Heather A. Boger

A partial GDNF depletion leads to earlier age-related deterioration of motor function and tyrosine hydroxylase expression in the substantia nigra

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for peripheral organs, spinal cord, and midbrain dopamine (DA) neurons. Levels of GDNF deteriorate in the substantia nigra in Parkinsons disease (PD). A heterozygous mouse model was created to assess whether chronic reductions in this neurotrophic factor impact motor function and the nigrostriatal dopamine system during the aging process. Due to the important role GDNF plays in kidney development, kidney function and histology were assessed and were found to be normal in both wild-type (WT) and GDNF+/- mice up to 22 months of age. Further, the animals of both genotypes had similar weights throughout the experiment. Locomotor activity was assessed for male WT and GDNF+/- mice at 4-month intervals from 4 to 20 months of age. Both GDNF+/- and WT mice exhibited an age-related decline in horizontal activity, although this was found 4 months earlier in GDNF+/- mice, at 12 months of age. Comparison of young (8 month old) and aged (20 month old) GDNF+/- and WT mice on an accelerating rotarod apparatus established a deficiency for aged but not young GDNF+/- mice, while aged WT mice performed as well as young WT mice on this task. Finally, both WT and GDNF+/- mice exhibited an age-related decrease in substantia nigra TH immunostaining, which was accelerated in the GDNF+/- mice. These behavioral and histological alterations suggest that GDNF may be an important factor for maintenance of motor coordination and spontaneous activity as well as DA neuronal function during aging, and further suggest that GDNF+/- mice may serve as a model for neuroprotective or rescue studies.

Effects of long-term memantine on memory and neuropathology in Ts65Dn mice, a model for Down syndrome.

Memantine is a partial NMDA receptor antagonist that has been shown to improve learning and memory in several animal models, and is approved for the treatment of Alzheimers disease (AD). Chronic treatments using memantine in animal models of Alzheimers disease show disease-modifying effects and suggest a potential neuroprotective function. The present study assessed the effects of both short- and long-term memantine treatment in a mouse model of Down syndrome (DS), the Ts65Dn mouse. The Ts65Dn mouse contains a partial trisomy of murine chromosome 16, and exhibits hippocampal-dependent memory deficits, as well as progressive degeneration of basal forebrain cholinergic neurons (BCFNs). Ts65Dn mice were treated with memantine for a period of 6 months, beginning at 4 months of age. At the end of treatment the mice underwent memory testing using novel object recognition and water radial arm maze tasks, and then histologically analyzed for markers of neurodegeneration. Memantine treatment improved spatial and recognition memory performance in the Ts65Dn mice, though not to the level of normosomic littermate controls. Despite these memory improvements, histological analysis found no morphological signs of neuroprotection of basal forebrain cholinergic or locus coeruleus neurons in memantine-treated Ts65Dn mice. However, memantine treatment of Ts65Dn mice gave rise to elevated brain-derived neurotrophic factor expression in the hippocampus and frontal cortex, suggesting a mechanism of behavioral modification. Thus, our findings provide further evidence for memory facilitation of memantine, but suggest pharmacological rather than neuroprotective effects of memantine both after acute and chronic treatment in this mouse model.

Synaptic Glutamate Spillover Due to Impaired Glutamate Uptake Mediates Heroin Relapse

Reducing the enduring vulnerability to relapse is a therapeutic goal in treating drug addiction. Studies with animal models of drug addiction show a marked increase in extrasynaptic glutamate in the core subcompartment of the nucleus accumbens (NAcore) during reinstated drug seeking. However, the synaptic mechanisms linking drug-induced changes in extrasynaptic glutamate to relapse are poorly understood. Here, we discovered impaired glutamate elimination in rats extinguished from heroin self-administration that leads to spillover of synaptically released glutamate into the nonsynaptic extracellular space in NAcore and investigated whether restoration of glutamate transport prevented reinstated heroin seeking. Through multiple functional assays of glutamate uptake and analyzing NMDA receptor-mediated currents, we show that heroin self-administration produced long-lasting downregulation of glutamate uptake and surface expression of the transporter GLT-1. This downregulation was associated with spillover of synaptic glutamate to extrasynaptic NMDA receptors within the NAcore. Ceftriaxone restored glutamate uptake and prevented synaptic glutamate spillover and cue-induced heroin seeking. Ceftriaxone-induced inhibition of reinstated heroin seeking was blocked by morpholino-antisense targeting GLT-1 synthesis. These data reveal that the synaptic glutamate spillover in the NAcore results from reduced glutamate transport and is a critical pathophysiological mechanism underling reinstated drug seeking in rats extinguished from heroin self-administration.

