The boundlessness of behavioral neuroscience: A look across 30 years.

It has been more than thirty years since the two inaugural IBNS presidents sat down at a larger neuroscience conference and decided that there should be more to behavioral neuroscience than a single theme at a meeting. The progeny of these conversations is the International Behavioral Neuroscience Society (IBNS) and this year will be its thirty year anniversary. We reflect back on the last thirty years of the research career of the society's second president, Paul R. Sanberg, as an example of how behavioral neuroscience research has changed these last few decades.

Semen quality and infertility status can be identified through measures of oxidation-reduction potential.

Standard analyses for evaluating semen quality require technical expertise and are interpretive in nature. Oxidative stress (OS) alters many of the semen parameters; thus, a measure of OS could be an indicator of semen quality. Static oxidation-reduction potential (sORP) is a universal measure of OS traditionally used in environmental applications but is increasingly used in biomedical studies. sORP was measured to determine how well it associates with semen quality and if it differentiates semen from infertile patients and fertile donors. All study participants (Infertile, n = 365 and Fertile, n = 50) underwent standard semen analyses, and sORP was measured in unprocessed semen. In infertile patients, sORP increased with decreased total sperm number, motility and morphology. sORP values were higher in samples with abnormal quality (low number, motility and/or normal morphology) compared with those of normal quality. Infertile patients had higher sOPR values compared to fertile donors. A sORP cut-off value of 1.38 mV/106  sperm/ml can differentiate normal from abnormal semen samples, while a cut-off value of 1.41 mV/106 sperm/ml, can differentiate between infertile and fertile semen samples. In conclusion, sORP provides a quick and unbiased indicator of semen quality that can be a beneficial addition to semen analysis to determine semen quality and fertility status.

Neural stem cells reduce hippocampal tau and reelin accumulation in aged Ts65Dn Down syndrome mice.

Tau accumulation, in the form of neurofibrillary tangles (NFT), is an early neuropathological characteristic of Alzheimer's disease (AD) and early onset AD frequently seen in Down syndrome (DS). We investigated the presence of tau accumulation in the brains of aging DS mice using the Ts65Dn mouse model. All aged mice appeared to have substantial clusters of extracellular granules that were positive for tau and reelin, but not for amyloid-ß or APP. These clusters were found primarily in CA1 of the hippocampus. In addition, the aged trisomic DS mice had a significantly greater accumulation of extracellular tau/reelin granular deposits compared to disomic littermates. These granules were similar to those described by others who also found extracellular proteinous granules in the brains of non-DS mice engineered to model aging and/or AD. When neural stem cells (NSC) were implanted unilaterally into the hippocampus of the Ts65Dn mice, the tau/reelin-positive granules were significantly reduced in both trisomic and disomic mice. Our findings indicate that changes in tau/reelin-positive granules could be used as an index for neuropathological assessment in aging DS and AD. Furthermore, changes in granule density could be used to test the efficacy of novel treatments, such as NSC implantation. Lastly, it is speculated that the unique abilities of NSC to migrate and express growth factors might be a contributing factor to reducing tau/reelin accumulation in aging DS and AD.

Biocompatibility of poly(ethylene glycol)-based hydrogels in the brain: an analysis of the glial response across space and time.

Poly(ethylene glycol) or PEG-based hydrogels provide a useful methodology for tissue engineering and the controlled-release of drugs within the central nervous system (CNS). To be successful, the local neuroinflammatory response to an implant must be well understood. Toward this end, the focus was to examine the localized recruitment and activation of microglia and astrocytes following implantation of PEG-based hydrogels in the brain. Because they are of clinical relevance and may impact brain tissue differently, hydrogels with different mass loss profiles were examined. At all time points, a needle penetration in sham animals evoked a greater astrocytic response than hydrogel conditions. The astrocyte response that ensued varied with degradation rate. An attenuated response was present in more slowly degrading and nondegrading conditions. Relative to sham, hydrogel conditions attenuated the acute microglial response during the week after implant. By 56 days, microglial levels in shams decreased below the observed response in slowly degrading and nondegradable gels, which remained constant overtime. Although the inflammatory response to PEG-based hydrogels was complex depending on degradation rates, the magnitude of the acute microglia response and the long-term astrocyte response were attenuated suggesting the use of these materials for drug and cell delivery to the CNS.

Embryonic substantia nigra grafts in the mesencephalon send neurites to the host striatum in non-human primate after overexpression of GDNF.

