Current Perspectives: Obesity and Neurodegeneration - Links and Risks.

Obesity is increasing in prevalence across all age groups. Long-term obesity can lead to the development of metabolic and cardiovascular diseases through its effects on adipose, skeletal muscle, and liver tissue. Pathological mechanisms associated with obesity include immune response and inflammation as well as oxidative stress and consequent endothelial and mitochondrial dysfunction. Recent evidence links obesity to diminished brain health and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Both AD and PD are associated with insulin resistance, an underlying syndrome of obesity. Despite these links, causative mechanism(s) resulting in neurodegenerative disease remain unclear. This review discusses relationships between obesity, AD, and PD, including clinical and preclinical findings. The review then briefly explores nonpharmacological directions for intervention.

Forelimb Resistance Exercise Protects Against Neuromuscular Junction Denervation in the SOD1-G93A Rat Model of ALS.

Introduction: The symptoms of Amyotrophic Lateral Sclerosis (ALS) include muscle weakness and eventual paralysis. These symptoms result from denervation of the neuromuscular junction (NMJ) and motor neuron cell death in the brain and spinal cord. Due to the "dying back" pattern of motor neuron degeneration, protecting NMJs should be a therapeutic priority. Although exercise has the potential to protect against NMJ denervation, its use in ALS has been controversial. Most preclinical studies have focused on aerobic exercise, which report that exercise can be beneficial at moderate intensities. The effects of resistance exercise on NMJ preservation in limb muscles have not been explored. Methods: We trained male SOD1-G93A rats, which model ALS, to perform a unilateral isometric forelimb resistance exercise task. This task allows within-animal comparisons of trained and untrained forelimbs. We then determined the effects of isometric resistance exercise on NMJ denervation and AMP kinase (AMPK) activation in forelimb muscles. Results: Our results revealed that SOD1-G93A rats were able to learn and perform the task similarly to wildtype rats, even after loss of body weight. SOD1-G93A rats exhibited significantly greater NMJ innervation in their trained vs their untrained forelimb biceps muscles. Measures of activated (phosphorylated) AMPK (pAMPK) were also greater in the trained vs untrained forelimb triceps muscles. Discussion: These results demonstrate that isometric resistance exercise may protect against NMJ denervation in ALS. Future studies are required to determine the extent to which our findings generalize to female SOD1-G93A rats and to other subtypes of ALS.

MGE-Like Neural Progenitor Cell Survival and Expression of Parvalbumin and Proenkephalin in a Jaundiced Rat Model of Kernicterus.

Kernicterus is a permanent condition caused by brain damage from bilirubin toxicity. Dystonia is one of the most debilitating symptoms of kernicterus and results from damage to the globus pallidus (GP). One potential therapeutic strategy to treat dystonia in kernicterus is to replace lost GP neurons and restore basal ganglia circuits through stem cell transplantation. Toward this end, we differentiated human embryonic stem cells (hESCs) into medial ganglion eminence (MGE; the embryological origin of most of the GP neurons)-like neural precursor cells (NPCs). We determined neurochemical phenotype in cell culture and after transplanting into the GP of jaundiced Gunn rats. We also determined grafted cell survival as well as migration, distribution, and morphology after transplantation. As in the GP, most cultured MGE-like NPCs expressed γ-aminobutyric acid (GABA), with some co-expressing markers for parvalbumin (PV) and others expressing markers for pro-enkephalin (PENK). MGE-like NPCs survived in brains at least 7 weeks after transplantation, with most aggregating near the injection site. Grafted cells expressed GABA and PV or PENK as in the normal GP. Although survival was low and the maturity of grafted cells varied, many cells produced neurite outgrowth. While promising, our results suggest the need to further optimize the differentiation protocol for MGE-like NPC for potential use in treating dystonia in kernicterus.

Reduced Liver-Specific PGC1a Increases Susceptibility for Short-Term Diet-Induced Weight Gain in Male Mice.

