/Cr on either side., Conclusion: NAA/Cr and FA can help diagnose ALS. Regional brain NAA/Cr and FA values could not assess the ALSFRS-R or disease progression rate., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bulbospinal neurons in the ventral medulla play important roles in the regulation of sympathetic outflow. Physiological evidence suggests that these neurons are activated by N-methyl-D-aspartate (NMDA) and non-NMDA subtypes of glutamate receptors. In this study, we examined bulbospinal neurons in the ventral medulla for the presence of immunoreactivity for the NMDA NR1 subunit, which is essential for NMDA receptor function. Rats received bilateral injections of cholera toxin B into the tenth thoracic spinal segment to label bulbospinal neurons. Triple immunofluorescent labeling was used to detect cholera toxin B with a blue fluorophore, NR1 with a red fluorophore, and either tyrosine hydroxylase or tryptophan hydroxylase with a green fluorophore. In the rostral ventrolateral medulla, NR1 occurred in all bulbospinal tyrosine hydroxylase-positive neurons and 96% of bulbospinal tyrosine hydroxylase-negative neurons, which were more common in sections containing the facial nucleus. In the raphe pallidus, the parapyramidal region, and the marginal layer, 98% of bulbospinal tryptophan hydroxylase-positive neurons contained NR1 immunoreactivity. NR1 was also present in all of the bulbospinal tryptophan hydroxylase-negative neurons, which comprised 20% of bulbospinal neurons in raphe pallidus and the parapyramidal region. These results show that virtually all bulbospinal tyrosine hydroxylase and non-tyrosine hydroxylase neurons in the rostral ventrolateral medulla and virtually all bulbospinal serotonin and non-serotonin neurons in raphe pallidus and the parapyramidal region express NR1, the obligatory subunit of the NMDA receptor. NMDA receptors on bulbospinal neurons in the rostral ventral medulla likely influence sympathoexcitation in normal and pathological conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Neurons in the rostral ventromedial medulla (RVM) are thought to modulate nociceptive transmission via projections to spinal and trigeminal dorsal horns. The cellular substrate for this descending modulation has been studied with regard to projections to spinal dorsal horn, but studies of the projections to trigeminal dorsal horn have been less complete. In this study, we combined anterograde tracing from RVM with immunocytochemical detection of the GABAergic synthetic enzyme, GAD67, to determine if the RVM sends inhibitory projections to trigeminal dorsal horn. We also examined the neuronal targets of this projection using immunocytochemical detection of NeuN. Finally, we used electron microscopy to verify cellular targets. We compared projections to both trigeminal and spinal dorsal horns. We found that RVM projections to both trigeminal and spinal dorsal horn were directed to postsynaptic profiles in the dorsal horn, including somata and dendrites, and not to primary afferent terminals. We found that RVM projections to spinal dorsal horn were more likely to contact neuronal somata and were more likely to contain GAD67 than projections from RVM to trigeminal dorsal horn. These findings suggest that RVM neurons send predominantly GABAergic projections to spinal dorsal horn and provide direct input to postsynaptic neurons such as interneurons or ascending projection neurons. The RVM projection to trigeminal dorsal horn is more heavily targeted to dendrites and is only modestly GABAergic in nature. These anatomical features may underlie differences between trigeminal and spinal dorsal horns with regard to the degree of inhibition or facilitation evoked by RVM stimulation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background and Purpose: Pentobarbital is known to affect cerebral metabolism; pentobarbital sedation is, however, frequently used for MR imaging and MR spectroscopy, especially in children. Accurate assessment of the brain metabolite levels is important, particularly in neonates with suspected brain injury. We investigated whether pentobarbital sedation has any effect on the ratios of spectral metabolites lactate, N-acetylaspartate, or choline in a group of premature neonates., Materials and Methods: MR spectroscopy was performed in 43 premature neonates, all with normal concurrent MR imaging and normal neurodevelopmental outcome at 12 months of age. Of those neonates, 14 (33%) required pentobarbital (Nembutal 1 mg/kg) sedation during MR spectroscopy; the remaining 29 neonates did not receive any sedation. Ratios of lactate, choline, and N-acetylaspartate were calculated in the basal ganglia, thalami, and corticospinal tracts and compared between those neonates with and without sedation., Results: Small amounts of brain lactate were detected in all of the premature neonates. The basal ganglia lactate/choline and lactate/N-acetylaspartate ratios were significantly lower, by 17% and 25% respectively, in the neonates with pentobarbital sedation compared with the age-matched neonates without sedation (P < .05). Sedation did not affect the lactate level in the thalami or the corticospinal tracts. The N-acetylaspartate/choline ratios were unaffected by pentobarbital sedation., Conclusion: Pentobarbital sedation is associated with lower lactate/choline and lactate/N-acetylaspartate ratios in the basal ganglia of premature neonates, as determined by proton MR spectroscopy. Investigators should be aware of this phenomenon for accurate interpretation of their MR spectroscopy results.