In spite of partial success in treating Parkinson's disease by using ectopically placed grafts of dopamine-producing cells, restoration of the original neuroanatomical circuits, if possible, might work better. Previous evidence of normal anatomic projections from ventral mesencephalic (VM) grafts placed in the substantia nigra (SN) has been limited to neonatal rodents and double grafting or bridging procedures. This study attempted to determine whether injection of a potent growth-promoting factor, glial cell line-derived neurotrophic factor (GDNF), into the target regions or placement of fetal striatal co-grafts in the nigrostriatal pathway might elicit neuritic outgrowth to the caudate nucleus. Four adult St. Kitts green monkeys received embryonic VM grafts into the rostral mesencephalon near the host SN, and injections of adeno-associated virus 2 (AAV2)/GDNF or equine infectious anemia virus (EIAV)/GDNF into the caudate. Three adult monkeys were co-grafted with fetal VM tissue near the SN and fetal striatal grafts (STR) 2.5 mm rostral in the nigrostriatal pathway. Before sacrifice, the striatal target regions were injected with the retrograde tracer Fluoro-Gold (FG). FG label was found in tyrosine hydroxylase-labeled neurons in VM grafts in the SN of only those monkeys that received AAV2/GDNF vector injections into the ipsilateral striatum. All monkeys showed FG labeling in the host SN when FG labeling was injected on the same side. These data show that grafted dopaminergic neurons can extend neurites to a distant target releasing an elevated concentration of GDNF, and suggest that grafted neurons can be placed into appropriate loci for potential tract reconstruction.

Development of porous PEG hydrogels that enable efficient, uniform cell-seeding and permit early neural process extension.

Three-dimensional polymer scaffolds are useful culture systems for neural cell growth and can provide permissive substrates that support neural processes as they extend across lesions in the brain and spinal cord. Degradable poly(ethylene) glycol (PEG) gels have been identified as a particularly promising scaffold material for this purpose; however, process extension within PEG gels is limited to late stages of hydrogel degradation. Here we demonstrate that earlier process extension can be achieved from primary neural cells encapsulated within PEG gels by creating a network of interconnected pores throughout the gel. Our method of incorporating these pores involves co-encapsulating a cell solution and a fibrin network within a PEG gel. The fibrin is subsequently enzymatically degraded under cytocompatible conditions, leaving behind a network of interconnected pores within the PEG gel. The primary neural cell population encapsulated in the gel is of mixed composition, containing differentiated neurons, and multipotent neuronal and glial precursor cells. We demonstrate that the initial presence of fibrin does not influence the cell-fate decisions of the encapsulated precursor cells. We also demonstrate that this fabrication approach enables simple, efficient and uniform seeding of viable cells throughout the entire porous scaffold.

Biocompatibility of PEG-based hydrogels in primate brain.

Degradable polymers have been used successfully in a wide variety of peripheral applications from tissue regeneration to drug delivery. These polymers induce little inflammatory response and appear to be well accepted by the host environment. Their use in the brain, for neural tissue reconstruction or drug delivery, also could be advantageous in treating neurodegenerative disorders. Because the brain has a unique immune response, a polymer that is compatible in the body may not be so in the brain. In the present study, polyethylene glycol (PEG)-based hydrogels were implanted into the striatum and cerebral cortex of nonhuman primates. Four months after implantation, brains were processed to evaluate the extent of astrogliosis and scaring, the presence of microglia/macrophages, and the extent of T-cell infiltration. Hydrogels with 20% w/v PEG implanted into the brain stimulated a slight increase in astrocytic and microglial/macrophage presence, as indicated by a small increase in glial fibrillary acidic protein (GFAP) and CD68 staining intensity. This increase was not substantially different from that found in the sham-implanted hemispheres of the brain. Staining for CD3+ T cells indicated no presence of peripheral T-cell infiltration. No gliotic scarring was seen in any implanted hemisphere. The combination of low density of GFAP-positive cells and CD68-positive cells, the absence of T cells, and the lack of gliotic scarring suggest that this level of immune response is not indicative of immunorejection and that the PEG-based hydrogel has potential to be used in the primate brain for local drug delivery or neural tissue regeneration.

Embryonic substantia nigra grafts show directional outgrowth to cografted striatal grafts and potential for pathway reconstruction in nonhuman primate.

