Cindy Moore

Circadian rhythm of intraocular pressure in the rat

To define the characteristics of the diurnal variation of intraocular pressure (IOP) in eyes of awake rats, ten male brown Norway rats were entrained to a 12-hour light:12-hour dark (12L:12D) lighting schedule and were conditioned to IOP measurement with the TonoPen XL tonometer while awake, using only 0.5% proparacaine HCl anesthesia. The IOP measurements were performed in 4 experiments: Preliminary-IOP was measured at 6-hour intervals in both eyes of each animal, to determine correlation between right and left eyes; Light:Dark-lighting remained the same as in the preliminary experiment, but the measurement schedule was altered so that measurements were obtained at 4-hour intervals in alternating eyes, over two 24-hour light cycles; Dark:Dark-animals were placed in constant dark (0L:24D) and, after 72 h, measurements were obtained at 4-hour intervals in alternating eyes. Animals were then re-entrained to the previous 12L:12D schedule for 7 days, after which they were returned to constant dark and the experiment was repeated; and Dark:Light-animals were entrained to a reversed light:dark cycle (12D:12L) for 28 days, after which measurements were obtained in the same fashion as in the Light:Dark experiment. Close agreement was found between right- and left-eye IOPs. Animals on a 12L:12D schedule exhibited lowest IOP while the lights were on (19.3 +/- 1.9 mm Hg), and highest (31.3 +/- 1.3 mm Hg) while the lights were off. Pressure changes anticipated the change from light to dark and dark to light. This pattern persisted in constant dark, and was reversed when the cycle was changed to 12D:12L. Brown Norway rats possess a regular rhythm of IOP that is entrained by the cycle of light and dark, and persistence of this rhythm in constant dark establishes it as a circadian rhythm. Furthermore, our results indicate that reliable and physiologically meaningful IOP measurements can be obtained in awake rats using the TonoPen XL tonometer.

Loss of Leucine-rich Repeat Kinase 2 (LRRK2) in Rats Leads to Progressive Abnormal Phenotypes in Peripheral Organs

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson’s disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype. In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats [1] and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson’s disease.

Long-term non-invasive measurement of intraocular pressure in the rat eye

To study the optic neuropathy associated with glaucoma, a system for accurate, reliable, and non-invasive monitoring of intraocular pressure (IOP) is required. Of particular interest is the effect of sampling frequency on IOP. To address this issue, ten adult male brown Norway rats (group 1) were acclimatized to a 12-h/12-h light/dark cycle. On 20 days over a 30-day period, rats were anesthetized with short-acting isoflurane (Forane) inhalant anesthesia and IOP for each eye was determined by averaging 15 valid individual readings obtained with a TonoPen 2 tonometer. The last 12 measurement sessions were performed on a daily basis. To determine the minimum tolerable interval between IOP measurement sessions, a second group of 10 animals (group 2) was acclimatized in the same manner as group 1, and IOP was measured every 4 days over a period of 80 days. Next, IOP was measured every 4 days over a period of 28 days, and finally, every 2 days over a period of 19 days. For all group 1 measurements, there was no statistically significant difference between the right and left eye IOP, 14.75 +/- 1.08 (SEM) and 14.90 +/- 1.09 mm Hg, respectively. However, daily measurements produced a steady decrease in IOP and gradual weight loss. For group 2, overall mean right and left eye IOPs were 15.24 +/- 1.28 (SEM) and 15.12 +/- 1.26, respectively and were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Astroglial plasticity and glutamate function in a chronic mouse model of Parkinson's disease.

Astrocytes play a major role in maintaining low levels of synaptically released glutamate, and in many neurodegenerative diseases, astrocytes become reactive and lose their ability to regulate glutamate levels, through a malfunction of the glial glutamate transporter-1. However, in Parkinsons disease, there are few data on these glial cells or their regulation of glutamate transport although glutamate cytotoxicity has been blamed for the morphological and functional decline of striatal neurons. In the present study, we use a chronic mouse model of Parkinsons disease to investigate astrocytes and their relationship to glutamate, its extracellular level, synaptic localization, and transport. C57/bl mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). From 4 to 8 weeks after treatment, these mice show a significant loss of dopaminergic terminals in the striatum and a significant increase in the size and number of GFAP-immunopositive astrocytes. However, no change in extracellular glutamate, its synaptic localization, or transport kinetics was detected. Nevertheless, the density of transporters per astrocyte is significantly reduced in the MPTP/p-treated mice when compared to controls. These results support reactive gliosis as a means of striatal compensation for dopamine loss. The reduction in transporter complement on individual cells, however, suggests that astrocytic function may be compromised. Although reactive astrocytes are important for maintaining homeostasis, changes in their ability to regulate glutamate and its associated synaptic functions could be important for the progressive nature of the pathophysiology associated with Parkinsons disease.