Inhibition of RhoA has been shown to enhance axonal regeneration following spinal cord injury. Here we mapped mRNA expression patterns of RhoA, B, and C, Rac1, Cdc42, and Tc10 in spinal cord, sensory ganglia, and sensorimotor cortex in uninjured rats, and following spinal cord injury or sham laminectomy. In the intact spinal cord, neurons displayed high levels of Rac1, Cdc42, and Tc10 mRNA hybridization signal. GFAP-immunoreactive astrocytes expressed primarily RhoB and Rac1, while oligodendrocyte-like cells expressed RhoA, Rac1, and Cdc42. Injury caused profound, long-lasting upregulation of RhoA, Rac1, Cdc42, and Tc10 mRNA in the spinal cord, while RhoB was modestly increased and RhoC did not change. GFAP-immunoreactive reactive astrocytes exhibited a dramatic increase of RhoA mRNA expression along with increases of Rac1 and Cdc42. Injury also led to elevation of RhoA, Cdc42, and Tc10 in neurons and modest increases of RhoA, Rac1, and Tc10 in oligodendrocyte-like cells. Laminectomy caused similar, but less pronounced alterations of investigated mRNA species. In dorsal root ganglia neuronal RhoA, Rac1, Cdc42, and Tc10 mRNA levels were increased similarly by spinal cord injury and sham surgery. The CST pyramidal cells expressed Tc10 mRNA and the CST itself was Tc10-immunoreactive. Tc10-immunoreactivity disappeared distal to injury. We conclude that there are gene-specific patterns of expression of the six different Rho-GTPases in normal spinal cord and dorsal root ganglia, and that specific changes of temporal and spatial expression patterns occur in response to spinal cord injury, suggesting different roles of these GTPases in the cellular sequelae of CNS injury., (Copyright 2005 Wiley-Liss, Inc.)

We report here the pattern of axonal branching for 11 descending cell types in the larval brainstem; eight of these cell types are individually identified neurons. Large numbers of brainstem neurons were retrogradely labeled in living larvae by injecting Texas-red dextran into caudal spinal cord. Subsequently, in each larva a single identified cell was injected in vivo with Alexa 488 dextran, using fluorescence microscopy to guide the injection pipette to the targeted cell. The filling of cells via pressure pulses revealed distinct and often extensive spinal axon collaterals for the different cell types. Previous fills of the Mauthner cell had revealed short, knob-like collaterals. In contrast, the two segmental homologs of the Mauthner cell, cells MiD2cm and MiD3cm, showed axon collaterals with extensive arbors recurring at regular intervals along nearly the full extent of spinal cord. Furthermore, the collaterals of MiD2cm crossed the midline at frequent intervals, yielding bilateral arbors that ran in the rostral-caudal direction. Other medullary reticulospinal cells, as well as cells of the nucleus of the medial longitudinal fasciculus (nMLF), also exhibited extensive spinal collaterals, although the patterns differed for each cell type. For example, nMLF cells had extensive collaterals in caudal medulla and far-rostral spinal cord, but these collaterals became sparse more caudally. Two cell types (CaD and RoL1) showed arbors projecting ventrally from a dorsally situated stem axon. Additional cell-specific features that seemed likely to be of physiological significance were observed. The rostral-caudal distribution of axon collaterals was of particular interest because of its implications for the descending control of the larva's locomotive repertoire. Because the same individual cell types can be identified from fish to fish, these anatomical observations can be directly linked to data obtained in other kinds of experiments. For example, 9 of the 11 cell types examined here have been shown to be active during escape behaviors., (Copyright 2003 Wiley-Liss, Inc.)