Transplantation of embryonic dopamine (DA) neurons has been tested as a therapy for Parkinson's disease. Most studies placed DA neurons into the striatum instead of the substantia nigra (SN). Reconstruction of this DA pathway could serve to establish a more favorable environment for control of DA release by grafted neurons. To test this we used cografts of striatum to stimulate growth of DA axons from embryonic SN that was implanted adjacent to the host SN in African green monkeys. Embryonic striatum was implanted at one of three progressive distances rostral to the SN. Immunohistochemical analysis revealed DA neuron survival and neuritic outgrowth from the SN grafts at 12-36 weeks after grafting. Each animal showed survival of substantial numbers of DA neurons. Most fibers that exited SN grafts coursed rostrally. Striatal grafts showed evidence of target-directed outgrowth and contained dense patterns of DA axons that could be traced from their origin in the SN grafts. A polarity existed for DA neurites that exited the grafts; that is, those seen caudal to the grafts did not appear to be organized into a directional outflow while those on the rostral side were arranged in linear profiles coursing toward the striatal grafts. Some TH fibers that reached the striatal grafts appeared to arise from the residual DA neurons of the SN. These findings suggest that grafted DA neurons can extend neurites toward a desired target over several millimeters through the brain stem and caudal diencephalon of the monkey brain, which favors the prospect of circuit reconstruction from grafted neurons placed into appropriate locations in their neural circuitry. Further study will assess the degree to which this approach can be used to restore motor balance in the nonhuman primate following neural transplantation.

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons.

Neural transplantation offers the potential of treating Parkinson's disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson's disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced overexpression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson's disease.

Infant mice with glutaric acidaemia type I have increased vulnerability to 3-nitropropionic acid toxicity.

Glutaric acidaemia type I (GA I) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH) and is characterized clinically by striatal degeneration that almost always occurs in early childhood. A murine knockout model of GA I has the organic aciduria seen in the human disorder, but this model does not develop striatal degeneration spontaneously. 3-Nitropropionic acid (3NP), a succinic dehydrogenase inhibitor with specificity for the striatum, was investigated as a potential initiator of striatal degeneration in GCDH-deficient mice. This study shows that GCDH-deficient mouse pups are more susceptible to 3NP than their wild-type littermates, and that all mouse pups are more sensitive to 3NP as infants than as adolescents and adults. Increased sensitivity to 3NP early in life may model the developmental window for the striatal damage observed in human GA I.

IGF-1 and bFGF reduce glutaric acid and 3-hydroxyglutaric acid toxicity in striatal cultures.

Glutaric acid (GA) and 3-hydroxyglutaric acid (3GA) are thought to contribute to the degeneration of the caudate and putamen that is seen in some children with glutaric acidaemia type I, a metabolic disorder caused by a glutaryl-CoA dehydrogenase deficiency. This study assessed the neurotoxicity of GA and 3GA (0-50 mmol/L) compared to quinolinic acid (QUIN) in striatal and cortical cultures. All three acids were neurotoxic in a dose-dependent manner; however, GA and 3GA were both more toxic than QUIN. The neurotoxic effects of low concentrations of GA or 3GA were additive to QUIN toxicity. A series of hormones and growth factors were tested for protection against GA and 3GA toxicity. Insulin (5-500 microU /ml), basic fibroblast growth factor (bFGF; 10 ng/ml), insulin-like growth factor (IGF-1; 50 ng/ml), brain-derived neurotrophic factor (BDNF; 10 ng/ml), glial-derived neurotrophic factor (GDNF; 10 ng/ml), and two glutamate antagonists were evaluated in brain cultures to which 7 mmol/L GA or 3GA were added. GA and 3GA neurotoxicities were prevented by bFGF. Attenuation of 3GA-induced neurotoxicity was seen with insulin (5 microU/ml) and IGF-1. BDNF and GDNF had no effects on neuronal survival. Glutamate antagonists MK801 (10 micromol/L) and NBQX (10 micromol/L) failed to prevent GA or 3GA neurotoxicity. We conclude that GA and 3GA are neurotoxic in cultures of embryonic rat striatum and cortex. Striatal neurons were rescued from death by bFGF and IGF-1 but not by glutamate antagonist, suggesting that toxicity in this embryonic system is not necessarily mediated by glutamate receptors.

Age at symptom onset predicts severity of motor impairment and clinical outcome of glutaric acidemia type 1.

OBJECTIVES: In patients with glutaric acidemia type 1 (GAI), biochemical and molecular markers fail to predict the course of individual patients; therefore we sought to identify nonbiochemical variables that correlate with severity of motor deficits or overall clinical outcome. STUDY DESIGN: Archival data was collected from 42 published articles describing 115 patients with GA1. A forward, stepwise, multiple regression analysis was used to find predictors for outcome. RESULTS: Analyses show that in patients who did not have a precipitating illness before the first appearance of motor symptoms, the age at onset was significantly associated with the severity of motor impairments and overall clinical outcome. In patients who had a precipitating illness, the age at onset did not predict the outcome. In both groups of patients, basal ganglia degeneration, enlargement of spaces containing cerebrospinal fluid, and white matter abnormalities were indicative of a poorer prognosis. Treatment given after the appearance of symptoms was not associated with a better clinical outcome or fewer motor deficits. CONCLUSION: Because the age at symptom onset can significantly predict the severity of motor deficits and the overall outcome, it is important to identify patients with GA1 as early as possible. Several studies suggest that presymptomatic treatment may prevent or postpone the onset of symptoms.

