George R. Breese

Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration

Inflammation is implicated in the progressive nature of neurodegenerative diseases, such as Parkinsons disease, but the mechanisms are poorly understood. A single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) or tumor necrosis factor alpha (TNFα, 0.25 mg/kg, i.p.) injection was administered in adult wild‐type mice and in mice lacking TNFα receptors (TNF R1/R2−/−) to discern the mechanisms of inflammation transfer from the periphery to the brain and the neurodegenerative consequences. Systemic LPS administration resulted in rapid brain TNFα increase that remained elevated for 10 months, while peripheral TNFα (serum and liver) had subsided by 9 h (serum) and 1 week (liver). Systemic TNFα and LPS administration activated microglia and increased expression of brain pro‐inflammatory factors (i.e., TNFα, MCP‐1, IL‐1β, and NF‐κB p65) in wild‐type mice, but not in TNF R1/R2−/− mice. Further, LPS reduced the number of tyrosine hydroxylase‐immunoreactive neurons in the substantia nigra (SN) by 23% at 7‐months post‐treatment, which progressed to 47% at 10 months. Together, these data demonstrate that through TNFα, peripheral inflammation in adult animals can: (1) activate brain microglia to produce chronically elevated pro‐inflammatory factors; (2) induce delayed and progressive loss of DA neurons in the SN. These findings provide valuable insight into the potential pathogenesis and self‐propelling nature of Parkinsons disease.

Depletion of brain noradrenaline and dopamine by 6‐hydroxydopamine

1 After intracisternal administration, 6‐hydroxydopamine had a greater effect on brain noradrenaline than on dopamine. 2 Administration of two doses of 6‐hydroxydopamine increased the depletion of noradrenaline but not of dopamine. 3 Small doses of 6‐hydroxydopamine decreased the concentration of noradrenaline with little or no effect on dopamine. Tyrosine hydroxylase activity was not reduced with these treatments. 4 While pargyline pretreatment offered no advantage in the depletion of brain noradrenaline after 6‐hydroxydopamine, depletion of brain dopamine was greatly potentiated by this treatment. The reduction of striatal tyrosine hydroxylase activity observed after 6‐hydroxydopamine was also potentiated by pargyline pretreatment. 5 The amounts of labelled noradrenaline and dopamine formed from 3H‐tyrosine were greatly reduced by 6‐hydroxydopamine treatment. After 3H‐DOPA, formation of noradrenaline was greatly reduced while formation of labelled dopamine was only moderately reduced suggesting that decarboxylation of DOPA can occur in other than catecholamine containing neurones. 6 Desmethylimipramine and imipramine inhibited depletion of noradrenaline produced by 6‐hydroxydopamine but did not alter depletion of dopamine. Reserpine did not inhibit depletion of catecholamines produced by 6‐hydroxydopamine. 7 Administration of 6‐hydroxydopamine to developing rats lowered both noradrenaline and dopamine concentrations as well as the tyrosine hydroxylase activity in the brains of these animals.

Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector.

Safe, long-term gene expression is a primary criteria for effective gene therapy in the brain, so studies were initiated to evaluate adeno-associated virus (AAV) vector transfer of a reporter gene into specific sites of the rat brain. In the 4 day old rat, site infusions of AAV-CMV-lacZ (1 microliter; 5 x 10(4) particles) produced neuronal beta-galactosidase gene expression 3 weeks later in the hippocampus and inferior colliculus, but not in the cerebral cortex. Seven days after infusion of AAV-CMV-lacZ viral vectors (1 microliter) in the adult rat, beta-galactosidase gene expression was found in the olfactory tubercle, caudate, hippocampus, piriform cortex and inferior colliculus. primarily in multipolar neurons close to the infusion site. Three months after vector microinfusion, similar levels of gene expression remained in the olfactory tubercle and the inferior colliculus, with some reduction found in the caudate, but substantial reductions in beta-galactosidase gene expression occurred in the hippocampus and piriform cortex. In no case were obvious signs of toxicity noted. Therefore, AAV vectors can transfer foreign genes into the adult and neonatal CNS, but the pattern and longevity of gene expression depends upon the area of brain being studied.