The central integration of peripheral neural signals is one mechanism by which systemic energy homeostasis is regulated. Previously, increased acute food intake following the chemical reduction of hepatic fatty acid oxidation and ATP levels was prevented by common hepatic branch vagotomy (HBV). However, possible offsite actions of the chemical compounds confound the precise role of liver energy metabolism. Herein, we used a hepatocyte PGC1a heterozygous (LPGC1a) mouse model, with associated reductions in mitochondrial fatty acid oxidation and respiratory capacity, to assess the role of liver energy metabolism in systemic energy homeostasis. LPGC1a male, but not female, mice had a 70% greater high-fat/high-sucrose (HFHS) diet-induced weight gain compared to wildtype (WT) mice (p < 0.05). The greater weight gain was associated with altered feeding behavior and lower activity energy expenditure during the HFHS diet in LPGC1a males. WT and LPGC1a mice underwent sham surgery or HBV to assess whether vagal signaling was involved in the HFHS-induced weight gain of male LPGC1a mice. HBV increased HFHS-induced weight gain (85%, p < 0.05) in male WT mice, but not LPGC1a mice. These data demonstrate a sex-specific role of reduced liver energy metabolism in acute diet-induced weight gain, and the need for a more nuanced assessment of the role of vagal signaling in short-term diet-induced weight gain.

Intrinsic Aerobic Capacity Affects Hippocampal pAkt and HSP72 Response to an Acute High Fat Diet and Heat Treatment in Rats.

BACKGROUND: Aerobic capacity is associated with metabolic, cardiovascular, and neurological health. Low-capacity runner (LCR) rats display low aerobic capacity, metabolic dysfuction, and spatial memory deficits. A heat treatment (HT) can improve metabolic dysfunction in LCR peripheral organs after high fat diet (HFD). Little is known about metabolic changes in the brains of these rats following HT. OBJECTIVE: Our objective was to examine the extent to which high or low aerobic capacity impacts Akt (a protein marker of metabolism) and heat shock protein 72 (HSP72, a marker of heat shock response) after HFD and HT in hippocampus. METHODS: We measured phosphorylated Akt (pAkt) in the striatum and hippocampus, and HSP72 in the hippocampus, of HFD-fed and chow-fed LCR and high-capacity runner (HCR) rats with and without HT. RESULTS: pAkt was lower in the hippocampus of chow-fed LCR than HCR rats. HFD resulted in greater pAkt in LCR but not HCR rats, but HT resulted in lower pAkt in the LCR HFD group. HSP72 was greater in both HCR and LCR rat hippocampus after HT. The HFD blunted this effect in LCR compared to HCR hippocampus. CONCLUSION: The abnormal phosphorylation of Akt and diminished HSP response in the hippocampus of young adult LCR rats might indicate early vulnerability to metabolic challenges in this key brain region associated with learning and memory.

RNA Sequencing of Human Peripheral Nerve in Response to Injury: Distinctive Analysis of the Nerve Repair Pathways.

The development of regenerative therapies for central nervous system diseases can likely benefit from an understanding of the peripheral nervous system repair process, particularly in identifying potential gene pathways involved in human nerve repair. This study employed RNA sequencing (RNA-seq) technology to analyze the whole transcriptome profile of the human peripheral nerve in response to an injury. The distal sural nerve was exposed, completely transected, and a 1 to 2 cm section of nerve fascicles was collected for RNA-seq from six participants with Parkinson's disease, ranging in age between 53 and 70 yr. Two weeks after the initial injury, another section of the nerve fascicles of the distal and pre-degenerated stump of the nerve was dissected and processed for RNA-seq studies. An initial analysis between the pre-lesion status and the postinjury gene expression revealed 3,641 genes that were significantly differentially expressed. In addition, the results support a clear transdifferentiation process that occurred by the end of the 2-wk postinjury. Gene ontology (GO) and hierarchical clustering were used to identify the major signaling pathways affected by the injury. In contrast to previous nonclinical studies, important changes were observed in molecular pathways related to antiapoptotic signaling, neurotrophic factor processes, cell motility, and immune cell chemotactic signaling. The results of our current study provide new insights regarding the essential interactions of different molecular pathways that drive neuronal repair and axonal regeneration in humans.