Long-Term Consequences of Methamphetamine Exposure in Young Adults Are Exacerbated in Glial Cell Line-Derived Neurotrophic Factor Heterozygous Mice

Methamphetamine abuse in young adults has long-term deleterious effects on brain function that are associated with damage to monoaminergic neurons. Administration of glial cell line-derived neurotrophic factor (GDNF) protects dopamine neurons from the toxic effects of methamphetamine in animal models. Therefore, we hypothesized that a partial GDNF gene deletion would increase the susceptibility of mice to methamphetamine neurotoxicity during young adulthood and possibly increase age-related deterioration of behavior and dopamine function. Two weeks after a methamphetamine binge (4 × 10 mg/kg, i.p., at 2 h intervals), GDNF+/− mice had a significantly greater reduction of tyrosine hydroxylase immunoreactivity in the medial striatum, a proportionally greater depletion of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the striatum, and a greater increase in activated microglia in the substantia nigra than wild-type mice. At 12 months of age, methamphetamine-treated GDNF+/− mice exhibited less motor activity and lower levels of tyrosine hydroxylase-immunoreactivity, dopamine, DOPAC, and serotonin than wild-type mice. Greater striatal dopamine transporter activity in GDNF+/− mice may underlie their differential response to methamphetamine. These data suggest the possibility that methamphetamine use in young adults, when combined with lower levels of GDNF throughout life, may precipitate the appearance of parkinsonian-like behaviors during aging.

Gq-DREADD Selectively Initiates Glial Glutamate Release and Inhibits Cue-induced Cocaine Seeking.

BACKGROUND Glial cells of the central nervous system directly influence neuronal activity by releasing neuroactive small molecules, including glutamate. Long-lasting cocaine-induced reductions in extracellular glutamate in the nucleus accumbens core (NAcore) affect synaptic plasticity responsible for relapse vulnerability. METHODS We transduced NAcore astrocytes with an adeno-associated virus vector expressing hM3D designer receptor exclusively activated by a designer drug (DREADD) under control of the glial fibrillary acidic protein promoter in 62 male Sprague Dawley rats, 4 dominant-negative soluble N-ethylmaleimide-sensitive factor attachment protein receptor mice, and 4 wild-type littermates. Using glutamate biosensors, we measured NAcore glutamate levels following intracranial or systemic administration of clozapine N-oxide (CNO) and tested the ability of systemic CNO to inhibit reinstated cocaine or sucrose seeking following self-administration and extinction training. RESULTS Administration of CNO in glial fibrillary acidic protein-hM3D-DREADD transfected animals increased NAcore extracellular glutamate levels in vivo. The glial origin of released glutamate was validated by an absence of CNO-mediated release in mice expressing a dominant-negative soluble N-ethylmaleimide-sensitive factor attachment protein receptor variant in glia. Also, CNO-mediated release was relatively insensitive to N-type calcium channel blockade. Systemic administration of CNO inhibited cue-induced reinstatement of cocaine seeking in rats extinguished from cocaine but not sucrose self-administration. The capacity to inhibit reinstated cocaine seeking was prevented by systemic administration of the group II metabotropic glutamate receptor antagonist LY341495. CONCLUSIONS DREADD-mediated glutamate gliotransmission inhibited cue-induced reinstatement of cocaine seeking by stimulating release-regulating group II metabotropic glutamate receptor autoreceptors to inhibit cue-induced synaptic glutamate spillover.