Objective: Examining the unresolved relationship between the lower motor neuron disorder progressive muscular atrophy (PMA) and ALS is important in clinical practice because of emerging therapies., Methods: Spinal and brainstem tissues donated from patients with ALS/motor neuron disorder (n = 81) were examined. Using retrospective case note review, the authors assigned patients into three categories: PMA (12), PMA progressing to ALS (6), and ALS ab initio (63). Conventional stains for long tract degeneration and immunocytochemistry for ubiquitin and the macrophage marker CD68 were examined., Results: Rapid progression and typical ubiquitinated inclusions in lower motor neurons were present in 77 (95%) of the cases. Immunocytochemistry for CD68 was a more sensitive marker of long tract pathology in comparison with conventional stains. Half of the cases with PMA showed corticospinal tract degeneration by CD68., Conclusion: Patients with PMA frequently have undetected long tract pathology and most have ubiquitinated inclusions typical of ALS. A patient presenting with PMA with rapid clinical evolution likely has the pathology and pathophysiology of ALS whether or not upper motor neuron signs evolve.

Feedback controlling is an important element in the sensory processing in the auditory system. It has been long recognized that the inferior colliculus (IC) sends direct ascending projections to the medial geniculate body (MGB), but receives feedback regulation from the auditory cortex. In the present study we probed the shorter extracortical projections to the IC, including the direct descending pathway from the MGB. In the rat, the fluorescence retrograde tracers Fluorogold, True Blue or Rhodamine latex microspheres were injected into the IC, and the auditory thalamus and surrounding regions were examined for fluorescent neurones. We did not find any retrograde labelling in the ventral division of the MGB. However, retrogradely labelled neurones were found in the medial and suprageniculate nuclei of the MGB. We also observed densely packed groups of fluorescent neurones in the peripeduncular nucleus and numerous labelled neurones in the nucleus of the brachium of the IC. The existence of a direct descending pathway to the IC from at least some auditory thalamic nuclei challenges the perception of the colliculo-thalamic relationship as one-way traffic and suggests more direct involvement of the auditory thalamus in the feedback regulation of the incoming acoustic signals.

L1 cell adhesion molecule (L1CAM) gene mutations are associated with X-linked 'recessive' neurological syndromes characterized by spasticity of the legs. L1CAM knock-out mice show hypoplasia of the corticospinal tract and failure of corticospinal axonal decussation and projection beyond the cervical spinal cord. The aim of this study was to determine if similar neuropathology underlies the spastic diplegia of males hemizygous for L1CAM mutations. Studies were performed on eight carrier females and 10 hemizygous males. Transcranial magnetic stimulation excited the corticospinal tract and responses were recorded in biceps brachii and quadriceps femoris. In contralateral biceps and quadriceps the responses had high thresholds and delayed onset compared with normal subjects. Ipsilateral responses in biceps were smaller, with higher thresholds and delayed onsets relative to contralateral responses. Subthreshold corticospinal conditioning of the stretch reflex of biceps and quadriceps was abnormal in both hemizygous males and carrier females suggesting there may also be a reduced projection to inhibitory interneurones. Histological examination of post-mortem material from a 2-week-old male with an L1CAM mutation revealed normal corticospinal decussation and axonal projections to lumbar spinal segments. These data support a role for L1CAM in corticospinal tract development in hemizygous males and 'carrier' females, but do not support a critical role for L1CAM in corticospinal axonal guidance.