Nicotine alters striatal glutamate function and decreases the apomorphine-induced contralateral rotations in 6-OHDA-lesioned rats

The overall goal of this study was to determine the effects of subchronic nicotine (0.4 mg/kg) treatment for 7 or 14 days on striatal glutamate function in both naïve and in 6-hydroxydopamine (6-OHDA)-treated rats in which the nigrostriatal dopamine pathway was lesioned. In lesioned animals, the effect of nicotine on apomorphine-induced contralateral rotations was also assessed. In naïve rats, once daily nicotine administration for 7 or 14 days resulted in a decrease and then an increase, respectively, in the basal extracellular level of striatal glutamate compared to the saline-treated group. Ultrastructurally, 14-day treatment with nicotine resulted in an increase in the density of striatal glutamate immunolabeling within nerve terminals making an asymmetrical synaptic contact compared to the saline-treated group. In 6-OHDA-lesioned animals, coadministration of nicotine with apomorphine or nicotine alone for 7 days resulted in an increase in the density of nerve terminal glutamate immunolabeling, compared to the apomorphine- or saline-treated groups. However, coadministration of nicotine with apomorphine for 14 days resulted in a decrease in the density of nerve terminal glutamate immunolabeling compared to the nicotine-treated group. Following subchronic treatment of 6-OHDA-lesioned rats with apomorphine for 7 or 14 days, there was an increase in the number of apomorphine-induced contralateral rotations compared to the saline treated group. There was a decrease in the number of apomorphine-induced contralateral rotations in the group coadministered nicotine with apomorphine for 7 or 14 days compared to the apomorphine treated group. The data suggests that in this 6-OHDA lesion model of Parkinsons disease, treatment with nicotine may be useful in counteracting the increased behavioral effect (i.e., contralateral rotations) observed after treatment with a dopamine agonist, such as apomorphine.

Effects of Subthalamic Nucleus Lesions and Stimulation upon Corticostriatal Afferents in the 6-Hydroxydopamine-Lesioned Rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinsons disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission.

Intervention with 7,8-dihydroxyflavone blocks further striatal terminal loss and restores motor deficits in a progressive mouse model of Parkinson’s disease☆

Parkinsons disease (PD) is a progressive neurological disorder and current therapies help alleviate symptoms, but are not disease modifying. In the flavonoid class of compounds, 7,8-dihydroxyflavone (7,8-DHF) has been reported to elicit tyrosine kinase receptor B (TrkB) dimerization and autophosphorylation that further stimulates signaling cascades to promote cell survival/growth, differentiation, and plasticity. In this study we investigated if 7,8-DHF could prevent further loss of dopaminergic cells and terminals if introduced at the midpoint (i.e. intervention) of our progressive mouse model of PD. In our model, 1-methyl-4phenyl-1,2,3,6-tetrahyrdopyridine (MPTP) is administered with increased doses each week (8, 16, 24, 32-kg/mg) over a 4-week period. We found that despite 4 weeks of MPTP treatment, animals administered 7,8-DHF starting at the 2-week time period maintained 54% of the tyrosine hydroxylase (TH) levels within the dorsolateral (DL) striatum compared to the vehicle group, which was comparable to animals treated with MPTP for 2 weeks and was significantly greater compared to animals treated with MPTP for the full 4 weeks. Animals treated with MPTP and 7,8-DHF also demonstrated increased levels of, a sprouting-associated protein, superior cervical ganglion-10 (SCG10), phosphorylated TrkB (pTrkB), and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) within the DL striatum and substantia nigra (SN) compared to the 4-week MPTP-treated animals. In addition, motor deficits seen in the 2- and 4-week MPTP-treated animals were restored following administration of 7,8-DHF. We are reporting here for the first time that intervention with 7,8-DHF blocks further loss of dopaminergic terminals and restores motor deficits in our progressive MPTP mouse model. Our data suggest that 7,8-DHF has the potential to be a translational therapy in PD.