METABOLISM OF NORMETANEPHRINE-H3 IN RAT BRAIN—IDENTIFICATION OF CONJUGATED 3-METHOXY-4-HYDROPHENYLGLYCOL AS THE MAJOR METABOLITE

Abstract Normetanephrine-H 3 injected into the cisterna magna of rats is rapidly metabolized and disappears from brain with an initial half-life of about 12 min. Monoamine oxidase inhibition prevents almost completely the conversion of normetanephrine-H 3 to other metabolites and markedly diminishes the rate of disappearance of radioactivity from brain ( T 1 2 = 2·4 hr ) . These data show that normetanephrine is normally metabolized primarily by monoamine oxidase and that unaltered normetanephrine does not readily pass out of the brain. Free 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3-methoxy-4-hydroxymandelic acid (VMA) formed from intracisternally injected normetanephrine-H 3 represent only a small fraction of the radioactivity in brain. The major metabolite was identified as the sulfate conjugate of MHPG. After intracisternal administration of norepinephrine-H 3 , 3-methoxy-4-hydroxyphenylglycol sulfate (MHPG-SO 4 ) was also found to be the major metabolite present in brain. These findings suggest that deamination, reduction, and subsequent conjugation with sulfate is the primary route of metabolism of normetanephrine in rat brain and that norepinephrine is also metabolized to this sulfate conjugate.

Neuroanatomical characterization of Fos induction in rat behavioral models of anxiety

Immunohistochemical staining for Fos-like immunoreactivity (Fos-LI) was used to map functional activation in discrete brain regions of rats processed in three empirical models of anxiety: foot shock avoidance responding in a shuttle box, the elevated plus maze, and an air puff-induced ultrasonic vocalization test. The avoidance test and elevated plus maze induced prominent Fos-LI in select brain regions, including the medial prefrontal, cingulate, and ventrolateral orbital cortices, taenia tecta, nucleus accumbens, paraventricular nucleus of the hypothalamus, medial nucleus of the amygdala and lateral septum. Air puff stimuli that produced ultrasonic vocalizations induced Fos-LI to a more limited extent compared to the plus maze and avoidance test, with only the medial prefrontal cortex, medial nucleus of the amygdala, and lateral septum being significantly affected by air-puff. Even though the sensory stimuli and environmental conditions associated with the three anxiety models were markedly different, specific common forebrain regions were affected, i.e. the medial prefrontal cortex, medial amygdala, and lateral septum. It is hypothesized that these regions are components of a circuit in the rat brain related to anxiety or distress. To determine the potential relationship between generalized arousal and the observed induction of Fos-LI in the anxiety models, rats were tested in a non-aversive situation involving marked behavioral activation. Accordingly, after vigorous bar pressing behavior for reinforcement with sweetened condensed milk, induction of Fos-LI was minimal and comparable to that in unhandled control rats. These latter data indicate that the distinctive neuroanatomical patterns of Fos-LI observed in the paradigms related to anxiety were not simply due to generalized behavioral activation. In summary, select common brain regions were identified that express Fos-LI in empirical models of anxiety. These data provide a functional framework to explore neuroanatomical sites of action of psychotherapeutic drugs that influence behavioral responses in these tasks.

Effects of ethanol on ion channels.

Ion channels play critical roles in nervous system function, from initiating rapid synaptic activity to propagation of action potentials. Studies have indicated that many of the effects of ethanol on the nervous system are likely caused by the actions of ethanol on ion channels. Ion channels are multimeric structures that gate ions through subtle changes in tertiary structure. Ethanol readily enters molecular sites within multimeric ion channels, modifying intermolecular forces and bonds that are important for the open-close-inactivation kinetic properties of channels. The diversity of channel composition caused by the multimeric structure results in subtypes of channels that have a spectrum of sensitivity to ethanol that translates into brain regional differences in ethanol sensitivity, in part caused by differences in ion channel subunit composition. Ethanol has been shown to affect both receptor-activated ion channels and voltage-gated ion channels. The acute intoxicating and incoordinating effects of ethanol are probably related to inhibition of subtypes of NMDA-glutamate receptor ion channels and potentiation of certain subtypes of GABAA receptor ion channels. Effects on these channels, as well as glycine, nicotinic cholinergic, serotonergic, and other ion channels, likely contribute to the euphoric, sedative, and other acute actions of ethanol. Changes in ion channel subunit composition, density, and properties probably also contribute to ethanol tolerance, dependence, withdrawal hyperexcitability, and neurotoxicity. A substantial number of studies have implicated glutamate NMDA receptor, GABAA, and L-type voltage-gated calcium channels in the adaptive changes in the brain during chronic ethanol exposure. The diversity of ion channels subunits, their prominent role in brain function, and ethanol action are likely to make them important contributors to alcoholism and alcohol abuse.