After traumatic injury, adult central nervous system (CNS) axons fail to regenerate. We have previously shown that one major impediment for axon regeneration is the basement membrane (BM) forming at the lesion center, by means of a wound healing collagenous scar, after lesioning the postcommisural fornix of the adult rat [Eur. J. Neurosci. 11 (1999) 632] [6]. This BM consists of a supramolecular network of collagen type IV, laminin (LN), nidogen, and associated proteoglycans [Crit. Rev. Biochem. Mol. Biol. 27 (1992) 93] [5]. Following axotomy, axons of the proximal stump of the transected postcommissural fornix fail to cross the lesion site. This regenerative failure is spatially and temporally highly correlated with the appearance of BM in the lesion site [Restor. Neurol. Neurosci. 15 (1999) 1] [7]. However, if the deposition of BM is prevented, the injured axons: (i) regenerate in their former pathway, (ii) are conductive across and behind the lesion site, and (iii) form chemical synapses in their target area, the mammillary body [Eur. J. Neurosci. 11 (1999) 632]. The developing BM is surrounded by neuropil and can easily be stained immunohistochemically using anti-collagen IV antibodies on fresh frozen sections (10 microm). To examine a clinically more relevant model of traumatic CNS injuries we developed a transection model of the thoracic dorsal corticospinal tract (CST) in the rat spinal cord. In contrast to fornix lesion this transection is performed in close proximity to the meninges. This involves the BM being completely washed out if fresh frozen tissue is used on slides. If the animals are sacrificed by perfusion with aldehydes the collagen IV and the LN antigen are masked by the fixative. To restore the correct immunohistochemical staining pattern (BM, blood vessels) a special protocol including an enzyme digestion is necessary. If thick sections are stained free floating, the tissue is destroyed due to the enzyme treatment. Here we present a method to prevent loss of the lesion-induced BM and to perform the correct immunohistochemical stainings of BM proteins in the traumatically injured spinal cord.

The devastating consequences of spinal cord injury (SCI) result primarily from damage to long tracts in the spinal white matter. To elucidate the secondary injury processes occurring after SCI, we investigated the relationship between apoptosis and Wallerian degeneration in spinal white matter tracts. In the rat spinal cord, the corticospinal tract (CST) and the dorsal ascending tract (DAT) are separated from each other in the dorsal column and relay information in opposite directions. A dorsal column cordotomy at the eighth thoracic (T8) level simultaneously induces Wallerian degeneration in the CST caudal to and in the DAT rostral to the injury. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, we demonstrate that apoptosis occurred in areas of Wallerian degeneration in both tracts throughout the length of the cord segments studied (from T3 to T12). This delayed cell death, more apparent in the DAT, began at 7 days after injury and peaked at 14 days for the DAT and 28 days for the CST. Although a few TUNEL+ cells, slightly above the noninjury control level, were found in intact areas of both tracts, statistically significant differences in the number of TUNEL+ cells were found between the intact and the lesioned tract segments (CST, F < 0.01; DAT, F < 0.001). Within a particular spinal segment, a mean number of 64 and 939 TUNEL+ cells in the degenerating CST and DAT, respectively, were estimated stereologically at 14 days postinjury. TUNEL+ cells in degenerating tracts outnumber their intact counterparts by 3.8:1 in the CST and 4.1:1 in the DAT, although a statistically significant difference between the two was only found in the DAT at this time point (P < 0.05). Finally, we demonstrated that oligodendrocytes, the myelin-forming cells in the central nervous system, constitute at least a portion of the cells undergoing apoptosis within areas of Wallerian degeneration.

It is widely accepted that the devastating consequences of spinal cord injury are due to the failure of lesioned CNS axons to regenerate. The current study of the spontaneous tissue repair processes following dorsal hemisection of the adult rat spinal cord demonstrates a phase of rapid and substantial nerve fibre in-growth into the lesion that was derived largely from both rostral and caudal spinal tissues. The response was characterized by increasing numbers of axons traversing the clearly defined interface between the lesion and the adjacent intact spinal cord, beginning by 5 days post operation (p.o.). Having penetrated the lesion, axons became associated with a framework of NGFr-positive non-neuronal cells (Schwann cells and leptomeningeal cells). Surprisingly few of these axons were derived from CGRP- or SP-immunoreactive dorsal root ganglion neurons. At the longest survival time (56 days p.o.), there was a marked shift in the overall orientation of fibres from a largely rostro-caudal to a dorso-ventral axis. Attempts to identify which recognition molecules may be important for these re-organizational processes during attempted tissue repair demonstrated the widespread and intense expression of the cell adhesion molecules (CAM) L1 and N-CAM. Double immunofluorescence suggested that both Schwann cells and leptomeningeal cells contributed to the pattern of CAM expression associated with the cellular framework within the lesion.

