Nonphosphorylated Neurofilament Protein Research Articles

Glycine is a transmitter in the human and chimpanzee cochlear nuclei.

Auditory information is relayed from the cochlea via the eighth cranial nerve to the dorsal and ventral cochlear nuclei (DCN, VCN). The organization, neurochemistry and circuitry of the cochlear nuclei (CN) have been studied in many species. It is well-established that glycine is an inhibitory transmitter in the CN of rodents and cats, with glycinergic cells in the DCN and VCN. There are, however, major differences in the laminar and cellular organization of the DCN between humans (and other primates) and rodents and cats. We therefore asked whether there might also be differences in glycinergic neurotransmission in the CN. We studied brainstem sections from humans, chimpanzees, and cats. We used antibodies to glycine receptors (GLYR) to identify neurons receiving glycinergic input, and antibodies to the neuronal glycine transporter (GLYT2) to immunolabel glycinergic axons and terminals. We also examined archival sections immunostained for calretinin (CR) and nonphosphorylated neurofilament protein (NPNFP) to try to locate the octopus cell area (OCA), a region in the VCN that rodents has minimal glycinergic input. In humans and chimpanzees we found widespread immunolabel for glycine receptors in DCN and in the posterior (PVCN) and anterior (AVCN) divisions of the VCN. We found a parallel distribution of GLYT2-immunolabeled fibers and puncta. The data also suggest that, as in rodents, a region containing octopus cells in cats, humans and chimpanzees has little glycinergic input. Our results show that glycine is a major transmitter in the human and chimpanzee CN, despite the species differences in DCN organization. The sources of the glycinergic input to the CN in humans and chimpanzees are not known.

Shades of gray in human white matter.

Anatomists have long expressed interest in neurons of the white matter, which is by definition supposed to be free of neurons. Hypotheses regarding their biochemical signature and physiological function are mainly derived from animal models. Here, we investigated 15 whole-brain human postmortem specimens, including cognitively normal cases and those with pathologic Alzheimer's disease (AD). Quantitative and qualitative methods were used to investigate differences in neuronal size and density, and the relationship between neuronal processes and vasculature. Double staining was used to evaluate colocalization of neurochemicals. Two topographically distinct populations of neurons emerged: one appearing to arise from developmental subplate neurons and the other embedded within deep, subcortical white matter. Both populations appeared to be neurochemically heterogeneous, showing positive reactivity to acetylcholinesterase (AChE) [but not choline acetyltransferase (ChAT)], neuronal nuclei (NeuN), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), microtubule-associated protein 2 (MAP-2), somatostatin (SOM), nonphosphorylated neurofilament protein (SMI-32), and calcium-binding proteins calbindin-D28K (CB), calretinin (CRT), and parvalbumin (PV). PV was more richly expressed in superficial as opposed to deep white matter neurons (WMNs); subplate neurons were also significantly larger than their deeper counterparts. NADPH-d, a surrogate for nitric oxide synthase, allowed for the striking morphological visualization of subcortical WMNs. NADPH-d-positive subcortical neurons tended to embrace the outer walls of microvessels, suggesting a functional role in vasodilation. The presence of AChE positivity in these neurons, but not ChAT, suggests that they are cholinoceptive but noncholinergic. WMNs were also significantly smaller in AD compared to control cases. These observations provide a landscape for future systematic investigations.

Cortical Layer Markers Expression and Increased Synaptic Density in Interstitial Neurons of the White Matter from Drug-Resistant Epilepsy Patients.

The interstitial neurons in the white matter of the human and non-human primate cortex share a similar developmental origin with subplate neurons and deep-layer cortical neurons. A subset of interstitial neurons expresses the molecular markers of subplate neurons, but whether interstitial neurons express cortical layer markers in the adult human brain remains unexplored. Here we report the expression of cortical layer markers in interstitial neurons in the white matter of the adult human brain, supporting the hypothesis that interstitial neurons could be derived from cortical progenitor cells. Furthermore, we found increased non-phosphorylated neurofilament protein (NPNFP) expression in interstitial neurons in the white matter of drug-resistant epilepsy patients. We also identified the expression of glutamatergic and g-aminobutyric acid (GABAergic) synaptic puncta that were distributed in the perikarya and dendrites of interstitial neurons. The density of glutamatergic and GABAergic synaptic puncta was increased in interstitial neurons in the white matter of drug-resistant epilepsy patients compared with control brain tissues with no history of epilepsy. Together, our results provide important insights of the molecular identity of interstitial neurons in the adult human white matter. Increased synaptic density of interstitial neurons could result in an imbalanced synaptic network in the white matter and participate as part of the epileptic network in drug-resistant epilepsy.

