Sharon Vinsant

Complete Dissociation of Motor Neuron Death from Motor Dysfunction by Bax Deletion in a Mouse Model of ALS

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Programmed Cell Death of Adult-Generated Hippocampal Neurons Is Mediated by the Proapoptotic Gene Bax

In the dentate gyrus (DG) of the adult mouse hippocampus, a substantial number of new cells are generated daily, but only a subset of these survive and differentiate into mature neurons, whereas the majority undergo programmed cell death (PCD). However, neither the intracellular machinery required for adult stem cell-derived neuronal death nor the biological implications of the significant loss of these newly generated cells have been examined. Several markers for apoptosis failed to reveal cell death in Bax-deficient mice, and this, together with a progressive increase in neuron number in the DG of the Bax knock-out, indicates that Bax is critical for the PCD of adult-generated hippocampal neurons. Whereas the proliferation of neural progenitor cells was not altered in the Bax-knock-out, there was an accumulation of doublecortin, calretinin+, and neuronal-specific nuclear protein+ postmitotic neurons, suggesting that Bax-mediated PCD of adult-generated neurons takes place during an early phase of differentiation. The absence of PCD in the adult also influenced the migration and maturation of adult-generated DG neurons. These results suggest that PCD in the adult brain plays a significant role in the regulation of multiple aspects of adult neurogenesis.

Quantitative changes in hippocampal structure following long-term exposure to Δ9-tetrahydrocannabinol: possible mediation by glucocorticoid systems

Although cannabinoids exert strong effects on brain function, there have been no extensive analyses of the long-term effects of cannabinoids on mammalian brain structure. Consequently, we conducted quantitative light and electron microscopic studies on the brains of rats treated chronically with delta 9-tetrahydrocannabinol (THC) (5 X weekly for 8 months--approximately 30% of the life-span). In these studies, we found significant THC-induced changes in hippocampal structure: specifically, THC-treated animals exhibited decreased neuronal density and increased glial cell reactivity (i.e. an increase of cytoplasmic inclusions). In addition, we confirmed prior reports of THC-induced increases in adrenal-pituitary activity, since both adrenocorticotropic hormone (ACTH) and corticosterone were elevated substantially during an acute stress. However, the animals appeared to be only minimally affected behaviorally by the doses used (highest dose: 8 mg/kg) and no effects of THC were observed on several ultrastructural variables, including synaptic density. The observed hippocampal morphometric effects of chronic THC are similar to apparent glucocorticoid-dependent changes that previously have been found to develop in rat hippocampus during normal aging. Given that cannabinoids and steroids are similar in chemical structure in several respects, therefore, the present results seem to raise the possibility that chronic THC exposure may alter hippocampal anatomical structure by interactions with, or mimicry of, adrenal steroid activity.

Effect of cocaine self‐administration on dopamine D2 receptors in rhesus monkeys

The present study used autoradiography to examine the effects of chronic self‐administration of cocaine on the density of dopamine D2 receptors in nonhuman primates. Three rhesus monkeys intravenously self‐administered an average of 1.35 mg/kg cocaine per day for 18–22 months until they were euthanized immediately after a self‐administration session. Binding site density of the D2 ligand [3H]raclopride (2 nM) was assessed in these monkeys as well as three untreated controls, using quantitativein vitro receptor autoradiography. As compared to untreated controls, D2 binding site density was significantly lower in the animals that self‐administered cocaine in all regions of the striatum rostral to the anterior commissure. These regions include the anterior and central regions of the caudate nucleus, putamen, olfactory tubercle, and both the shell and core of the nucleus accumbens. Within the substantia nigra and ventral tegmental area, by contrast, no differences were found in the density of D2 binding sites. These findings suggest a pervasive effect of cocaine on the regulation of D2 receptors in the striatum. The lack of change within the ventral midbrain, however, suggests a differential regulation of D2 receptors in the striatum and ventral midbrain. This study confirms and extends our knowledge of the neurobiological changes in the mesolimbic dopamine system that result from chronic exposure to cocaine. Synapse 30:88–96, 1998.