RC3 (neurogranin; BICKS) is a neuron-specific calmodulin-binding protein kinase C substrate. Thus far, immunohistochemical studies on the localization of RC3 revealed its presence in all neuronal phenotypes, which were restricted to specific areas in the neostriatum, the neocortex, and the hippocampus. RC3 was mostly found in cell bodies and dendrites, with some infrequent presence in axonal profiles, i.e. in the internal capsule. Until now, RC3 expression was reported to be absent in the adult rat spinal cord. RC3 might, however, act as an intermediate of protein kinase C-mediated signaling pathways during synaptic development and plasticity. We hypothesized a role for this 78-amino-acid protein in dendritic plasticity occurring after spinal cord injury. To our surprise, an immunohistological analysis of the uninjured adult rat spinal cord revealed the presence of RC3-positive cell bodies and dendrites in specific regions in the gray matter. Interestingly, axon-containing structures, such as the dorsal and ventral corticospinal tract, were also found to be RC3-positive. This axonal labeling was confirmed by preembedding electron microscopy. In conclusion, we demonstrate here that RC3 is present in the adult rat spinal cord in pre- and postsynaptic structures., (Copyright 2000 Wiley-Liss, Inc.)

Objective: To seek regional metabolite abnormalities in patients with Kennedy disease (KD) using proton magnetic resonance spectroscopy., Design: Nine patients with KD showing the typical phenotype without clinical signs of upper motor neuron involvement were compared with 17 male, age-matched, healthy control subjects. Relative metabolite concentrations for N-acetyl (NA) groups, choline-containing groups (Cho), phosphocreatine (Cr), and lactate (Lac) were determined in the brainstem and the motor region., Results: Pathologic Lac signals suggesting impaired energy metabolism were absent in patients and controls. In the brainstem area, patients with KD showed a significant reduction in the NA/Cho metabolite ratio (P = .01). In the motor region, NA/Cho (P = .04) and NA/Cr (P = .03) ratios were significantly reduced. The reduction of the NA/Cho ratio in the motor region mainly resulted from decreased metabolite ratios in 3 patients. Changes in metabolite ratios did not correlate with the number of trinucleotide cytosine-adenine-guanine repeats from leukocytes. Because of the relatively small sample size due to the rarity of KD, these results should be considered preliminary., Conclusions: Spectroscopic data fail to provide further evidence for altered energy metabolism in KD. Metabolite changes in the brainstem indicate a reduction of the neuronal marker NA or elevated Cho. These findings may reflect neuronal loss or gliosis consistent with the known pathologic features. In a subset of patients, altered metabolite ratios best explained by neuronal loss suggest subclinical involvement of the motor region. The extent of metabolite changes does not correlate with the trinucleotide repeat length.