Differential electrophysiological and morphological alterations of thalamostriatal and corticostriatal projections in the R6/2 mouse model of Huntington's disease

Huntingtons disease (HD) is a fatal genetic disorder characterized by cell death of medium-sized spiny neurons (MSNs) in the striatum, traditionally attributed to excessive glutamate inputs and/or receptor sensitivity. While changes in corticostriatal projections have typically been studied in mouse models of HD, morphological and functional alterations in thalamostriatal projections have received less attention. In this study, an adeno-associated virus expressing channelrhodopsin-2 under the calcium/calmodulin-dependent protein kinase IIα promoter was injected into the sensorimotor cortex or the thalamic centromedian-parafascicular nuclear complex in the R6/2 mouse model of HD, to permit selective activation of corticostriatal or thalamostriatal projections, respectively. In symptomatic R6/2 mice, peak amplitudes and areas of corticostriatal glutamate AMPA and NMDA receptor-mediated responses were reduced. In contrast, although peak amplitudes of AMPA and NMDA receptor-mediated thalamostriatal responses also were reduced, the areas remained unchanged due to an increase in response decay times. Blockade of glutamate reuptake further increased response areas and slowed rise and decay times of NMDA responses. These effects appeared more pronounced at thalamostriatal synapses of R6/2 mice, suggesting increased activation of extrasynaptic NMDA receptors. In addition, the probability of glutamate release was higher at thalamostriatal than corticostriatal synapses, particularly in R6/2 mice. Morphological studies indicated that the density of all excitatory synaptic contacts onto MSNs was reduced, which matches the basic electrophysiological findings of reduced amplitudes. There was a consistent reduction in the area of spines but little change in presynaptic terminal size, indicating that the postsynaptic spine may be more significantly affected than presynaptic terminals. These results highlight the significant and differential contribution of the thalamostriatal projection to glutamate excitotoxicity in HD.

Exercise in an animal model of Parkinson’s disease: Motor recovery but not restoration of the nigrostriatal pathway

Many clinical studies have reported on the benefits of exercise therapy in patients with Parkinsons disease (PD). Exercise cannot stop the progression of PD or facilitate the recovery of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) (Bega et al., 2014). To tease apart this paradox, we utilized a progressive MPTP (1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine) mouse model in which we initiated 4weeks of treadmill exercise after the completion of toxin administration (i.e., restoration). We found in our MPTP/exercise (MPTP+EX) group several measures of gait function that recovered compared to the MPTP only group. Although there was a small recovery of tyrosine hydroxylase (TH) positive DA neurons in the SNpc and terminals in the striatum, this increase was not statistically significant. These small changes in TH could not explain the improvement of motor function. The MPTP group had a significant 170% increase in the glycosylated/non-glycosylated dopamine transporter (DAT) and a 200% increase in microglial marker, IBA-1, in the striatum. The MPTP+EX group showed a nearly full recovery of these markers back to the vehicle levels. There was an increase in GLT-1 levels in the striatum due to exercise, with no change in striatal BDNF protein expression. Our data suggest that motor recovery was not prompted by any significant restoration of DA neurons or terminals, but rather the recovery of DAT and dampening the inflammatory response. Although exercise does not promote recovery of nigrostriatal DA, it should be used in conjunction with pharmaceutical methods for controlling PD symptoms.

Dietary therapy restores glutamatergic input to orexin/hypocretin neurons after traumatic brain injury in mice

Study ObjectivesnIn previous work, dietary branched-chain amino acid (BCAA) supplementation, precursors to de novo glutamate and γ-aminobutyric acid (GABA) synthesis, restored impaired sleep-wake regulation and orexin neuronal activity following traumatic brain injury (TBI) in mice. TBI was speculated to reduce orexin neuronal activity through decreased regional excitatory (glutamate) and/or increased inhibitory (GABA) input. Therefore, we hypothesized that TBI would decrease synaptic glutamate and/or increase synaptic GABA in nerve terminals contacting orexin neurons, and BCAA supplementation would restore TBI-induced changes in synaptic glutamate and/or GABA.nnnMethodsnBrain tissue was processed for orexin pre-embed diaminobenzidine labeling and glutamate or GABA postembed immunogold labeling. The density of glutamate and GABA immunogold within presynaptic nerve terminals contacting orexin-positive lateral hypothalamic neurons was quantified using electron microscopy in three groups of mice (n = 8 per group): Sham/noninjured controls, TBI without BCAA supplementation, and TBI with BCAA supplementation (given for 5 days, 48 hr post-TBI). Glutamate and GABA were also quantified within the cortical penumbral region (layer VIb) adjacent to the TBI lesion.nnnResultsnIn the hypothalamus and cortex, TBI decreased relative glutamate density in presynaptic terminals making axodendritic contacts. However, BCAA supplementation only restored relative glutamate density within presynaptic terminals contacting orexin-positive hypothalamic neurons. BCAA supplementation did not change relative glutamate density in presynaptic terminals making axosomatic contacts, or relative GABA density in presynaptic terminals making axosomatic or axodendritic contacts, within either the hypothalamus or cortex.nnnConclusionsnThese results suggest TBI compromises orexin neuron function via decreased glutamate density and highlight BCAA supplementation as a potential therapy to restore glutamate density to orexin neurons.