A Noradrenergic Lesion Exacerbates Neurodegeneration in a Down Syndrome Mouse Model

Individuals with Down syndrome (DS) acquire Alzheimers-like dementia (AD) and associated neuropathology earlier and at significantly greater rates than age-matched normosomic individuals. However, biological mechanisms have not been discovered and there is currently limited therapy for either DS- or AD-related dementia. Segmental trisomy 16 (Ts65Dn) mice provide a useful model for many of the degenerative changes which occur with age in DS including cognitive deficits, neuroinflammation, and degeneration of basal forebrain cholinergic neurons. Loss of noradrenergic locus coeruleus (LC) neurons is an early event in AD and in DS, and may contribute to the neuropathology. We report that Ts65Dn mice exhibit progressive loss of norepinephrine (NE) phenotype in LC neurons. In order to determine whether LC degeneration contributes to memory loss and neurodegeneration in Ts65Dn mice, we administered the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 2 doses of 50 mg/kg, i.p.) to Ts65Dn mice at four months of age, prior to working memory loss. At eight months of age, Ts65Dn mice treated with DSP-4 exhibited an 80% reduction in hippocampal NE, coupled with a marked increase in hippocampal neuroinflammation. Noradrenergic depletion also resulted in accelerated cholinergic neuron degeneration and a further impairment of memory function in Ts65Dn mice. In contrast, DSP-4 had minimal effects on normosomic littermates, suggesting a disease-modulated vulnerability to NE loss in the DS mouse model. These data suggest that noradrenergic degeneration may play a role in the progressive memory loss, neuroinflammation, and cholinergic loss occurring in DS individuals, providing a possible therapeutic avenue for future clinical studies.

Effects of brain-derived neurotrophic factor on dopaminergic function and motor behavior during aging

Brain‐derived neurotrophic factor (BDNF) is critical in synaptic plasticity and in the survival and function of midbrain dopamine neurons. In this study, we assessed the effects of a partial genetic deletion of BDNF on motor function and dopamine (DA) neurotransmitter measures by comparing Bdnf+/− with wildtype mice (WT) at different ages. Bdnf+/− and WT mice had similar body weights until 12 months of age; however, at 21 months, Bdnf+/− mice were significantly heavier than WT mice. Horizontal and vertical motor activity was reduced for Bdnf+/− compared to WT mice, but was not influenced by age. Performance on an accelerating rotarod declined with age for both genotypes and was exacerbated for Bdnf+/− mice. Body weight did not correlate with any of the three behavioral measures studied. Dopamine neurotransmitter markers indicated no genotypic difference in striatal tyrosine hydroxylase, DA transporter (DAT) or vesicular monoamine transporter 2 (VMAT2) immunoreactivity at any age. However, DA transport via DAT (starting at 12 months) and VMAT2 (starting at 3 months) as well as KCl‐stimulated DA release were reduced in Bdnf+/− mice and declined with age suggesting an increasingly important role for BDNF in the release and uptake of DA with the aging process. These findings suggest that a BDNF expression deficit becomes more critical to dopaminergic dynamics and related behavioral activities with increasing age.

Cocaine Self-Administration and Extinction Leads to Reduced Glial Fibrillary Acidic Protein Expression and Morphometric Features of Astrocytes in the Nucleus Accumbens Core

BACKGROUND As a more detailed picture of nervous system function emerges, diversity of astrocyte function becomes more widely appreciated. While it has been shown that cocaine experience impairs astroglial glutamate uptake and release in the nucleus accumbens (NAc), few studies have explored effects of self-administration on the structure and physiology of astrocytes. We investigated the effects of extinction from daily cocaine self-administration on astrocyte characteristics including glial fibrillary acidic protein (GFAP) expression, surface area, volume, and colocalization with a synaptic marker. METHODS Cocaine or saline self-administration and extinction were paired with GFAP Westerns, immunohistochemistry, and fluorescent imaging of NAc core astrocytes (30 saline-administering and 36 cocaine-administering male Sprague Dawley rats were employed). Imaging was performed using a membrane-tagged lymphocyte protein tyrosine kinase-green fluorescent protein (Lck-GFP) driven by the GFAP promoter, coupled with synapsin I immunohistochemistry. RESULTS GFAP expression was significantly reduced in the NAc core following cocaine self-administration and extinction. Similarly, we observed an overall smaller surface area and volume of astrocytes, as well as reduced colocalization with synapsin I, in cocaine-administering animals. Cocaine-mediated reductions in synaptic contact were reversed by the β-lactam antibiotic ceftriaxone. CONCLUSIONS Multiple lines of investigation indicate that NAc core astrocytes exist in a hyporeactive state following cocaine self-administration and extinction. Decreased association with synaptic elements may be particularly meaningful, as cessation of chronic cocaine use is associated with changes in synaptic strength and resistance to the induction of synaptic plasticity. We hypothesize that the reduced synaptic colocalization of astrocytes represents an important maladaptive cellular response to cocaine and the mechanisms underlying relapse vulnerability.