Neurogenesis and neuronal differentiation in the postnatal frontal cortex in Down syndrome.

Although Down syndrome (DS), the most common developmental genetic cause of intellectual disability, displays proliferation and migration deficits in the prenatal frontal cortex (FC), a knowledge gap exists on the effects of trisomy 21 upon postnatal cortical development. Here, we examined cortical neurogenesis and differentiation in the FC supragranular (SG, II/III) and infragranular (IG, V/VI) layers applying antibodies to doublecortin (DCX), non-phosphorylated heavy-molecular neurofilament protein (NHF, SMI-32), calbindin D-28K (Calb), calretinin (Calr), and parvalbumin (Parv), as well as β-amyloid (APP/Aβ and Aβ1–42) and phospho-tau (CP13 and PHF-1) in autopsy tissue from age-matched DS and neurotypical (NTD) subjects ranging from 28-weeks (wk)-gestation to 3 years of age. Thionin, which stains Nissl substance, revealed disorganized cortical cellular lamination including a delayed appearance of pyramidal cells until 44 wk of age in DS compared to 28 wk in NTD. SG and IG DCX-immunoreactive (-ir) cells were only visualized in the youngest cases until 83 wk in NTD and 57 wk DS. Strong SMI-32 immunoreactivity was observed in layers III and V pyramidal cells in the oldest NTD and DS cases with few appearing as early as 28 wk of age in layer V in NTD. Small Calb-ir interneurons were seen in younger NTD and DS cases compared to Calb-ir pyramidal cells in older subjects. Overall, a greater number of Calb-ir cells were detected in NTD, however, the number of Calr-ir cells were comparable between groups. Diffuse APP/Aβ immunoreactivity was found at all ages in both groups. Few young cases from both groups presented non-neuronal granular CP13 immunoreactivity in layer I. Stronger correlations between brain weight, age, thionin, DCX, and SMI-32 counts were found in NTD. These findings suggest that trisomy 21 affects postnatal FC lamination, neuronal migration/neurogenesis and differentiation of projection neurons and interneurons that likely contribute to cognitive impairment in DS.

Individual variability in the size and organization of the human arcuate nucleus of the medulla.

The arcuate nucleus (Arc) of the medulla is found in almost all human brains and in a small percentage of chimpanzee brains. It is absent in the brains of other mammalian species including mice, rats, cats, and macaque monkeys. The Arc is classically considered a precerebellar relay nucleus, receiving input from the cerebral cortex and projecting to the cerebellum via the inferior cerebellar peduncle. However, several studies have found aplasia of the Arc in babies who died of SIDS (Sudden Infant Death Syndrome), and it was suggested that the Arc is the locus of chemosensory neurons critical for brainstem control of respiration. Aplasia of the Arc, however, has also been reported in adults, suggesting that it is not critical for survival. We have examined the Arc in closely spaced Nissl-stained sections in thirteen adult human cases to acquire a better understanding of the degree of variability of its size and location in adults. We have also examined immunostained sections to look for neurochemical compartments in this nucleus. Caudally, neurons of the Arc are ventrolateral to the pyramidal tracts (py); rostrally, they are ventro-medial to the py and extend up along the midline. In some cases, the Arc is discontinuous, with a gap between sections with the ventrolaterally located and the ventromedially located neurons. In all cases, there is some degree of left-right asymmetry in Arc position, size, and shape at all rostro-caudal levels. Somata of neurons in the Arc express calretinin (CR), neuronal nitric oxide synthase (nNOS), and nonphosphorylated neurofilament protein (NPNFP). Calbindin (CB) is expressed in puncta whereas there is no expression of parvalbumin (PV) in somata or puncta. There is also immunostaining for GAD and GABA receptors suggesting inhibitory input to Arc neurons. These properties were consistent among cases. Our data show differences in location of caudal and rostral Arc neurons and considerable variability among cases in the size and shape of the Arc. The variability in size suggests that "hypoplasia" of the Arc is difficult to define. The discontinuity of the Arc in many cases suggests that establishing aplasia of the Arc requires examination of many closely spaced sections through the brainstem.

