Anna Josephson

NOGO mRNA Expression in Adult and Fetal Human and Rat Nervous Tissue and in Weight Drop Injury

Nogo is a myelin-associated protein known to inhibit growth of neurites. In order to understand possible physiological roles of Nogo, we performed in situ hybridization using rat and human probes complementary to a Nogo-A-specific sequence and a sequence shared by all known Nogo transcripts recognizing nogo-A, -B, and -C. We studied the cellular distribution of nogo-mRNA in fetal and adult human and rat tissues, with a focus on the spinal cord and ganglia. Rat mRNA expression was also studied in a spinal cord weight-drop model and in animals exposed to kainic acid. In human fetal tissue, nogo-A was strongly expressed in the ventral two-thirds of the spinal cord, the dorsal root ganglia, and autonomic ganglia. Similarly, nogo-A mRNA expression was observed in the adult human spinal cord and ganglia. High levels of nogo-A message were observed in neurons, such as motor neurons and sensory ganglia neurons. The distribution of nogo message in rats resembled that seen in human tissues. Thus, nogo mRNA was expressed in neurons and oligodendrocytes, but not astrocytes or Schwann cells. In addition, expression of nogo-A mRNA was observed in human and rat developing muscle tissue. High level of nogo-mRNA were also expressed in the rat trigeminal ganglion and trigeminal pontine nucleus. In fetal rats the adrenal gland and cell clusters in the liver were positive for the nogo-ABC pan-probe, but negative for the nogo-A probe. While neurons in the adult rat brain were generally positive, very prominent nogo-A mRNA and nogo-ABC mRNA signals were obtained from neurons of the hippocampus, piriform cortex, the red nucleus, and the oculomotor nucleus. Nogo-A mRNA expression was markedly reduced in the epicenter of a lesion in the spinal cord of adult rats 6 and 24 h after a weight-drop injury, while no perifocal upregulation of nogo mRNA was seen. No obvious change of nogo expression was detected in kainic acid exposed animals. In conclusion our in situ hybridization study has demonstrated widespread expression of nogo mRNA in the fetal, developing and adult nervous system of rat and man. In addition to oligodendroglial cells, high levels of nogo-A mRNA expression were found in neurons, raising important questions about the function of neuronal nogo mRNA. No obvious regulation of nogo was detected following injury.

Nogo-receptor gene activity: Cellular localization and developmental regulation of mRNA in mice and humans

Nogo (reticulon‐4) is a myelin‐associated protein that is expressed in three different splice variants, Nogo‐A, Nogo‐B, and Nogo‐C. Nogo‐A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo‐66, and a Nogo‐66‐receptor (Nogo‐R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo‐R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo‐R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo‐R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium‐sized neurons of the dorsal root ganglia. Nogo‐R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo‐R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo‐R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo‐R mRNA at detectable levels. In summary, Nogo‐66‐R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. J. Comp. Neurol. 453:292–304, 2002.

Activity-induced and developmental downregulation of the Nogo receptor

Abstract.The three axon growth inhibitory proteins, myelin associated glycoprotein, oligodendrocyte-myelin glycoprotein and Nogo-A, can all bind to the Nogo-66 receptor (NgR). This receptor is expressed by neurons with high amounts in regions of high plasticity where Nogo expression is also high. We hypothesized that simultaneous presence of high levels of Nogo and its receptor in neurons confers a locked state to hippocampal and cortical microcircuitry and that one or both of these proteins must be effectively and temporarily downregulated to permit plastic structural changes underlying formation of long-term memory. Hence, we subjected rats to kainic acid treatment and exposed rats to running wheels and measured NgR mRNA levels by quantitative in situ hybridization at different time points. We also studied spinal cord injuries and quantified NgR mRNA levels in spinal cord and ganglia during a critical postnatal period using real-time PCR. Strikingly, kainic acid led to a strong transient downregulation of NgR mRNA levels in gyrus dentatus, hippocampus, and neocortex during a time when BDNF mRNA was upregulated instead. Animals exposed to running wheels for 3 and 7, but not 1 or 21, days showed a significant downregulation of NgR mRNA in cortex, hippocampus and the dentate gyrus. NgR mRNA levels decreased from high to low expression in spinal cord and ganglia during the first week of life. No robust regulation of NgR was observed in the spinal cord following spinal cord injury. Together, our data show that NgR levels in developing and adult neurons are regulated in vivo under different conditions. Strong, rapid and transient downregulation of NgR mRNA in response to kainic acid and after wheel running in cortex and hippocampus suggests a role for NgR and Nogo-A in plasticity, learning and memory.

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFRα‐1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT‐3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFRα‐1, GFRα‐2, or GFRα‐3 was 79, 20, 51, and 32%, respectively. GFRα‐2 was expressed selectively in small, GFRα‐3 principally in small and GFRα‐1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT‐3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFRα‐2, NT‐3, and p75. Notably, GFRα‐2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord. J. Comp. Neurol. 440:204–217, 2001.

Nogo receptor 1 regulates formation of lasting memories

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.

Functional MRI at 4.7 tesla of the rat brain during electric stimulation of forepaw, hindpaw, or tail in single- and multislice experiments.

