D. Eugene Redmond

CLONIDINE BLOCKS ACUTE OPIATE-WITHDRAWAL SYMPTOMS

In a double-blind, placebo-controlled, cross-over trial, clonidine eliminated objective signs and subjective symptoms of opiate withdrawal for 240--360 min in eleven addicts in a hospital setting. In an open pilot study of the effects of clonidine on longer-term opiate abstinence and symptoms, the same patients did well while taking clonidine for one week. There was only one documented instance of heroin use, in a patient who did not take clonidine after hospital discharge. 6 weeks or more after the study, four patients were back on reduced doses of methadone, one was on tricyclic antidepressants, and seven were off of all opiates. All eleven patients were doing well. These data suggest that opiate withdrawal is due to increased neuronal activity in areas such as the locus coeruleus which are regulated by both alpha-2 adrenergic and opiate receptors.

Yohimbine induced anxiety and increased noradrenergic function in humans: effects of diazepam and clonidine.

Yohimbine (30 mg) produced significant increases in subjective anxiety, autonomic symptoms, blood pressure, and plasma 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in ten healthy subjects. The effects of pretreatment with diazepam (10 mg) or clonidine (5 micrograms/kg) on these yohimbine induced changes was examined. Both diazepam and clonidine significantly antagonized yohimbine-induced anxiety, but only clonidine significantly attenuated the yohimbine induced increases in plasma MHPG, blood pressure, and autonomic symptoms. When given alone, clonidine significantly decreased plasma MHPG and blood pressure, whereas diazepam did not. These findings indicate that: (1) noradrenergic hyperactivity may be a factor in the production of some anxiety states; (2) the anti-anxiety effects of clonidine appear to result from its actions on receptors which decrease noradrenergic activity; (3) diazepam reverses yohimbine-induced anxiety without effects on several physiological or biochemical indicators of noradrenergic activity in humans.

Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinsons disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH+ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on α-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.

Noradrenergic agonists and antagonists: Effects on conditioned fear as measured by the potentiated startle paradigm

Clonidine (10–40 μg/kg) produced a dosedependent reduction of fear as measured by the potentiated startle effect (increased acoustic startle in the presence of a cue which had been previously paired with shock). The reduction of potentiated startle could not be accounted for entirely by a general depressant effect of clonidine on startle nor by an acceleration of extinction. Piperoxane and yohimbine, which are associated with anxiety in humans, increased potentiated startle, whereas propranolol and WB-4101 did not. These results provide further evidence that the potentiated startle paradigm in the rat is sensitive to drugs that alter anxiety in humans. Moreover, they support the hypothesis that norepinephrine transmission is important for the expression of fear or anxiety.

Estrogen is essential for maintaining nigrostriatal dopamine neurons in primates: implications for Parkinson's disease and memory.

There are sexual differences in several parameters of the nigrostriatal dopamine neurons, as well as in the progression of diseases associated with this system, e.g., Parkinsons disease and dementia. These differences, as well as direct experimental data in rodents, suggest that gonadal hormones play a role in modulating this system. To determine whether circulating estrogen might have long-term effects by altering the number of dopamine neurons, the density of dopamine neurons was calculated in the compact zone of the substantia nigra of male and intact female short- (10 d) and longer-term (30 d) ovariectomized and short- and longer-term ovariectomized but estrogen-replaced nonhuman primates (African green monkeys). Furthermore, the number of tyrosine hydroxylase-expressing neurons, the total number of all types of neurons, and the volume of the compact zone of the substantia nigra were calculated in 30 d ovariectomized and in 30 d ovariectomized and estrogen-replaced monkeys. Unbiased stereological analyses demonstrated that a 30 d estrogen deprivation results in an apparently permanent loss of >30% of the total number of substantia nigra dopamine cells. Furthermore, the density calculations showed that brief estrogen replacement restores the density of tyrosine hydroxylase-immunoreactive cells after a 10 d, but not after a 30 d, ovariectomy. Moreover, the density of dopamine cells is higher in females than in males. These observations show the essential role of estrogen in maintaining the integrity of the nigral dopamine system, suggest a new treatment strategy for patients with Parkinsons disease and with certain forms of memory-impairing disorders, and provide another rationale for estrogen replacement therapy for postmenopausal women.