Neurodevelopmental disorders of the prefrontal cortex in an evolutionary context.

The prefrontal cortex consists of several cytoarchitectonically defined areas that are involved in higher-order cognitive and emotional processing. The areas are highly variable in terms of organization of cortical layers and distribution of specific neuronal classes, and are affected in neurodevelopmental and psychiatric disorders. Here the focus is on microstructural anatomical characteristics of human prefrontal cortex in an evolutionary context with special emphasis on Williams syndrome. We include a pilot analysis of distribution of neurons labeled with an antibody to non-phosphorylated neurofilament protein (SMI-32) in the frontal pole of Williams syndrome to further examine microstructural characteristics of the prefrontal cortex in Williams syndrome and implications of the distribution of SMI-32 immunoreactive neurons for connectivity between the frontal pole and other cortical areas in the disorder.

Abstract TMP27: Tgfalpha Protects Oligodendrocytes Against Ischemic Injury and Promotes White Matter Integrity After Stroke

Introduction: The loss of white matter integrity after ischemic stroke contributes to long-term sensorimotor and cognitive deficits. Elucidating the mechanisms for white matter protection or recovery is therefore important to improve stroke outcomes. This study investigates the function of TGFα, a member of the epidermal growth factor family, in white matter recovery after stroke and elucidate the mechanisms of action. Methods: Cerebral ischemia was induced by transient (60 min) middle cerebral artery occlusion (tMCAO) in wild type and TGFα knockout mice. The spatiotemporal pattern of TGFα expression was evaluated by immunofluorescent staining at 1, 3, 5, 14, 21 and 35 days after tMCAO. Sensorimotor (rota-rod test, adhesive-remove test and open field test) and cognitive (Morris water-maze test) functions were assessed up to 35 days after stroke. White matter injury was quantified by MBP (myelin basic protein) and SMI32 (recognizes damaged non-phosphorylated neurofilament proteins) staining. The direct effect of TGFα on oligodendrocytes were evaluated in vitro using primary cultures. Live/dead cell staining and LDH (lactate dehydrogenase) assay were used to assess oligodendrocytes death after 90 min oxygen and glucose deprivation (OGD). Result: Cerebral ischemic induced an abrupt and transient increase of TGFα in the brain. The expression of TGFα was mainly increased in microglia and neurons. TGFα deficiency resulted in exacerbation in sensorimotor and cognitive deficits up to 35 days after tMCAO. MAP2 (microtubule-associated protein 2) staining at 3 days and 35 days showed lager tissue loss in TGFα knockout mice as compared with wild type stroke mice. Importantly. TGFα knockout mice also exhibited impaired white matter integrity, as revealed by the loss of MBP staining and elevation in SMI32 staining, at 35 days after stroke. Correlation analysis demonstrated positive correlation between white matter integrity and functional outcomes after stroke in TGFα knockout mice and wild type mice. In vitro study confirmed that TGFα (5, 10 or 20 ng/ml) protected oligodendrocyte precursor cells and oligodendrocytes against OGD. Conclusion: TGFα is important for maintaining white matter integrity and improving neurological recovery after stroke.

Altered neurofilament protein expression in the lateral vestibular nucleus in Parkinson's disease.

