Growth of new brainstem connections in adult monkeys with massive sensory loss

Somatotopic maps in the cortex and the thalamus of adult monkeys and humans reorganize in response to altered inputs. After loss of the sensory afferents from the forelimb in monkeys because of transection of the dorsal columns of the spinal cord, therapeutic amputation of an arm or transection of the dorsal roots of the peripheral nerves, the deprived portions of the hand and arm representations in primary somatosensory cortex (area 3b), become responsive to inputs from the face and any remaining afferents from the arm. Cortical and subcortical mechanisms that underlie this reorganization are uncertain and appear to be manifold. Here we show that the face afferents from the trigeminal nucleus of the brainstem sprout and grow into the cuneate nucleus in adult monkeys after lesions of the dorsal columns of the spinal cord or therapeutic amputation of an arm. This growth may underlie the large-scale expansion of the face representation into the hand region of somatosensory cortex that follows such deafferentations.

An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3.5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2–4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

Survival motor neuron protein modulates neuron-specific apoptosis

Spinal muscular atrophy (SMA) is attributed to mutations in the SMN1 gene, leading to loss of spinal cord motor neurons. The neurotropic Sindbis virus vector system was used to investigate a role for the survival motor neuron (SMN) protein in regulating neuronal apoptosis. Here we show that SMN protects primary neurons and differentiated neuron-like stem cells, but not cultured cell lines from virus-induced apoptotic death. SMN also protects neurons in vivo and increases survival of virus-infected mice. SMN mutants (SMNΔ7 and SMN-Y272C) found in patients with SMA not only lack antiapoptotic activity but also are potently proapoptotic, causing increased neuronal apoptosis and animal mortality. Full-length SMN is proteolytically processed in brains undergoing apoptosis or after ischemic injury. Mutation of an Asp-252 of SMN abolished cleavage of SMN and increased the antiapoptotic function of full-length SMN in neurons. Taken together, deletions or mutations of the C terminus of SMN that result from proteolysis, splicing (SMNΔ7), or germ-line mutations (e.g., Y272C), produce a proapoptotic form of SMN that may contribute to neuronal death in SMA and perhaps other neurodegenerative disorders.

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

Nociception in cyclooxygenase isozyme-deficient mice

Prostaglandins formed by cyclooxygenase-1 (COX-1) or COX-2 produce hyperalgesia in sensory nerve endings. To assess the relative roles of the two enzymes in pain processing, we compared responses of COX-1- or COX-2-deficient homozygous and heterozygous mice with wild-type controls in the hot plate and stretching tests for analgesia. Preliminary observational studies determined that there were no differences in gross parameters of behavior between the different groups. Surprisingly, on the hot plate (55°C), the COX-1-deficient heterozygous groups showed less nociception, because mean reaction time was longer than that for controls. All other groups showed similar reaction times. In the stretching test, there was less nociception in COX-1-null and COX-1-deficient heterozygotes and also, unexpectedly, in female COX-2-deficient heterozygotes, as shown by a decreased number of writhes. Measurements of mRNA levels by reverse transcription–PCR demonstrated a compensatory increase of COX-1 mRNA in spinal cords of COX-2-null mice but no increase in COX-2 mRNA in spinal cords of COX-1-null animals. Thus, compensation for the absence of COX-1 may not involve increased expression of COX-2, whereas up-regulation of COX-1 in the spinal cord may compensate for the absence of COX-2. The longer reaction times on the hot plate of COX-1-deficient heterozygotes are difficult to explain, because nonsteroid anti-inflammatory drugs have no analgesic action in this test. Reduction in the number of writhes of the COX-1-null and COX-1-deficient heterozygotes may be due to low levels of COX-1 at the site of stimulation with acetic acid. Thus, prostaglandins made by COX-1 mainly are involved in pain transmission in the stretching test in both male and female mice, whereas those made by COX-2 also may play a role in the stretching response in female mice.

A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and β-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the α-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, β-galactosidase and immunochemical stainings revealed differences in GFP and β-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense β-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

Creating a Web-accessible, point-of-care, team-based information system (PoinTIS): the librarian as publisher*

The Internet has created new opportunities for librarians to develop information systems that are readily accessible at the point of care. This paper describes the multiyear process used to justify, fund, design, develop, promote, and evaluate a rehabilitation prototype of a point-of-care, team-based information system (PoinTIS) and train health care providers to use this prototype for their spinal cord injury and traumatic brain injury patient care and education activities. PoinTIS is a successful model for librarians in the twenty-first century to serve as publishers of information created or used by their parent organizations and to respond to the opportunities for information dissemination provided by recent technological advances.

Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity

Ciliary Neurotrophic Factor (CNTF) was first characterized as a trophic factor for motor neurons in the ciliary ganglion and spinal cord, leading to its evaluation in humans suffering from motor neuron disease. In these trials, CNTF caused unexpected and substantial weight loss, raising concerns that it might produce cachectic-like effects. Countering this possibility was the suggestion that CNTF was working via a leptin-like mechanism to cause weight loss, based on the findings that CNTF acts via receptors that are not only related to leptin receptors, but also similarly distributed within hypothalamic nuclei involved in feeding. However, although CNTF mimics the ability of leptin to cause fat loss in mice that are obese because of genetic deficiency of leptin (ob/ob mice), CNTF is also effective in diet-induced obesity models that are more representative of human obesity, and which are resistant to leptin. This discordance again raised the possibility that CNTF might be acting via nonleptin pathways, perhaps more analogous to those activated by cachectic cytokines. Arguing strongly against this possibility, we now show that CNTF can activate hypothalamic leptin-like pathways in diet-induced obesity models unresponsive to leptin, that CNTF improves prediabetic parameters in these models, and that CNTF acts very differently than the prototypical cachectic cytokine, IL-1. Further analyses of hypothalamic signaling reveals that CNTF can suppress food intake without triggering hunger signals or associated stress responses that are otherwise associated with food deprivation; thus, unlike forced dieting, cessation of CNTF treatment does not result in binge overeating and immediate rebound weight gain.

F-spondin is a contact-repellent molecule for embryonic motor neurons

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural–functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis

We show that when telencephalic neural progenitors are briefly exposed to bone morphogenetic protein 2 (BMP2) in culture, their developmental fate is changed from neuronal cells to astrocytic cells. BMP2 significantly reduced the number of cells expressing microtubule-associated protein 2, a neuronal marker, and cells expressing nestin, a marker for undifferentiated neural precursors, but BMP2 increased the number of cells expressing S100-β, an astrocytic marker. In telencephalic neuroepithelial cells, BMP2 up-regulated the expression of negative helix–loop–helix (HLH) factors Id1, Id3, and Hes-5 (where Hes is homologue of hairy and Enhancer of Split) that inhibited the transcriptional activity of neurogenic HLH transcription factors Mash1 and neurogenin. Ectopic expression of either Id1 or Id3 (where Id is inhibitor of differentiation) inhibited neurogenesis of neuroepithelial cells, suggesting an important role for these HLH proteins in the BMP2-mediated changes in the neurogenic fate of these cells. Because gliogenesis in the brain and spinal cord, derived from implanted neural stem cells or induced by injury, is responsible for much of the failure of neuronal regeneration, this work may lead to a therapeutic strategy to minimize this problem.

Forebrain mechanisms of nociception and pain: Analysis through imaging

Pain is a unified experience composed of interacting discriminative, affective-motivational, and cognitive components, each of which is mediated and modulated through forebrain mechanisms acting at spinal, brainstem, and cerebral levels. The size of the human forebrain in relation to the spinal cord gives anatomical emphasis to forebrain control over nociceptive processing. Human forebrain pathology can cause pain without the activation of nociceptors. Functional imaging of the normal human brain with positron emission tomography (PET) shows synaptically induced increases in regional cerebral blood flow (rCBF) in several regions specifically during pain. We have examined the variables of gender, type of noxious stimulus, and the origin of nociceptive input as potential determinants of the pattern and intensity of rCBF responses. The structures most consistently activated across genders and during contact heat pain, cold pain, cutaneous laser pain or intramuscular pain were the contralateral insula and anterior cingulate cortex, the bilateral thalamus and premotor cortex, and the cerebellar vermis. These regions are commonly activated in PET studies of pain conducted by other investigators, and the intensity of the brain rCBF response correlates parametrically with perceived pain intensity. To complement the human studies, we developed an animal model for investigating stimulus-induced rCBF responses in the rat. In accord with behavioral measures and the results of human PET, there is a progressive and selective activation of somatosensory and limbic system structures in the brain and brainstem following the subcutaneous injection of formalin. The animal model and human PET studies should be mutually reinforcing and thus facilitate progress in understanding forebrain mechanisms of normal and pathological pain.