Impaired Cu-Zn Superoxide Dismutase (SOD1) and Calcineurin (Cn) Interaction in ALS: A Presumed Consequence for TDP-43 and Zinc Aggregation in Tg SOD1G93A Rodent Spinal Cord Tissue.

Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)- enriched phosphorylated TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1G93A protein does not properly bind zinc (Zn) in animal models; hence, mutant SOD1G93A-Cn interaction weakens. Consequently, unstable Cn fails to dephosphorylate TDP-43 that yields hyperphosphorylated TDP-43 aggregates. Our previous studies had suggested that Cn and SOD1 interaction was necessary to keep Cn enzyme functional. We have observed low Cn level, increased Zn concentrations, and increased TDP-43 protein levels in cervical, thoracic, lumbar, and sacral regions of the spinal cord tissue homogenates. This study further supports our previously published work indicating that Cn stability depends on functional Cn-SOD1 interaction because Zn is crucial for maintaining the Cn stability. Less active Cn did not efficiently dephosphorylate TDP-43; hence TDP-43 aggregations appeared in the spinal cord tissue.

Short Term Development and Fate of MGE-Like Neural Progenitor Cells in Jaundiced and Non-Jaundiced Rat Brain.

Neonatal hyperbilirubinemia targets specific brain regions and can lead to kernicterus. One of the most debilitating symptoms of kernicterus is dystonia, which results from bilirubin toxicity to the globus pallidus (GP). Stem cell transplantation into the GP to replace lost neurons and restore basal ganglia circuits function is a potential therapeutic strategy to treat dystonia in kernicterus. In this study we transplanted human medial ganglionic eminence (MGE)-like neural progenitor cells (NPCs) that we differentiated into a primarily gamma-aminobutyric acid (GABA)ergic phenotype, into the GP of non-immunosuppressed jaundiced (jj) and non-jaundiced (Nj) rats. We assessed the survival and development of graft cells at three time-points post-transplantation. While grafted MGE-like NPCs survived and generated abundant fibers in both jj and Nj brains, NPC survival was greater in the jj brain. These results were consistent with our previous finding that excitatory spinal interneuron-like NPCs exhibited a higher survival rate in the jj brain than in the Nj brain. Our findings further support our hypothesis that slightly elevated bilirubin levels in the jj brain served as an antioxidant and immunosuppressant to protect the transplanted cells. We also identified graft fibers growing toward brain regions that receive projections from the GP, as well as host fibers extending toward the graft. These promising findings suggest that MGE-like NPCs may have the capacity to restore the circuits connecting GP and other nuclei.

Region-specific differences in bioenergetic proteins and protein response to acute high fat diet in brains of low and high capacity runner rats.

Aerobic capacity is a strong predictor of mortality. Low capacity runner (LCR) rats exhibit reduced mitochondrial function in peripheral organs. A high fat diet (HFD) can worsen metabolic phenotype in LCR rats. Little is known about metabolic changes in the brains of these rats, however. This study examined protein markers of mitochondrial function and metabolism as a function of aerobic running capacity and an acute HFD in four brain regions: the striatum, hippocampus, hypothalamus, and substantia nigra. After 3 days HFD or chow diets, we measured peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1-α), nuclear respiratory factors 1 (Nrf-1), mitochondrial transcription factor A (TFAM), and phosphorylated (activated) AMP-activated protein kinase (p-AMPK) protein levels in the four brain regions. LCR rats exhibited lower levels of mitochondrial proteins (PGC1-α, Nrf-1, TFAM), and greater p-AMPK, in striatum, but not in the other brain regions. Mitochondrial protein levels were greater in HFD LCR striatum, while p-AMPK was lower in this group. Markers of lower mitochondrial biogenesis and increased metabolic demand were limited to the LCR striatum, which nevertheless maintained the capacity to respond to an acute HFD challenge.