Stimulation of peripheral nerves activates corresponding regions in sensorimotor cortex. We have applied functional magnetic resonance imaging (fMRI) techniques to monitor activated brain regions by means of measuring changes of blood oxygenation level-dependent contrast during electric stimulation of the forepaw, hindpaw, or tail in rats. During alpha-chloralose anesthesia, artificial respiration, and complete muscle relaxation, stimulations were delivered at 3 Hz via subcutaneous bipolar electrodes with 500-microseconds-current pulses of 0.2-2.0 mA. Single- or multislice gradient echo images were collected during recording sessions consisting of five alternating rest and stimulation periods. Stimulation of the right and left forepaws and hindpaws repeatedly led to robust activation of the contralateral sensorimotor cortex. There was a significant correlation (P < 0.05) between current pulse strength and amount of activation of the sensory cortex during forepaw stimulation. The center of the main cortical representation of the forepaw was situated 3.4 mm lateral to the midline and 5 mm posterior to the rhinal fissure. The main representation of the hindpaw was 2.0 mm lateral to the midline and 6 mm posterior to the rhinal fissure. Tail stimulation gave rise to a strikingly extended bilateral cortical activation, localized along the midline in medial parietal and frontal cortex 4 and 5 mm posterior to the rhinal fissure. In conclusion, the experiments provide evidence that peripheral nerve stimulation induces a fMRI signal in the respective division of the somatosensory cortex in a stimulus-related manner. The marked cortical activation elicited by tail stimulation underlines the key importance of the tail.

A Spinal Thecal Sac Constriction Model Supports the Theory That Induced Pressure Gradients in the Cord Cause Edema and Cyst Formation

OBJECTIVESpinal cord cysts are a devastating condition that occur secondary to obstructions of the spinal canal, which may be caused by congenital malformations, trauma, spinal canal stenosis, tumors, meningitis, or arachnoiditis. A hypothesis that could explain how spinal cord cysts form in these situations has been presented recently. Therefore, a novel spinal thecal sac constriction model was implemented to test various aspects of this hypothesis. METHODSThecal sac constriction was achieved by subjecting rats to an extradural silk ligature at the T8 spinal cord level. Rats with complete spinal cord transection served as a second model for comparison. The animals underwent high-resolution magnetic resonance imaging and histological analysis. RESULTSThecal sac constriction caused edema cranial and caudal to the ligation within 3 weeks, and cysts developed after 8 to 13 weeks. In contrast, cysts in rats with spinal cord transection were located predominantly in the cranial spinal cord. Histological sections of spinal cords confirmed the magnetic resonance imaging results. CONCLUSIONMagnetic resonance imaging provided the specific advantage of enabling characterization of events as they occurred repeatedly over time in the spinal cords of individual living animals. The spinal thecal sac constriction model proved useful for investigation of features of the cerebrospinal fluid pulse pressure theory. Edema and cyst distributions were in accordance with this theory. We conclude that induced intramedullary pressure gradients originating from the cerebrospinal fluid pulse pressure may underlie cyst formation in the vicinity of spinal canal obstructions and that cysts are preceded by edema.

MED NORD–A tool for measuring medical students’ well-being and study orientations

Background: The relationship between medical students’ well-being, motivation, and their conceptions of learning and knowledge has not been previously explored. Aims: This study aimed to validate a research instrument intending to measure medical students’ (n = 280) (1) experiences of stress, anxiety and disinterest, (2) motivational (thinking) strategies, (3) conceptions of learning and knowledge (epistemologies), and (4) approaches to learning. Methods: We developed an instrument, MED NORD, which is a composition of scales measuring different theoretical constructs that previously have shown good predictive value, validity and reliability. A principal component analysis with Varimax-rotation was performed in order to see how the scales related to each other. Results: The internal consistency reliability was found to be satisfactory or good for each scale. The results showed five factors: Dysfunctional Orientation, Collaborative Knowledge Building Orientation, Cookbook Orientation, Social Orientation, and Individual Abilities Orientation. These study orientations were related to how medical students perceived their learning environment. Conclusions: The new tool showed consistency and validity and was judged appropriate for future use in measuring medical students’ well-being and study orientations.

The anatomy of learning anatomy

The experience of clinical teachers as well as research results about senior medical students’ understanding of basic science concepts has much been debated. To gain a better understanding about how this knowledge-transformation is managed by medical students, this work aims at investigating their ways of setting about learning anatomy. Second-year medical students were interviewed with a focus on their approach to learning and their way of organizing their studies in anatomy. Phenomenographic analysis of the interviews was performed in 2007 to explore the complex field of learning anatomy. Subjects were found to hold conceptions of a dual notion of the field of anatomy and the interplay between details and wholes permeated their ways of studying with an obvious endeavor of understanding anatomy in terms of connectedness and meaning. The students’ ways of approaching the learning task was characterized by three categories of description; the subjects experienced their anatomy studies as memorizing, contextualizing or experiencing. The study reveals aspects of learning anatomy indicating a deficit in meaningfulness. Variation in approach to learning and contextualization of anatomy are suggested as key-elements in how the students arrive at understanding. This should be acknowledged through careful variation of the integration of anatomy in future design of medical curricula.

Neuronal activity-induced regulation of Lingo-1.

Axonal regeneration after injury can be limited in the adult CNS by the presence of inhibitory proteins such as Nogo. Nogo binds to a receptor complex that consists of Nogo receptor (NgR), p75NTR, and Lingo-1. Nogo binding activates RhoA, which inhibits axonal outgrowth. Here we assessed Lingo-1 and NgR mRNA levels after delivery of BDNF into the rat hippocampal formation, Lingo-1 mRNA levels in rats subjected to kainic acid (KA) and running in running wheels. Lingo-1 mRNA was not changed by running. However, we found that Lingo-1 mRNA was strongly up-regulated while NgR mRNA was down-regulated in the dentate gyrus in both the BDNF and the KA experiments. Our data demonstrate inverse regulation of NgR and Lingo-1 in these situations, suggesting that Lingo-1 up-regulation is one characteristic of activity-induced neural plasticity responses.