A major cause of morbidity in Parkinson's disease (PD) is postural instability. The neuropathology underlying postural instability is unknown. Postural control is mediated by Deiters' neurons of the lateral vestibular nucleus (LVN), which are the brainstem origin of descending vestibulospinal reflexes. Deiters' neurons express the cytostructural protein, non-phosphorylated neurofilament protein (NPNFP). In PD, reduced expression of NPNFP in substantia nigra (SN) neurons is believed to contribute to dysfunction. It was the aim of this study to determine if there is altered expression of NPNFP in the LVN in PD. We immunolabeled NPNFP in brainstem sections of six aged controls (mean age 92yo) and six PD donors (mean age 83yo). Our results show there was a ~50% reduction in NPNFP-positive Deiters' neurons compared to controls (13±2.0/section vs 25.7±3.0/section; p<0.01, repeated measures ANOVA). In contrast, there was no difference in NPNFP-positive counts in the facial nucleus between control and PD. The normalized intensity of NPNFP labeling in LVN was also reduced in PD (0.87±0.05 vs 1.09±0.03; p<0.01). There was a 35% concurrent reduction in NPNFP-positive neuropil in PD relative to controls (p<0.01). We also show there was an 84% increase (p<0.05) in somatic lipofuscin in PD patients compared to control. Lipofuscin aggregation has been shown to increase not only with age but also with neurodegeneration. Furthermore, decreased NPNFP intensity was strongly correlated with increasing lipofuscin autofluorescence across all cases (R 2=0.81, p<0.01). These results show two alterations in cellular content with PD, reduced expression and intensity of NPNFP and increased lipofuscin aggregation in Deiter's neurons. These changes may contribute to degeneration of postural reflexes observed in PD.

Differential activation of neuronal and glial STAT3 in the spinal cord of the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Signal transducer and activator of transcription (STAT) proteins are activated by phosphorylation in the spinal cord of patients suffering from amyotrophic lateral sclerosis (ALS). The major scope of our study is a comprehensive histological characterization of the mechanisms underlying neuronal and glial STAT3 activation in the pathogenesis of ALS using SOD1G93A mice. We calculated the fold changes (FCs, ratios vs. appropriate controls) of the numerical densities of the following phosphorylated STAT3-positive (pSTAT3)+ cells - choline acetyltransferase (ChAT)+ α-motoneurons, ionized calcium-binding adapter molecule 1 (Iba1)+ microglia, and S100β+ astrocytes in SOD1G93A mice. The FCs of pSTAT3+ microglia and pSTAT3+ astrocytes were increased from 9 to 15weeks of age and then plateaued until 21weeks. In contrast, the FCs of pSTAT3+ α-motoneurons peaked at 9weeks and then decreased until 21weeks. The immunoreactivity for nonphosphorylated neurofilament protein (SMI-32), a marker of axonal impairment, was decreased in pSTAT3+ α-motoneurons compared with pSTAT3- α-motoneurons at 9weeks of age. We then compared the following pharmacological models - the chronic administration of 3,3'-iminodipropionitrile (IDPN), which models axonal impairment, and the acute administration of lipopolysaccharide (LPS), which is a model of neuroinflammation. The FCs of pSTAT3+ α-motoneurons were increased in IDPN-treated mice, while those of pSTAT3+ microglia were increased in LPS-treated mice. The FCs of pSTAT3+ astrocytes were higher in SOD1G93A mice at 9weeks compared with IDPN- and LPS-treated mice. Our results indicate that axonopathy and neuroinflammation may trigger the respective activation of neuronal and glial STAT3, which is observed during ALS pathogenesis.

Immunohistochemical characteristics of the substantia nigra neurons of the human

To determine the cytochemical characteristics of unchanged neurons of the human substantia nigra using a wide range of immunocytochemical markers some of which (glutamate decarboxylase-65, PGP 9.5, non-phosphorylated neurofilament proteins, alpa-tubulin) have never been used for study of human dopaminergic neurons. Fragments of human midbrain (17 men and women, aged from 28 to 78 years) from the archives of the Department of General and Specific Morphology of the Institute of Experimental Medicine were used. The study was performed using classical neurohistological techniques and immunocytochemistry using antibodies to 15 different proteins. Most neurons in substantia nigra exhibited a reduced expression of common neuronal markers such as neuronal nuclear protein NeuN, PGP 9.5 protein, and neuron-specific enolase. GABAergic (GAD65-immunopositive) neurons were not found in the substantia nigra. Single cholinergic neurons without neuromelanin were identified in the dorsal part of the substantia nigra. Calcium-binding proteins calbindin and calretinin were not found in the majority of nigral cells although calbindin was rarely seen in some neurons of the dorsal part and calretinin in the ventral one. Nitric oxide synthase (NOS) was present in the substantia nigra both in neuropil and neuronal bodies. The results suggest the unique cytochemical properties of the nigral neurons, which may be related to their increased susceptibility to lesion and degeneration.