CPI-1189 prevents apoptosis and reduces glial fibrillary acidic protein immunostaining in a TNF-alpha infusion model for AIDS dementia complex.

AIDS dementia complex (ADC) is characterized by increased apoptosis, gliosis, and oxidative stress in the CNS, as well as a compromised blood-brain barrier. TNF-alpha has been shown to be elevated in AIDS dementia complex brains and may contribute to AIDS dementia complex. To model elevated TNF-alpha in AIDS dementia complex, TNF-alpha was infused ICV bilaterally into rats for 3 days. TNF-alpha treatment increased apoptosis around the infusion site and selectively in the septum and corpus callosum. Co-administration of the synthetic antioxidant CPI-1189 prevented TNF-alpha induced apoptosis. Both TNF-alpha and CPI-1189 treatment suppressed glial fibrillary acidic protein (GFAP) staining at the infusion site. TNF-alpha did not significantly affect the integrity of the blood-brain barrier, but CPI-1189 treatment increased blood-brain barrier integrity at the infusion site. No effect of TNF-alpha or CPI-1189 treatment was found on measures of oxidative stress. These results support TNF-alpha as a key agent for increasing apoptosis in AIDS dementia complex. Additionally, CPI-1189 treatment may protect against TNF-alpha induced apoptosis and astrogliosis in AIDS dementia complex. Lastly, the toxic effect of TNF-alpha and the protective effect of CPI-1189 may not be mediated primarily through manipulation of classic reactive oxygen species.

Intravascular beta-amyloid infusion increases blood pressure: implications for a vasoactive role of beta-amyloid in the pathogenesis of Alzheimer's disease.

Hypertension has been recognized as a risk factor for Alzheimer's disease (AD). Moreover, serum beta-amyloid (A beta) levels are elevated in several mutations linked to familial AD, as well as in some sporadic AD individuals. To determine the in vivo effects of A beta on blood pressure, A beta(1-40) was infused intra-arterially into anesthetized rats. For all animals, strong correlations exist between pre-infusion mean arterial blood pressure (MA beta) and post-arterial infusion increases in blood pressure. In spontaneously hypotensive animals, A beta infusion resulted in substantial increases in MA beta compared to vehicle distilled water infusion. A beta(1-40) was also able to accelerate MA beta return from induced hypotension, but infusion of A beta(1-42), or rat amylin had no such effect. These results provide evidence that circulating A beta(1-40) can exert vasopressor actions in vivo. Moreover, they suggest a pathophysiologic role for vascular A beta in AD that precedes A beta deposition and dementia onset.

In vitro and in vivo studies investigating possible antioxidant actions of nicotine: relevance to Parkinson's and Alzheimer's diseases.

An inverse relationship appears to exist between cigarette smoking and the risk of Parkinson's and Alzheimer's diseases. Since both diseases are characterized by enhanced oxidative stress, we investigated the antioxidant potential of nicotine, a primary component of cigarette smoke. Initial chromatographic studies suggest that nicotine can affect the formation of the neurotoxin 6-hydroxydopamine resulting from the addition of dopamine to Fenton's reagent (i.e., Fe2+ and H2O2). Thus, under certain circumstances, nicotine can strongly affect the course of the Fenton reaction. In in vivo studies, adult male rats being treated with nicotine showed greater memory retention than controls in a water maze task. However, neurochemical analysis of neocortex, hippocampus, and neostriatum from these same animals revealed that nicotine treatment had no effect on the formation of reactive oxygen species or on lipid peroxidation for any brain region studied. In an in vitro study, addition of various concentrations of nicotine to rat neocortical homogenates had no effect on lipid peroxidation compared to saline controls. The results of these studies suggest that the beneficial/protective effects of nicotine in both Parkinson's disease and Alzheimer's disease may be, at least partly, due to antioxidant mechanisms.

Intravascular infusions of soluble beta-amyloid compromise the blood-brain barrier, activate CNS glial cells and induce peripheral hemorrhage.