Supraspinal contributions to hyperalgesia

Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central (spinal) neurons, termed central sensitization. Nociceptor sensitization and central sensitization are considered to underlie, respectively, development of primary hyperalgesia and secondary hyperalgesia. Because central sensitization is considered to reflect plasticity at spinal synapses, the spinal cord has been the principal focus of studies of mechanisms of hyperalgesia. Not surprisingly, glutamate, acting at a spinal N-methyl-d-aspartate (NMDA) receptor, has been implicated in development of secondary hyperalgesia associated with somatic, neural, and visceral structures. Downstream of NMDA receptor activation, spinal nitric oxide (NO⋅), protein kinase C, and other mediators have been implicated in maintaining such hyperalgesia. Accumulating evidence, however, reveals a significant contribution of supraspinal influences to development and maintenance of hyperalgesia. Spinal cord transection prevents development of secondary, but not primary, mechanical and/or thermal hyperalgesia after topical mustard oil application, carrageenan inflammation, or nerve-root ligation. Similarly, inactivation of the rostral ventromedial medulla (RVM) attenuates hyperalgesia and central sensitization in several models of persistent pain. Inhibition of medullary NMDA receptors or NO⋅ generation attenuates somatic and visceral hyperalgesia. In support, topical mustard oil application or colonic inflammation increases expression of NO⋅ synthase in the RVM. These data suggest a prominent role for the RVM in mediating the sensitization of spinal neurons and development of secondary hyperalgesia. Results to date suggest that peripheral injury and persistent input engage spinobulbospinal mechanisms that may be the prepotent contributors to central sensitization and development of secondary hyperalgesia.

Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors

The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

Implications of immune-to-brain communication for sickness and pain

This review presents a view of hyperalgesia and allodynia not typical of the field as a whole. That is, exaggerated pain is presented as one of many natural consequences of peripheral infection and injury. The constellation of changes that results from such immune challenges is called the sickness response. This sickness response results from immune-to-brain communication initiated by proinflammatory cytokines released by activated immune cells. In response to signals it receives from the immune system, the brain orchestrates the broad array of physiological, behavioral, and hormonal changes that comprise the sickness response. The neurocircuitry and neurochemistry of sickness-induced hyperalgesia are described. One focus of this discussion is on the evidence that spinal cord microglia and astrocytes are key mediators of sickness-induced hyperalgesia. Last, evidence is presented that hyperalgesia and allodynia also result from direct immune activation, rather than neural activation, of these same spinal cord glia. Such glial activation is induced by viruses such as HIV-1 that are known to invade the central nervous system. Implications of exaggerated pain states created by peripheral and central immune activation are discussed.

Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions

Compelling evidence has accumulated over the last several years from our laboratory, as well as others, indicating that central hyperactive states resulting from neuronal plastic changes within the spinal cord play a critical role in hyperalgesia associated with nerve injury and inflammation. In our laboratory, chronic constriction injury of the common sciatic nerve, a rat model of neuropathic pain, has been shown to result in activation of central nervous system excitatory amino acid receptors and subsequent intracellular cascades including protein kinase C translocation and activation, nitric oxide production, and nitric oxide-activated poly(ADP ribose) synthetase activation. Similar cellular mechanisms also have been implicated in the development of tolerance to the analgesic effects of morphine. A recently observed phenomenon, the development of “dark neurons,” is associated with both chronic constriction injury and morphine tolerance. A site of action involved in both hyperalgesia and morphine tolerance is in the superficial laminae of the spinal cord dorsal horn. These observations suggest that hyperalgesia and morphine tolerance may be interrelated at the level of the superficial laminae of the dorsal horn by common neural substrates that interact at the level of excitatory amino acid receptor activation and subsequent intracellular events. The demonstration of interrelationships between neural mechanisms underlying hyperalgesia and morphine tolerance may lead to a better understanding of the neurobiology of these two phenomena in particular and pain in general. This knowledge may also provide a scientific basis for improved pain management with opiate analgesics.