Clinical, imaging, and immunohistochemical characteristics of focal cortical dysplasia Type II extratemporal epilepsies in children: analyses of an institutional case series.

OBJECTIVE Focal cortical dysplasia (FCD) Type II is divided into 2 subgroups based on the absence (IIA) or presence (IIB) of balloon cells. In particular, extratemporal FCD Type IIA and IIB is not completely understood in terms of clinical, imaging, biological, and neuropathological differences. The aim of the authors was to analyze distinctions between these 2 formal entities and address clinical, MRI, and immunohistochemical features of extratemporal epilepsies in children. METHODS Cases formerly classified as Palmini FCD Type II nontemporal epilepsies were identified through the prospectively maintained epilepsy database at the British Columbia Children's Hospital in Vancouver, Canada. Clinical data, including age of seizure onset, age at surgery, seizure type(s) and frequency, affected brain region(s), intraoperative electrocorticographic findings, and outcome defined by Engel's classification were obtained for each patient. Preoperative and postoperative MRI results were reevaluated. H & E-stained tissue sections were reevaluated by using the 2011 International League Against Epilepsy classification system and additional immunostaining for standard cellular markers (neuronal nuclei, neurofilament, glial fibrillary acidic protein, CD68). Two additional established markers of pathology in epilepsy resection, namely, CD34 and α-B crystallin, were applied. RESULTS Seven nontemporal FCD Type IIA and 7 Type B cases were included. Patients with FCD Type IIA presented with an earlier age of epilepsy onset and slightly better Engel outcome. Radiology distinguished FCD Types IIA and IIB, in that Type IIB presented more frequently with characteristic cortical alterations. Nonphosphorylated neurofilament protein staining confirmed dysplastic cells in dyslaminated areas. The white-gray matter junction was focally blurred in patients with FCD Type IIB. α-B crystallin highlighted glial cells in the white matter and subpial layer with either of the 2 FCD Type II subtypes and balloon cells in patients with FCD Type IIB. α-B crystallin positivity proved to be a valuable tool for confirming the histological diagnosis of FCD Type IIB in specimens with rare balloon cells or difficult section orientation. Distinct nonendothelial cellular CD34 staining was found exclusively in tissue from patients with MRI-positive FCD Type IIB. CONCLUSIONS Extratemporal FCD Types IIA and IIB in the pediatric age group exhibited imaging and immunohistochemical characteristics; cellular immunoreactivity to CD34 emerged as an especially potential surrogate marker for lesional FCD Type IIB, providing additional evidence that FCD Types IIA and IIB might differ in their etiology and biology. Although the sample number in this study was small, the results further support the theory that postoperative outcome-defined by Engel's classification-is multifactorial and determined by not only histology but also the extent of the initial lesion, its location in eloquent areas, intraoperative electrocorticographic findings, and achieved resection grade.

The influence of aging on the number of neurons and levels of non-phosporylated neurofilament proteins in the central auditory system of rats.