The K-INBRE symposium: a 10-institution collaboration to improve undergraduate education.

The Kansas-IDeA Network of Biomedical Research Excellence (K-INBRE) is an infrastructure-building program funded by the National Institute of General Medical Sciences. Undergraduate education, through undergraduate research, is a key component of the program. The K-INBRE network includes 10 higher education institutions in Kansas and northern Oklahoma, with over 1,000 student participants in 16 yr. Since 2003, the K-INBRE has held an annual state-wide research symposium that includes national and regional speakers and provides a forum for undergraduates to give platform and poster presentations. The symposium is well attended by K-INBRE participants and has grown to a size of over 300 participants per year from all 10 K-INBRE schools. Two surveys were distributed to students and mentors to assess the impact of the symposium on student learning. Surveys (153) were distributed to students who participated in K-INBRE from 2013 through 2015 with a 51% response rate. Mentors were surveyed with a response of 111 surveys out of 161. Survey results indicate that students and mentors alike find the symposium to be beneficial and enriching of the student experience. Almost 80% of student respondents indicated that their participation in the symposium fostered appreciation of research. In short, the K-INBRE symposium provides a unique opportunity for students to gain experience in collecting, preparing, and communicating research in a professional environment. The collaborative experience of the annual K-INBRE symposium, the impact it has on student learning, and how it has influenced the research culture at our 10 institutions will be described.

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Fate of Neural Progenitor Cells Transplanted Into Jaundiced and Nonjaundiced Rat Brains.

High levels of bilirubin in infants can cause kernicterus, which includes basal ganglia damage and dystonia. Stem cell transplantation may be an effective treatment for this disease. In this study, we transplanted human neural progenitor cells differentiated toward propriospinal interneurons into the striatum of 20-day-old spontaneously jaundiced (jj) Gunn rats and nonjaundiced (Nj) littermates. Using immunohistochemical methods, we found that grafted cells survived and grew fibers in jj and Nj brains 3 weeks after transplantation. Grafted cells had a higher survival rate in jj than in Nj brains, suggesting that slightly elevated bilirubin may protect graft survival due to its antioxidative and immunosuppressive effects. Despite their survival, only a small portion of grafted neurons expressed GAD-6 or ChAT, which mark GABAergic and cholinergic neurons, respectively, and are the cells that we are attempting to replace in kernicterus. Thus, NPCs containing large populations of GABAergic and cholinergic neurons should be used for further study in this field.

Effects of Tongue Force Training on Bulbar Motor Function in the Female SOD1-G93A Rat Model of Amyotrophic Lateral Sclerosis.

BACKGROUND: The use of exercise in amyotrophic lateral sclerosis (ALS) is controversial. Although moderate exercise appears to be beneficial for limb muscles in ALS, the effects of exercise on bulbar muscles such as the tongue have not been studied. OBJECTIVE: To determine the effects of tongue force training on bulbar motor function in the SOD1-G93A rat model of ALS. METHODS: We compared the effects of tongue force training on bulbar motor function and neuromuscular junction innervation in female SOD1-G93A rats and age-matched female wild-type controls. Half of each group underwent afternoon tongue force training sessions, and all rats were tested under minimal force conditions in the mornings. RESULTS: Tongue force did not differ between the SOD1-G93A rats and healthy controls during the morning testing sessions, nor was it affected by training. Surprisingly, decreases in tongue motility, the number of licks per session, and body weight were greater in the tongue force-trained SOD1-G93A rats. Forelimb grip force, survival, and denervation of the genioglossus (GG) muscle did not differ between the trained and untrained SOD1-G93A rats. GG innervation was correlated with changes in tongue force but not tongue motility in SOD1-G93A rats at end stage. CONCLUSIONS: The results indicate a potential deleterious effect of tongue force training on tongue motility in female SOD1-G93A rats. The lack of a relationship between GG innervation and tongue motility suggests that factors other than lower-motor neuron integrity likely accounted for this effect.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aß1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

A high fat diet alters metabolic and bioenergetic function in the brain: A magnetic resonance spectroscopy study.