Characterization of early pathogenesis in the SOD1G93A mouse model of ALS: part II, results and discussion

Pathological events are well characterized in amyotrophic lateral sclerosis (ALS) mouse models, but review of the literature fails to identify a specific initiating event that precipitates disease pathology. There is now growing consensus in the field that axon and synapses are first cellular sites of degeneration, but controversy exists over whether axon and synapse loss is initiated autonomously at those sites or by pathology in the cell body, in nonneuronal cells or even in nonmotoneurons (MNs). Previous studies have identified pathological events in the mutant superoxide dismutase 1 (SOD1) models involving spinal cord, peripheral axons, neuromuscular junctions (NMJs), or muscle; however, few studies have systematically examined pathogenesis at multiple sites in the same study. We have performed ultrastructural examination of both central and peripheral components of the neuromuscular system in the SOD1G93A mouse model of ALS. Twenty percent of MNs undergo degeneration by P60, but NMJ innervation in fast fatigable muscles is reduced by 40% by P30. Gait alterations and muscle weakness were also found at P30. There was no change in axonal transport prior to initial NMJ denervation. Mitochondrial morphological changes are observed at P7 and become more prominent with disease progression. At P30 there was a significant decrease in excitatory axo‐dendritic and axo‐somatic synapses with an increase in C‐type axo‐somatic synapses. Our study examined early pathology in both peripheral and central neuromuscular system. The muscle denervation is associated with functional motor deficits and begins during the first postnatal month in SOD1G93A mice. Physiological dysfunction and pathology in the mitochondria of synapses and MN soma and dendrites occur, and disease onset in these animals begins more than 2 months earlier than originally thought. This information may be valuable for designing preclinical trials that are more likely to impact disease onset and progression.

Impaired Migration in the Rostral Migratory Stream But Spared Olfactory Function after the Elimination of Programmed Cell Death in Bax Knock-Out Mice

Rats and mice exhibit neurogenesis of olfactory bulb (OB) interneurons throughout adulthood. To homeostatically maintain stable neuron numbers, it is necessary to continuously remove a subset of OB neurons by programmed cell death (PCD). Here we demonstrate that Bax is critical for the elimination of OB neurons by showing that Bax-KO mice exhibit greatly reduced PCD in the OB. Despite the reduction of PCD, however, proliferation of progenitors and the size of the OB were virtually unaffected in Bax-knock-out (KO) mice. However, reducing PCD by Bax deletion affected the migration of a subset of adult-produced neurons by the disruption of glial tube formation as well as by premature detachment of neuroblasts from the migratory chain. Rescued cells aberrantly remained in the subventricular zone (SVZ)-rostral migratory stream (RMS), in which they differentiated into calretinin+ or GABA-expressing interneurons. Because of the migratory deficit, OB cell homeostasis involving new cell entry and PCD (neuronal turnover) was virtually absent in adult Bax-KO mice. Despite this, Bax-KO mice exhibited normal olfactory behaviors such as odor discrimination and olfactory memory which are thought to be influenced by adult neurogenesis. These results demonstrate that PCD is involved in the regulation of RMS migration and differentiation after OB neurogenesis, but that animals maintain normal olfactory function in the absence of PCD.

Effect of cocaine self-administration on striatal dopamine D1 receptors in rhesus monkeys

An array of evidence indicates that long‐term exposure to cocaine alters several components of the brain dopamine system. Because the release of dopamine in the nucleus accumbens (NAc) has been implicated in mediating the reinforcing effects of cocaine, changes in dopamine function can have profound effects on drug‐seeking and drug‐taking behavior. The present study examined the effects of the chronic self‐administration of cocaine on the D1 family of dopamine receptors in the rhesus monkey. The brains of three rhesus monkeys that had intravenously self‐administered an average of 1.35 mg/kg cocaine per day for 18–22 months were compared to the brains of three cocaine‐naive controls. The in vitro quantitative autoradiographic technique was used to quantify binding densities of the D1 ligand [3H]SCH‐23390 on cryostat‐cut sections of fresh frozen tissue. In animals that self‐administered cocaine, the density of D1 binding was significantly lower in the regions of the striatum at the level where the nucleus accumbens is most fully developed. The shell of the NAc showed the largest difference with significantly lower D1 binding also detected in adjacent regions of the caudate nucleus and the putamen. No differences were found in the rostral pole of the NAc or the dorsal striatum at that level. These findings suggest that chronic self‐administration of cocaine can modulate the density of dopamine D1 receptors in specific portions of the primate striatum. Such changes might underlie some of the behavioral consequences, like drug dependence and craving, of long‐term cocaine use. Synapse 28:1–9, 1998.