In the present study, an unbiased stereological method was used to determine the number of all neurons in Nissl stained sections of the inferior colliculus (IC), medial geniculate body (MGB), and auditory cortex (AC) in rats (strains Long Evans and Fischer 344) and their changes with aging. In addition, using the optical fractionator and western blot technique, we also evaluated the number of SMI-32-immunoreactive (-ir) neurons and levels of non-phosphorylated neurofilament proteins in the IC, MGB, AC, and visual cortex of young and old rats of the two strains. The SMI-32 positive neuronal population comprises about 10% of all neurons in the rat IC, MGB, and AC and represents a prevalent population of large neurons with highly myelinated and projecting processes. In both Long Evans and Fischer 344 rats, the total number of neurons in the IC was roughly similar to that in the AC. With aging, we found a rather mild and statistically non-significant decline in the total number of neurons in all three analyzed auditory regions in both rat strains. In contrast to this, the absolute number of SMI-32-ir neurons in both Long Evans and Fischer 344 rats significantly decreased with aging in all the examined structures. The western blot technique also revealed a significant age-related decline in the levels of non-phosphorylated neurofilaments in the auditory brain structures, 30–35%. Our results demonstrate that presbycusis in rats is not likely to be primarily associated with changes in the total number of neurons. On the other hand, the pronounced age-related decline in the number of neurons containing non-phosphorylated neurofilaments as well as their protein levels in the central auditory system may contribute to age-related deterioration of hearing function.

Laminar and neurochemical organization of the dorsal cochlear nucleus of the human, monkey, cat, and rodents.

The dorsal cochlear nucleus (DCN) is a brainstem structure that receives input from the auditory nerve. Many studies in a diversity of species have shown that the DCN has a laminar organization and identifiable neuron types with predictable synaptic relations to each other. In contrast, studies on the human DCN have found a less distinct laminar organization and fewer cell types, although there has been disagreement among studies in how to characterize laminar organization and which of the cell types identified in other animals are also present in humans. We have reexamined DCN organization in the human using immunohistochemistry to analyze the expression of several proteins that have been useful in delineating the neurochemical organization of other brainstem structures in humans: nonphosphorylated neurofilament protein (NPNFP), nitric oxide synthase (nNOS), and three calcium-binding proteins. The results for humans suggest a laminar organization with only two layers, and the presence of large projection neurons that are enriched in NPNFP. We did not observe evidence in humans of the inhibitory interneurons that have been described in the cat and rodent DCN. To compare humans and other animals directly we used immunohistochemistry to examine the DCN in the macaque monkey, the cat, and three rodents. We found similarities between macaque monkey and human in the expression of NPNFP and nNOS, and unexpected differences among species in the patterns of expression of the calcium-binding proteins.

Altered neuronal architecture and plasticity in the visual cortex of adult MMP-3-deficient mice.

Matrix metalloproteinases (MMPs) are Zn(2+)-dependent endopeptidases considered to be essential for normal brain development and neuroplasticity by modulating extracellular matrix proteins, receptors, adhesion molecules, growth factors and cytoskeletal proteins. Specifically, MMP-3 has recently been implicated in synaptic plasticity, hippocampus-dependent learning and neuronal development and migration in the cerebellum. However, the function(s) of this enzyme in the neocortex is understudied. Therefore, we explored the phenotypical characteristics of the neuronal architecture and the capacity for experience-dependent cortical plasticity in the visual cortex of adult MMP-3-deficient (MMP-3(-/-)) mice. Golgi-Cox stainings revealed a significant reduction in apical dendritic length and an increased number of apical obliques for layer V pyramidal neurons in the visual cortex of adult MMP-3(-/-) mice compared to wild-type (WT) animals. In addition, a significant upregulation of both phosphorylated and non-phosphorylated neurofilament protein (NF)-high, phosphorylated NF-medium, NF-low and α-internexin was detected in the visual cortex of MMP-3(-/-) mice. To assess the effect of MMP-3 deficiency on cortical plasticity, we monocularly enucleated adult MMP-3(-/-) mice and analyzed the reactivation of the contralateral visual cortex 7 weeks post-enucleation. In contrast to previous results in C57Bl/6J adult mice, activity remained confined to the binocular zone and did not expand into the monocular regions indicative for an aberrant open-eye potentiation. Permanent hypoactivity in the monocular cortex lateral and medial to V1 also indicated a lack of cross-modal plasticity. These observations demonstrate that genetic inactivation of MMP-3 has profound effects on the structural integrity and plasticity response of the visual cortex of adult mice.