Diet-induced obesity and associated metabolic effects can lead to neurological dysfunction and increase the risk of developing Alzheimer's disease (AD) and Parkinson's disease (PD). Despite these risks, the effects of a high-fat diet on the central nervous system are not well understood. To better understand the mechanisms underlying the effects of high fat consumption on brain regions affected by AD and PD, we used proton magnetic resonance spectroscopy ((1)H-MRS) to measure neurochemicals in the hippocampus and striatum of rats fed a high fat diet vs. normal low fat chow. We detected lower concentrations of total creatine (tCr) and a lower glutamate-to-glutamine ratio in the hippocampus of high fat rats. Additional effects observed in the hippocampus of high fat rats included higher N-acetylaspartylglutamic acid (NAAG), and lower myo-inositol (mIns) and serine (Ser) concentrations. Post-mortem tissue analyses revealed lower phosphorylated AMP-activated protein kinase (pAMPK) in the striatum but not in the hippocampus of high fat rats. Hippocampal pAMPK levels correlated significantly with tCr, aspartate (Asp), phosphoethanolamine (PE), and taurine (Tau), indicating beneficial effects of AMPK activation on brain metabolic and energetic function, membrane turnover, and edema. A negative correlation between pAMPK and glucose (Glc) indicates a detrimental effect of brain Glc on cellular energy response. Overall, these changes indicate alterations in neurotransmission and in metabolic and bioenergetic function in the hippocampus and in the striatum of rats fed a high fat diet.

Effects of discontinuing a high-fat diet on mitochondrial proteins and 6-hydroxydopamine-induced dopamine depletion in rats.

Diet-induced obesity can increase the risk for developing age-related neurodegenerative diseases including Parkinson's disease (PD). Increasing evidence suggests that mitochondrial and proteasomal mechanisms are involved in both insulin resistance and PD. The goal of this study was to determine whether diet intervention could influence mitochondrial or proteasomal protein expression and vulnerability to 6-Hydroxydopamine (6-OHDA)-induced nigrostriatal dopamine (DA) depletion in rats' nigrostriatal system. After a 3 month high-fat diet regimen, we switched one group of rats to a low-fat diet for 3 months (HF-LF group), while the other half continued with the high-fat diet (HF group). A chow group was included as a control. Three weeks after unilateral 6-OHDA lesions, HF rats had higher fasting insulin levels and higher Homeostasis model assessment of insulin resistance (HOMA-IR), indicating insulin resistance. HOMA-IR was significantly lower in HF-LF rats than HF rats, indicating that insulin resistance was reversed by switching to a low-fat diet. Compared to the Chow group, the HF group exhibited significantly greater DA depletion in the substantia nigra but not in the striatum. DA depletion did not differ between the HF-LF and HF group. Proteins related to mitochondrial function (such as AMPK, PGC-1α), and to proteasomal function (such as TCF11/Nrf1) were influenced by diet intervention, or by 6-OHDA lesion. Our findings suggest that switching to a low-fat diet reverses the effects of a high-fat diet on systemic insulin resistance, and mitochondrial and proteasomal function in the striatum. Conversely, they suggest that the effects of the high-fat diet on nigrostriatal vulnerability to 6-OHDA-induced DA depletion persist.

Hyperactivity in the Gunn rat model of neonatal jaundice: age-related attenuation and emergence of gait deficits.