The nigrostriatal dopamine system of aging GFRα-1 heterozygous mice: neurochemistry, morphology and behavior

Given the established importance of glial cell line‐derived neurotrophic factor (GDNF) in maintaining dopaminergic neurotransmitter systems, the nigrostriatal system and associated behaviors of mice with genetic reduction of its high‐affinity receptor, GDNF receptor (GFR)α‐1 (GFRα‐1+/−), were compared with wild‐type controls. Motor activity and the stimulatory effects of a dopamine (DA) D1 receptor agonist (SKF 82958) were assessed longitudinally at 8 and 18 months of age. Monoamine concentrations and dopaminergic nerve terminals in the striatum and the number of dopaminergic neurons in the substantia nigra (SN) were assessed. The results support the importance of GFRα‐1 in maintaining normal function of the nigrostriatal dopaminergic system, with deficits being observed for GFRα‐1+/− mice at both ages. Motor activity was lower and the stimulatory effects of the DA agonist were enhanced for the older GFRα‐1+/− mice. DA in the striatum was reduced in the GFRα‐1+/− mice at both ages, and tyrosine hydroxylase‐positive cell numbers in the SN were reduced most substantially in the older GFRα‐1+/− mice. The combined behavioral, pharmacological probe, neurochemical and morphological measures provide evidence of abnormalities in GFRα‐1+/− mice that are indicative of an exacerbated aging‐related decline in dopaminergic system function. The noted deficiencies, in turn, suggest that GFRα‐1 is necessary for GDNF to maintain normal function of the nigrostriatal dopaminergic system. Although the precise mechanism(s) for the aging‐related changes in the dopaminergic system remain to be established, the present study clearly establishes that genetic reductions in GFRα‐1 can contribute to the degenerative changes observed in this system during the aging process.

Glial cell line-derived neurotrophic factor is essential for neuronal survival in the locus coeruleus-hippocampal noradrenergic pathway.

It has been shown that the noradrenergic (NE) locus coeruleus (LC)-hippocampal pathway plays an important role in learning and memory processing, and that the development of this transmitter pathway is influenced by neurotrophic factors. Although some of these factors have been discovered, the regulatory mechanisms for this developmental event have not been fully elucidated. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor influencing LC-NE neurons. We have utilized a GDNF knockout animal model to explore its function on the LC-NE transmitter system during development, particularly with respect to target innervation. By transplanting various combinations of brainstem (including LC) and hippocampal tissues from wildtype or GDNF knockout fetuses into the brains of adult wildtype mice, we demonstrate that normal postnatal development of brainstem LC-NE neurons is disrupted as a result of the GDNF null mutation. Tyrosine hydroxylase immunohistochemistry revealed that brainstem grafts had markedly reduced number and size of LC neurons in transplants from knockout fetuses. NE fiber innervation into the hippocampal co-transplant from an adjacent brainstem graft was also influenced by the presence of GDNF, with a significantly more robust innervation observed in transplants from wildtype fetuses. The most successful LC/hippocampal co-grafts were generated from fetuses expressing the wildtype GDNF background, whereas the most severely affected transplants were derived from double transplants from null-mutated fetuses. Our data suggest that development of the NE LC-hippocampal pathway is dependent on the presence of GDNF, most likely through a target-derived neurotrophic function.