Unique Features of the Human Brainstem and Cerebellum

The cerebral cortex is greatly expanded in the human brain. There is a parallel expansion of the cerebellum, which is interconnected with the cerebral cortex. We have asked if there are accompanying changes in the organization of pre-cerebellar brainstem structures. We have examined the cytoarchitectonic and neurochemical organization of the human medulla and pons. We studied human cases from the Witelson Normal Brain Collection, analyzing Nissl sections and sections processed for immunohistochemistry for multiple markers including the calcium-binding proteins calbindin, calretinin, and parvalbumin, non-phosphorylated neurofilament protein, and the synthetic enzyme for nitric oxide, nitric oxide synthase. We have also compared the neurochemical organization of the human brainstem to that of several other species including the chimpanzee, macaque and squirrel monkey, cat, and rodent, again using Nissl staining and immunohistochemistry. We found that there are major differences in the human brainstem, ranging from relatively subtle differences in the neurochemical organization of structures found in each of the species studied to the emergence of altogether new structures in the human brainstem. Two aspects of human cortical organization, individual differences and left–right asymmetry, are also seen in the brainstem (principal nucleus of the inferior olive) and the cerebellum (the dentate nucleus). We suggest that uniquely human motor and cognitive abilities derive from changes at all levels of the central nervous system, including the cerebellum and brainstem, and not just the cerebral cortex.

The nucleus pararaphales in the human, chimpanzee, and macaque monkey

The human cerebral cortex and cerebellum are greatly expanded compared to those of other mammals, including the great apes. This expansion is reflected in differences in the size and organization of precerebellar brainstem structures, such as the inferior olive. In addition, there are cell groups unique to the human brainstem. One such group may be the nucleus pararaphales (PRa); however, there is disagreement among authors about the size and location of this nucleus in the human brainstem. The name "pararaphales" has also been used for neurons in the medulla shown to project to the flocculus in the macaque monkey. We have re-examined the existence and status of the PRa in eight humans, three chimpanzees, and four macaque monkeys using Nissl-stained sections as well as immunohistochemistry. In the human we found a cell group along the midline of the medulla in all cases; it had the form of interrupted cell columns and was variable among cases in rostrocaudal and dorsoventral extent. Cells and processes were highly immunoreactive for non-phosphorylated neurofilament protein (NPNFP); somata were immunoreactive to the synthetic enzyme for nitric oxide, nitric oxide synthase, and for calretinin. In macaque monkey, there was a much smaller oval cell group with NPNFP immunoreactivity. In the chimpanzee, we found a region of NPNFP-immunoreactive cells and fibers similar to what was observed in macaques. These results suggest that the "PRa" in the human may not be the same structure as the flocculus-projecting cell group described in the macaque. The PRa, like the arcuate nucleus, therefore may be unique to humans.

Vector-mediated expression of erythropoietin improves functional outcome after cervical spinal cord contusion injury

We evaluated the therapeutic effect of erythropoietin (EPO) delivered by direct injection of a nonreplicating herpes simplex virus (HSV)-based vector coding for EPO (vEPO) in a model of cervical hemicord contusion at C7. At 1 h after spinal cord injury (SCI), either vEPO or control vector carrying a reporter gene (vC) was injected into the cord above and below the lesion. Animals injected with vEPO showed a statistically significant improvement in the ipsilateral forelimb function, as measured by open-field evaluation of motor performance, forelimb reaching in the cylinder test and misplacement in grid walk. This correlated with preservation of gray matter in the area of the lesion. There was also mild but significant improvement of hindlimb motor function measured by Basso-Beattie-Bresnahan score and computerized gait analysis in vEPO compared with control vector-injected animals. Microtubule-associated protein tau, phosphorylated and nonphosphorylated neurofilament protein and the synaptic proteins synaptophysin and PSD-95 were all significantly increased in the spinal cord of vEPO-treated animals compared with control vector-injected animals. These data suggest that gene transfer of EPO after cervical SCI by minimizing the injury size and enhancing tissue sparing preserves large-caliber axons and promotes synaptogenesis.