Traumatic brain injury (TBI) in animals and man generates widespread axonal injury characterized by focal axolemmal permeability changes, induction of calpain-mediated proteolysis, and neurofilament side-arm modification associated with neurofilament compaction (NFC) evolving to axonal disconnection. Recent observations have suggested that moderate hypothermia is neuroprotective in several models of TBI. Nevertheless, the pathway by which hypothermia prevents traumatic axonal injury (TAI) is still a matter of debate. The present study was conducted to evaluate the effects of moderate, early posttraumatic hypothermia on calpain-mediated spectrin proteolysis (CMSP), implicated in the pathogenesis of TAI. Using moderate (32 degrees C) hypothermia of 90 min duration without rewarming, the density of CMSP immunoreactive/damaged axons was quantified via LM analysis in vulnerable brain stem fiber tracts of hypothermic and normothermic rats subjected to impact acceleration TBI (90 min postinjury survival). To assess the influence of posthypothermic rewarming, a second group of animals was subjected to 90 min of hypothermia followed by 90 min of rewarming to normothermic levels when CMSP was analyzed to detect if any purported CMSP prevention persisted (180 min postinjury survival). Additionally, to determine if this protection translated into comparable cytoskeletal protection in the same foci showing decreased CMSP, antibodies targeting altered/compacted NF subunits were also employed. Moderate hypothermia applied in the acute postinjury period drastically reduced the number of damaged axons displaying CMSP at both time points and significantly reduced NFC immunoreactivity at 180 min postinjury. These results suggest that the neuroprotective effects of hypothermia in TBI are associated with the inhibition of axonal/cytoskeletal damage., (Copyright 1999 Academic Press.)

Mutations in the cell adhesion molecule L1 cause severe developmental anomalies in the human nervous system. Recent descriptions of L1 gene knock-out mice from three research groups demonstrate that these mice are strikingly similar to humans with mutations in the L1 gene. In both humans and mice there are defects in the development of the corticospinal tract and cerebellar vermis, hydrocephalus, and impaired learning. The production of a viable animal model for X-linked hydrocephalus suggests that unanswerable questions posed by the human disease will finally be approachable using modern experimental methods., (Copyright 1998 Academic Press.)

The temporal changes in intensity of myelination of the nervous pathways in 0 to 42-day-old Wistar rats were quantitatively analyzed by immunohistochemistry with anti-proteolipid protein and compared with that obtained by immunohistochemistry with anti-myelin basic protein. Immunohistochemistry was performed on paraffin-embedded tissue according to the standard ABC technique. Intensity of myelination was examined by an image analyzing system. We analyzed nine nervous pathways: corpus callosum, optic tract, internal capsule, spinal tract of the trigeminal nerve, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract, medial longitudinal fasciculus, and cuneate fasciculus. The presence of immunoreactive fibers for proteolipid protein (PLP) in the spinal tract of the trigeminal nerve, medial longitudinal fasciculus and cuneate fasciculus was noted on postnatal day 0. Those of the corpus callosum, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract and internal capsule were noted on day 7, and that of optic tract on day 14. The time required to reach the intensity of myelination of day 42 was day 14 for the cuneate fasciculus, day 21 for the spinal tract of the trigeminal nerve, inferior cerebellar peduncle and medial longitudinal fasciculus, day 28 for the optic and pyramidal tracts, day 35 for the corpus callosum and day 42 for the internal capsule and cerebellar white matter. The appearance of immunoreactive fibers for PLP was usually earlier than that for myelin basic protein (MBP) and the pattern of difference between PLP and MBP can be classified into three groups: (1) their time of appearance and progress are almost the same, as in the optic tract; (2) the appearance and progress of PLP occurs earlier than those of MBP, as in the pyramidal tract; (3) the appearance of PLP occurs earlier than that of MBP, but their progress is the same. Our findings revealed that the time of appearance and progress of myelination as measured by PLP are different among the nervous pathways, and that there is also a difference between PLP and MBP. This difference between PLP and MBP may indicate a functional difference between them.