Vascular wall levels of soluble beta-amyloid1-40 (Abeta1-40) are elevated in Alzheimer's disease (AD). Moreover, plasma Abeta levels are increased in familial AD, as well as in some cases of sporadic AD. To determine the histopathologic and behavioral consequences of elevated vascular Abeta levels, Abeta1-40 (50 micrograms in distilled water) or vehicle was intravenously infused twice daily into 3-month old male Sprague-Dawley rats for 2 weeks. Intravenous Abeta infusions impaired blood-brain barrier integrity, as indicated by substantial perivascular and parenchyma IgG immunostaining within the brain. Also evident in Abeta-infused animals was an increase in GFAP immunostaining around cerebral blood vessels, and an enhancement of OX-42 microglial immunostaining in brain white matter. Gross pulmonary hemorrhage was noted in most Abeta-infused animals. All the observed changes occurred in the absence of Congo red birefringence. No significant cognitive deficits were present in Abeta-infused animals during water maze acquisition and retention testing, which was conducted during the second week of treatment. These results indicate that circulating Abeta can: (1) induce vessel dysfunction/damage in both the brain and the periphery without complex Abeta fibril formation/deposition, and (2) induce an activation of brain astrocytes and microglia. Taken together, our results suggest that if circulating Abeta is elevated in AD, it is likely to have a pathophysiologic role.

Evidence that oxidative stress is associated with the pathophysiology of inherited hydrocephalus in the H-Tx rat model.

Oxidative stress can contribute to many neurological disease processes. Because many events known to involve oxidative stress (infection, hemorrhage, brain trauma) are accompanied by hydrocephalus, the present study sought to evaluate the relationship between oxidative stress and the progression of hydrocephalus. Assays for reactive oxygen species (ROS), using dichlorofluorescein (DCF) fluorescence, and lipid peroxidation, using malondialdehyde (MDA), were performed on brain tissue from the cerebral cortex, cerebellum, basal ganglia, and hippocampus of 4-, 10-, and 25-day-old normal and hydrocephalic H-Tx rats. These rats inherit hydrocephalus at a rate of 30-50% and represent a unique model for studying the progression of hydrocephalus. When hydrocephalic and normal H-Tx rats were compared, ROS levels were significantly higher in the cerebral cortex of 4-day-old and in the cerebellum and hippocampus of 4- and 10-day-old hydrocephalic rats. ROS levels also were significantly higher in the basal ganglia of 25-day-old hydrocephalic rats. MDA levels were significantly higher in the hippocampus and basal ganglia of 25-day-old hydrocephalic rats. There were no significant differences in MDA levels at younger ages. These results indicate that, in H-Tx rats, oxidative stress is associated with the progression and molecular pathophysiology of hydrocephalus. This association suggests that oxidative brain damage may represent an important factor resulting from or contributing to the pathogenesis of hydrocephalus.

Preventive actions of a synthetic antioxidant in a novel animal model of AIDS dementia.

Accumulating evidence indicates that the mechanism for causing AIDS dementia complex (ADC) involves the release of damaging inflammatory-related agents by HIV-infected microglia in the brain resulting in CNS oxidative damage. One such agent, tumor necrosis factor alpha (TNF-alpha) is consistently elevated in the brains of ADC patients compared to non-demented HIV patients. To model this aspect of ADC in rats, chronic ventricular infusions of TNF-alpha were given and found to induce several aspects of ADC, including weight loss, learning/memory impairment, enlarged lateral ventricles, and increased apoptosis. Concurrent oral treatment with the antioxidant CPI-1189 prevented all of these TNF-alpha induced effects. The results support TNF-alpha as a key toxic agent in ADC and provide the first in vivo evidence that chronic treatment with a synthetic antioxidant may protect HIV-infected patients against ADC. Our findings may also have implications in other neurological diseases where brain TNF-alpha levels are elevated and inflammation/oxidative stress is suspected to be a contributing cause, such as Alzheimer's disease and Parkinson's disease.

Age-dependent neurobehavioral responses by young and mature adult rats to systemic kainic acid.

Neurobehavioral effects caused by the excitotoxin kainic acid (KA) have been characterized by convulsions including 'wet dog shakes' (WDS) with accompanying hippocampal degeneration in experimental animals. Accordingly, this model has been proposed for putative excitotoxin-mediated disorders, such as the temporal lobe epilepsy. There have been reports on age-dependent neurobehavioral effects of KA; however, little is known about possible correlations between neuropathology and behavioral responses to KA. The present study demonstrates that mature adult rats (12 months old) injected subcutaneously (s.c.) with KA (12 mg/kg) had severer damage to the hippocampal formation, i.e. CA3 region, compared with KA-treated young adult rats (2 months old). The mature adult animals also exhibited an earlier onset of WDS, a significantly higher number of WDS (P > 0.01), and severer convulsions compared with young adult rats. These findings indicate a positive correlation between KA-induced hippocampal damage and behavioral responses in young and mature adult rats.