Characterization of early pathogenesis in the SOD1G93A mouse model of ALS: part I, background and methods

Charcot first described amyotrophic lateral sclerosis (ALS) in 1869; however, its causes remain largely unknown and effective, long‐term treatment strategies are not available. The first mouse model of ALS was developed after the identification of mutations in the superoxide dismutase 1 (SOD1) gene in 1993, and accordingly most of our knowledge of the etiology and pathogenesis of the disease comes from studies carried out using this animal model. Although numerous preclinical trials have been conducted in the mutant SOD1 mouse models, the results have been disappointing because they did not positively translate to clinical trials. One explanation may be that current understanding of when and where pathogenesis begins is insufficient to accurately guide preclinical trials. Further characterization of these early events may provide insight into disease onset, help in the discovery of presymptomatic diagnostic disease markers, and identify novel therapeutic targets. Here, we describe the rationale, approach, and methods for our extensive analysis of early changes that included an ultrastructural examination of central and peripheral components of the neuromuscular system in the SOD1G93A mouse and correlated these alterations with early muscle denervation, motor dysfunction, and motoneuron death. We also provide a discussion of published work to review what is known regarding early pathology in the SOD1 mouse model of ALS. The significance of this work is that we have examined early pathology simultaneously in both the spinal cord and peripheral neuromuscular system, and the results are presented in the companion paper (Part II, Results and Discussion). Our results provide evidence as to why a thorough characterization of animal models throughout the life span is critical for a strong foundation to design preclinical trials that may produce meaningful results.

Neuromuscular Development in the Absence of Programmed Cell Death: Phenotypic Alteration of Motoneurons and Muscle

The widespread, massive loss of developing neurons in the central and peripheral nervous system of birds and mammals is generally considered to be an evolutionary adaptation. However, until recently, models for testing both the immediate and long-term consequences of preventing this normal cell loss have not been available. We have taken advantage of several methods for preventing neuronal death in vivo to ask whether rescued neurons [e.g., motoneurons (MNs)] differentiate normally and become functionally incorporated into the nervous system. Although many aspects of MN differentiation occurred normally after the prevention of cell death (including the expression of several motoneuron-specific markers, axon projections into the ventral root and peripheral nerves, ultrastructure, dendritic arborization, and afferent axosomatic synapses), other features of the neuromuscular system (MNs and muscle) were abnormal. The cell bodies and axons of MNs were smaller than normal, many MN axons failed to become myelinated or to form functional synaptic contacts with target muscles, and a subpopulation of rescued cells were transformed from α- to γ-like MNs. Additionally, after the rescue of MNs in myogenin glial cell line-derived neurotrophic factor (MyoGDNF) transgenic mice, myofiber differentiation of extrafusal skeletal muscle was transformed and muscle physiology and motor behaviors were abnormal. In contrast, extrafusal myofiber phenotype, muscle physiology, and (except for muscle strength tests) motor behaviors were all normal after the rescue of MNs by genetic deletion of the proapoptotic gene Bax. However, there was an increase in intrafusal muscle fibers (spindles) in Bax knock-out versus both wild-type and MyoGDNF mice. Together, these data indicate that after the prevention of MN death, the neuromuscular system becomes transformed in novel ways to compensate for the presence of the thousands of excess cells.

Angiotensin-(1-7) immunoreactivity in the hypothalamus of the (mRen-2d)27 transgenic rat

The distribution of angiotensin-(1-7) immunoreactive neurons was compared to those of vasopressin-(VP) and oxytocin-(OT) immunoreactive (IR) neurons in the hypothalamus of adult (mRen-2d)27 transgenic hypertensive and Sprague-Dawley rats. In both strains, angiotensin (Ang)-(1-7)-IR cells were found in the supraoptic nucleus (SON), and in the anterior (ap-), medial (mp-), and lateral (lp-) parvocellular, and posterior magnocellular (pm-) subdivisions of the paraventricular (PVN) nucleus. Three-dimensional reconstructions showed that cells immunoreactive to Ang-(1-7) and VP were specifically co-distributed in the SON and in the pmPVN. Double-labeling neurons for both peptides revealed that both Ang-(1-7) and VP were colocalized in a subpopulation of neurons in the pmPVN and SON. In combination with previous studies, our results suggest that Ang-(1-7) and VP are colocalized, co-released and may have a combined action at a common target. In addition, the introduction of the mouse submandibular renin (mRen-2d) transgene into Sprague-Dawley rats does not appear to have altered the fundamental organization of hypothalamic peptide systems involved in fluid homeostasis.