FETAL NEURONAL GRAFTS IN MONKEYS GIVEN METHYLPHENYLTETRAHYDROPYRIDINE

Fetal substantia nigra cells of two different gestational ages were successfully transplanted into the brains of three methylphenyltetrahydropyridine-treated monkeys with severe parkinsonian motor and behavioural deficits. Functional improvement continued for 10 weeks after cell grafts into the striata of two monkeys with substantial numbers of tyrosine-hydroxylase-positive fetal neurons at necropsy. Behavioural improvement was correlated with increases in cerebrospinal fluid (CSF) homovanillic acid (HVA) concentrations after the transplants. A control monkey with inappropriately placed transplanted cells of an earlier gestational age remained severely parkinsonian and died during a similar period. CSF HVA fell slightly in this monkey from the low level seen before the transplants. Fetal dopamine neurons of two different gestational ages appear to survive transplantation in primates and have biochemical and functional effects.

Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

Dihydrexidine, a full dopamine D1 agonist, reduces MPTP-ioduced parkinsonism in monkeys

Since there are reasons to hypothetize that appropriate occupation of D 1 receptors may be an essential component of efficacious antiparkinsonian therapy (Brewster et al., 1990), we tested dihydrexidine in non-human primates pretreated with the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP), which results in a parkinsonian syndrome in humans and non-human primates

Survival and growth of fetal catecholamine neurons transplanted into primate brain

Dopamine and norepinephrine neuroblasts of the ventral mesencephalon, hypothalamus, and dorsolateral pons were transplanted from fetal African green monkeys into multiple brain sites in adult (host) African green monkeys. Tissue was grafted from both early and late gestational age fetuses. Immunohistochemical analysis, with antibodies to tyrosine hydroxylase, a marker of catecholamine-containing neurons, showed large numbers of transplanted catecholamine neurons in host cerebral cortex, corpus striatum and lateral ventricles up to 69 days after transplantation. Serial reconstructions revealed extensive outgrowth of neuronal processes from large numbers of transplanted neurons as well as expansion of the size of transplanted (solid) grafts of fetal brain tissue in the host brain. Some grafts extended from the caudate nucleus into the adjacent lateral ventricles or from the cerebral cortex into the underlying corpus callosum and ventricle. There were dense networks of varicose fibers emanating from the tyrosine hydroxylase positive neurons within intraparenchymal and intraventricular grafts. The size and shape of transplanted neurons retained characteristics common to catecholaminergic neurons from the dissected regions of fetal brain. Thus, a variety of fetal, catecholamine-containing neurons survive transplantation to primate brain and produce extensive neuritic outgrowths. Moreover, rejection of transplanted tissue was not apparent. These findings provide essential information on nerve cell grafting in a species closely related to humans as a prerequisite in the consideration of neural transplants as therapeutic measures in neurological disease.

Gonadal hormones are responsible for maintaining the integrity of spine synapses in the CA1 hippocampal subfield of female nonhuman primates

It is well established that gonadal hormonal manipulation results in morphologic changes in the rat hippocampus. The great similarities in the hippocampal formation between nonhuman primates and humans, as well as the differences in this structure between humans and rats, led to this investigation of whether hormonal manipulation in female subhuman primates influences pyramidal cell spine density in the CA1 hippocampal subfield, as it does in rats. African green monkeys (Cercopithecus aethiops sabaeus) were ovariectomized, and half of the animals received estrogen replacement therapy. One month later, the monkeys were killed. In the first group of experiments, pyramidal cell spines were analyzed on Golgi‐impregnated material taken from the CA1 hippocampal subfield. In the second experiment, unbiased electron microscopic stereologic calculations were performed to estimate the volumetric density of spine synapses in the same hippocampal subfield. Analysis of the Golgi‐impregnated material showed that the spine density of CA1 pyramidal cells is much lower in the ovariectomized animals than in ovariectomized and estrogen‐replaced monkeys. The unbiased, electron microscopic, stereologic calculation confirmed the light microscopic observation. The volumetric density (number of spine synapses/μ m3) of spine synapses was significantly lower (43.33%) in the ovariectomized animals than in ovariectomized and estrogen‐replaced monkeys. Because the hippocampus is involved in specific mnemonic functions, this observation highlights the importance of hormone replacement therapy in postmenopausal conditions. J. Comp. Neurol. 447:34–42, 2002.