Neurochemical and Structural Organization of the Principal Nucleus of the Inferior Olive in the Human

The inferior olive (IO) is the sole source of the climbing fibers that innervate the Purkinje cells of the cerebellar cortex. The IO comprises several subdivisions, the dorsal accessory olive (DAO), medial accessory olive (MAO), and principal nuclei of the IO (IOpr); the relative sizes of these subnuclei vary among species. In human, there is an expansion of the cerebellar hemispheres and a corresponding expansion of the IOpr. We have examined the structural and neurochemical organization of the human IOpr, using sections stained with cresyl violet (CV) or immunostained for the calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV), the synthetic enzyme for nitric oxide (nNOS), and nonphosphorylated neurofilament protein (NPNFP). We found qualitative differences in the folding patterns of the IOpr among individuals and between the two sides of the brainstem. Quantification of IOpr volumes and indices of folding complexity, however, did not reveal consistent left-right differences in either parameter. Single-label immunohistochemistry showed that populations of neurons in the IOpr express CB, CR, NPNFP, and nNOS. Individual fibers labeled for PV, CB, CR, NPNFP, and nNOS were also found. There was individual variability in the numbers and density of stained neurons in the human IOpr; such variability was not seen in other brainstem nuclei. These data are consistent with, and complement, earlier studies showing a dramatic age-related increase in lipofuscin and decrease in RNA in the human IOpr. The impact of these changes in the IOpr on cerebellar function is, however, not known.

The insula of Reil revisited: multiarchitectonic organization in macaque monkeys.

The insula of Reil represents a large cortical territory buried in the depth of the lateral sulcus and subdivided into 3 major cytoarchitectonic domains: agranular, dysgranular, and granular. The present study aimed at reinvestigating the architectonic organization of the monkey's insula using multiple immunohistochemical stainings (parvalbumin, PV; nonphosphorylated neurofilament protein, with SMI-32; acetylcholinesterase, AChE) in addition to Nissl and myelin. According to changes in density and laminar distributions of the neurochemical markers, several zones were defined and related to 8 cytoarchitectonic subdivisions (Ia1–Ia2/Id1–Id3/Ig1–Ig2/G). Comparison of the different patterns of staining on unfolded maps of the insula revealed: 1) parallel ventral to dorsal gradients of increasing myelin, PV- and AChE-containing fibers in middle layers, and of SMI-32 pyramidal neurons in supragranular layers, with merging of dorsal and ventral high-density bands in posterior insula, 2) definition of an insula “proper” restricted to two-thirds of the “morphological” insula (as bounded by the limiting sulcus) and characterized most notably by lower PV, and 3) the insula proper is bordered along its dorsal, posterodorsal, and posteroventral margin by a strip of cortex extending beyond the limits of the morphological insula and continuous architectonically with frontoparietal and temporal opercular areas related to gustatory, somatosensory, and auditory modalities.

Nonphosphorylated neurofilament protein is expressed by scattered neurons in the human vestibular brainstem

Vestibular information is critical for the maintenance of balance and posture and for the control of eye movements. The eighth nerve carries vestibular information to four brainstem nuclei called the vestibular nuclear complex (VNC); these nuclei relay vestibular signals to several additional brainstem nuclei. The structure, connections, effects of lesions and neuronal response properties of the vestibular brainstem have been studied in many nonhuman species. The development of bipedal locomotion in humans mandates differences in the vestibular control of balance and suggests that there may also be differences in the organization of the human vestibular brainstem. While the four nuclei of the VNC are described in human, there is a lot of variability among reports in their borders and extent. Further, there are several nuclei described in the human brainstem that are not present in other species. We have been using immunohistochemistry to study the patterns of expression of several different proteins to define and compare the organization of the vestibular brainstem in animals and humans. We here describe the expression of nonphosphorylated neurofilament protein (NPNFP) in the human vestibular brainstem. As in the cat, NPNFP is expressed by scattered cells within multiple regions of the vestibular brainstem and in cranial nerve nuclei. NPNFP expression in other cortical and subcortical regions suggests that it is expressed by projection neurons. For vestibular brainstem, these may be vestibulospinal, vestibulo-oculomotor or vestibulocerebellar neurons. Studies of other brain regions suggest that brainstem neurons expressing NPNFP may be especially vulnerable in different neurological disorders including Alzheimer's disease or to alterations in sensory input.