BACKGROUND: Neonatal jaundice resulting from elevated unconjugated bilirubin occurs in 60-80% of newborn infants. Although mild jaundice is generally considered harmless, little is known about its long-term consequences. Recent studies have linked mild bilirubin-induced neurological dysfunction (BIND) with a range of neurological syndromes, including attention-deficit hyperactivity disorder. The goal of this study was to measure BIND across the lifespan in the Gunn rat model of BIND. METHODS: Using a sensitive force plate actometer, we measured locomotor activity and gait in jaundiced (jj) Gunn rats versus their nonjaundiced (Nj) littermates. Data were analyzed for young adult (3-4 mo), early middle-aged (9-10 mo), and late middle-aged (17-20 mo) male rats. RESULTS: jj rats exhibited lower body weights at all ages and a hyperactivity that resolved at 17-20 mo of age. Increased propulsive force and gait velocity accompanied hyperactivity during locomotor bouts at 9-10 mo in jj rats. Stride length did not differ between the two groups at this age. Hyperactivity normalized, and gait deficits, including decreased stride length, propulsive force, and gait velocity, emerged in the 17-20-mo-old jj rats. CONCLUSION: These results demonstrate that, in aging, hyperactivity decreases with the onset of gait deficits in the Gunn rat model of BIND.

Differential effects of amphetamine and GBR-12909 on orolingual motor function in young vs aged F344/BN rats.

RATIONALE: Orolingual motor deficits, such as dysarthria and dysphagia, contribute to increased morbidity and mortality in the elderly. In preclinical studies, we and others have reported age-related decreases in tongue motility in both F344 and F344/BN rats. The fact that these deficits are associated with nigrostriatal dopamine (DA), tissue measures suggest that increasing dopamine function might normalize tongue motility. OBJECTIVE: The purpose of the current study was to determine whether two indirect dopamine agonists with locomotor-enhancing effects, d-amphetamine (amphetamine; 1 and 2 mg/kg) and GBR-12909 (5, 10, and 20 mg/kg), can improve tongue motility in aged F344/BN rats. METHODS: Young (6 months) and aged (30 months) F344/BN rats licked water from an isometric force disc so that tongue motility (licks/second) and tongue force could be measured as a function of age and drug dose. RESULTS: Consistent with our previous studies, tongue force was greater and tongue motility was lower in the aged group. Tongue motility was increased by amphetamine but not by GBR-12909. Amphetamine decreased peak tongue force, primarily in the young group. GBR-12909 did not affect tongue force. GBR-12909 increased the number of licks/session in the young group but not in the aged group, while amphetamine increased this measure in both groups. CONCLUSION: These results demonstrate differential effects of these drugs on orolingual motor function and suggest that blocking DA uptake is insufficient to increase tongue motility in aging.

Differential effects of intrastriatal 6-hydroxydopamine on cell number and morphology in midbrain dopaminergic subregions of the rat.

The midbrain dopaminergic perikarya are differentially affected in Parkinson׳s disease (PD). This study compared the effects of a partial unilateral intrastriatal 6-hydroxydopamine (6-OHDA) lesion model of PD on the number, morphology, and nucleolar volume of dopaminergic cells in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), and retrorubral field (RRF). Adult, male rats (n=10) underwent unilateral intrastriatal infusion of 6-OHDA (12.5µg). Lesions were verified by amphetamine-stimulated rotation 7 days post-infusion. Rats were euthanized 14 days after treatment with 6-OHDA and brains were stained with a tyrosine hydroxylase-silver nucleolar (TH-AgNOR) stain. Dopaminergic cell number and morphology in the lesioned and intact hemispheres were quantified using stereological methods. The magnitude of decrease in planimetric volume, neuronal number, cell density, and neuronal volume resulting from 6-OHDA lesion differed between regions, with the SNpc exhibiting the greatest loss of neurons (46%), but the smallest decrease in neuronal volume (13%). The lesion also resulted in a decrease in nucleolar volume that was similar in all three regions (22-26%). These findings indicate that intrastriatal 6-OHDA lesion differentially affects dopaminergic neurons in the SNpc, VTA, and RRF; however, the resulting changes in nucleolar morphology suggest a similar cellular response to the toxin in all three cell populations.