Activation of serotonin-1A receptors (5-HT(1A)R) in the medulla oblongata lowers sympathetic nerve discharge and blood pressure. Binding sites for 5-HT(1A)R ligands are present in ventral medullary nuclei [e.g., rostral ventrolateral medulla (RVLM), raphe pallidus (RPa), and parapyramidal region (PPR)] that project to sympathetic preganglionic neurons in the intermediolateral cell column (IML). However, the projections and the neurochemical contents of the ventral medullary neurons that are likely to be involved in the hypotensive actions of 5-HT(1A) agonists are unclear. Using a sheep antibody to a fragment of the third intracellular loop of the 5-HT(1A)R, we localized 5-HT(1A)R immunoreactivity (ir) to IML-projecting neurons that were retrogradely labeled with rhodamine beads injected into the IML of adult male rats. The percentages of IML-projecting neurons containing 5-HT(1A)R-ir were 49% in RPa, 34% in PPR, and 44% in RVLM. Using multiple-immunofluorescence labeling, we also demonstrated 5-HT(1A)R-ir in serotonergic (5-HT) and in catecholaminergic (tyrosine hydroxylase; TH-ir) neurons of the ventral medulla. The percentages of 5-HT-ir neurons containing 5-HT(1A)R-ir were 28% in RPa, 18% in PPR, and 31% in raphe obscurus. In addition, 5-HT(1A)R-ir was present in 14% of TH-ir neurons of the RVLM. Moreover, some IML-projecting neurons in the PPR and RPa were doubly immunolabeled for 5-HT(1A)R-ir and 5-HT, and some IML-projecting neurons in the RVLM were doubly immunolabeled for 5-HT(1A)R-ir and TH-ir. These data provide anatomical evidence for the presence of 5-HT(1A)R on serotonergic and catecholaminergic bulbospinal neurons and for their potential role in directly modifying the activity of these ventral medullary neurons.

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer Dil. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry.

The neural cell adhesion molecule N-CAM has been proposed to function in the guidance of outgrowing axons in the peripheral and central nervous system. Light microscopic observations have shown that the embryonic form of N-CAM (200-230 kDa) is present in the ventralmost part of the dorsal funiculus during corticospinal tract (CST) ingrowth in the first postnatal week (Joosten, Dev. Brain Res., 78 (1994) 226-236). Here, the subcellular localization of the embryonic form of N-CAM (E-NCAM) is determined by pre-embedding staining on vibratome sections and by postembedding immunogold-labelling on Epon embedded spinal cord sections. The electron microscopical observations indicate that E-NCAM is present on the outer membrane of CST growth cones as well as other unmyelinated axons which are present in the ventralmost part of the dorsal funiculus. Furthermore, E-NCAM is localized in an irregular patchy way on the outer side of the axonal membrane of small unmyelinated, later arriving CST axons. From these results it may be deduced that E-NCAM is involved in CST tract formation through guidance of outgrowing pioneer CST growth cones along other unmyelinated axons and through mediation of axon fasciculation of later arriving CST axons.

The coexistence of neurokinin A (NKA) with substance P (SP) and serotonin (5-HT) in ventral medullary neurons of the parapyramidal region and nucleus raphe pallidus of the rat was studied using multiple immunofluorescence labeling. Nearly all of the NKA-immunoreactive (IR) cells in the parapyramidal region and raphe pallidus were SP-IR nd 5-HT-IR, whereas about 70% of the SP-IR neurons and about 60% of the 5-HT-IR neurons contained NKA-IR. There were no apparent differences in the patterns of coexistence between parapyramidal and raphe pallidus neurons. NKA-IR neurons, which colocalized SP-IR and 5-HT-IR, were studied for projections to the lumbar and thoracic spinal cord by use of retrograde transport of fluorescent tracer. Whereas about 50% of the retrogradely labeled neurons of the parapyramidal region and raphe pallidus contained NKA-IR, nearly all of the NKA-IR neurons projected to the thoracic and lumbar spinal cord. In addition, some NKA-IR neurons in the ventral medulla were retrogradely labeled with tracer from localized injections into the thoracic intermediolateral cell column. In summary, this study demonstrated that NKA-IR is colocalized with SP-IR in bulbospinal serotonergic neurons of the parapyramidal region and raphe pallidus, which are known to regulate sensory, motor, and autonomic activities of the spinal cord.

Centrosomes are unique cytoplasmic structures which serve as microtubule organizing centers (MTOC). In most animal cells centrosomes consist of one or more pair of centrioles surrounded by electron dense amorphous pericentriolar material (PCM) responsible for nucleation of microtubules. In the present study we analyzed the pattern of induction and localization of proteins of the PCM at different stages of neuronal development in cell cultures prepared from the embryonic hippocampus. For this purpose we used a human polyclonal antibody that recognizes two proteins of the PCM (100 kd and 60 kd, respectively). The results indicate that in mature neurons, pericentriolar immunoreactive material is preferentially localized in dendritic processes, and that throughout the course of neurite development and differentiation it is systematically excluded from the neuron's axon. Western blot analysis showed that during neuronal development in situ, there is an increase in the immunoreactivity for both proteins recognized by this antibody. In contrast, in hippocampal pyramidal neurons that develop in culture, there is an increase in the 60 kd polypeptide, while the 100 kd one is not detected after 7 days in vitro.

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.

Using quantitative autoradiography, the anatomical distribution of the binding sites (kainate, N-methyl-D-aspartate and quisqualate) for the excitatory neurotransmitter glutamate has been established in the hippocampal formation from control and schizophrenic brains, post mortem. There is a loss of the kainate subtype particularly in schizophrenic hippocampi mainly from the CA4/CA3 mossy fibre termination zone of the cornu ammonis (CA4 and CA3; control and schizophrenic left hippocampus, respectively, 54.2 and 66.6 pmol/g; 18.3 and 17.9 pmol/g), as well as bilateral losses in the dentate gyrus (left 14.2 pmol/g and right 28.0 pmol/g; left 9.5 pmol/g and right 7.9 pmol/g, control and schizophrenic, respectively) and parahippocampal gyrus (left 50.8 pmol/g and right 41.7 pmol/g, left 27.7 pmol/g and right 25.3 pmol/g, control and schizophrenic, respectively). There is complete preservation of N-methy-D-aspartate sites in schizophrenic hippocampi, and a marginally significant loss of the quisqualate binding site in CA4/CA3 regions (left 249 fmol/g and right 306 fmol/g, left 157 fmol/g and right 148 fmol/g, control and schizophrenic, respectively). These findings reflect the possible importance of glutamate in the pathophysiology of schizophrenia and represent novel targets for therapeutic manipulation in schizophrenia.

A double electronmicroscopical (EM) staining was developed which enabled the ultrastructural localization of cell adhesion molecules on the outer axonal membrane of horseradish peroxidase (HRP)-labelled axons in the developing central nervous system (CNS). HRP was used to anterogradely trace outgrowing corticospinal tract (CST) axons in ten-day-old rats. After visualization of HRP using tetramethylbenzidine (TMB) as a chromogen and ammoniumheptamolybdate (AHM) as a stabilizer at pH 6.0 as described previously (Joosten et al. 1987, J Histochem Cytochem 35: 623-626) an additional diaminobenzindine (DAB)-Ni incubation was carried out for further stabilization. Subsequently a preembedding immunoperoxidase (DAB) staining was executed for detection of cell adhesion molecule L1. Using this procedure anterogradely HRP-labelled CST axons were recognizable by a granular black TMB-AHM-DABNi reaction product at the light microscopic (LM) level, which clearly contrasts to the relatively homogeneous brown L1-immunostaining. Electronmicroscopically HRP-labelled CST axons were characterized by the presence of an intracellular crystaloid TMB-AHM-DABNi reaction product which made identification of CST axons rather easy, whereas the L1-DAB precipitate could be noted on the outer axonal membrane of the HRP-labelled CST axons, marking the presence of the L1 cell adhesion molecule. In addition the procedure described in this report preserves ultrastructural details of developing neural tissue. In conclusion, the method presented can be employed in combined HRP-tracing and immunohistochemical electronmicroscopic studies.

AB/Jena, DBA/2Jena, C57B1/6 Jena inbred mice and ABD2F1- and B6D2F4-hybrid mice either were reared communally and weaned at day 30 or were reared by isolated mothers and weaned at day 21 (I21). On day 50 the total RNA content of pyramidal brain cells of male offspring was determined by cytophotometry. The studied brain areas were the frontal pole of cerebral cortex (FCC) and the CA1 region of the dorsal hippocampus (DHI). The DBA, C57 and ABD2 mice had a reduced FCC and DHI RNA-content under I21-rearing. Under these conditions the AB strain has the highest RNA-content in both substrates. The results call for the awareness of possible genetic influences when studying the effects of environmental enrichment/impoverishing.