Thyrotropin releasing hormone: Antagonism of pentobarbital in rodents

Abstract Thyrotropin releasing hormone (TRH) antagonizes the behavioral and temperature reducing effects of pentobarbital in rodents. The hormone is effective whether given before or after the barbiturate. This antagonism by TRH of the effects of pentobarbital probably does not depend upon thyroid hormone release as L-triiodothyronine administration is ineffective.

Ontogeny of D1a and D2 dopamine receptor subtypes in rat brain using in situ hybridization and receptor binding

The prenatal and postnatal ontogeny of D1A and D2 dopamine receptors was assessed by in situ hybridization of messenger RNAs encoding the receptors and by radioligand binding autoradiography. On gestational day 14, signals for D1A and D2 dopamine receptor messages were observed in selected regions in ventricular and subventricular zones which contain dividing neuroblasts, and in intermediate zones that contain maturing and migrating neurons. Specifically, D1A and D2 dopamine receptor message was observed in the developing caudate-putamen, olfactory tubercle, and frontal, cingulate, parietal and insular cortices. Additionally, D1A dopamine receptor messenger RNA was found in the developing epithalamus, thalamus, hypothalamus, pons, spinal cord and neural retina; D2 dopamine receptor messenger RNA was also observed in the mesencephalic dopaminergic nuclear complex. Gene expression of D1A and D2 dopamine receptor subtypes in specific cells as they differentiate precedes dopamine innervation and implies that receptor expression is an intrinsic property of these neurons. The early expression of dopamine receptor messenger RNA suggests a regulatory role for these receptors in brain development. While the signal for both messages increased in the intermediate zones on gestational day 16, it decreased in the ventricular and subventricular zones, and was no longer apparent in these zones by gestational day 18. By gestational day 18, abundant D1A or D2 dopamine receptor messenger RNA was observed in cell groups similar in location to those observed in the adult brain. On gestational day 18, D1A dopamine receptor message was noted in the neural retina, anterior olfactory nucleus, the insular, prefrontal, frontal, cingulate, parietal and retrosplenial cortices, the olfactory tubercle, caudate-putamen, lateral habenula, dorsolateral geniculate nucleus, ventrolateral and mediolateral thalamic nuclei, and the suprachiasmatic and ventromedial nuclei of the hypothalamus. D2 dopamine receptor message was observed on gestational day 18 in the insular, prefrontal, frontal and cingulate cortices, the olfactory tubercle, caudate-putamen, ventral tegmental area, substantia nigra, and the intermediate lobe of the pituitary. At birth, expression of messenger RNA for both dopamine receptor subtypes in the striatum approximated that seen in mature rats. In contrast, D1A and D2 receptor binding, measured with [3H]SCH-23390 and [3H]raclopride, respectively, was low at birth and progressively increased to reach adult levels between days 14 and 21. The in situ hybridization data showing early prenatal expression of messenger RNA for the D1A and D2 dopamine receptors are consistent with the hypothesis that these receptors have a regulatory role in neuronal development.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental characteristics of brain catecholamines and tyrosine hydroxylase in the rat: effects of 6-hydroxydopamine.

1 Brain noradrenaline, dopamine and tyrosine hydroxylase were found in rat brain a few days before birth and increased progressively until reaching adult values. The most rapid period of growth for these substances seemed to occur between 7 and 18 days. 2 The intracisternal administration of 6‐hydroxydopamine to rats 7 days of age reduced concentrations of noradrenaline, dopamine, and tyrosine hydroxylase by 72 hours. 3 The concentrations of noradrenaline, dopamine or tyrosine hydroxylase in rats that received 6‐hydroxydopamine at 7 or 14 days of age remained markedly reduced when determined at adulthood, indicating that fibres did not continue to develop after the administration of this compound. The rats treated at 7 days also showed diminished concentrations of noradrenaline in heart. 4 Rats injected with 6‐hydroxydopamine at 7 days had reduced body weight as well as a reduction of some organ weights. This growth deficit was not observed in animals that received this drug at 14 days of age. 5 The administration of ovine growth hormone to rats that received 6‐hydroxydopamine at 7 days did not reverse the growth deficiency in these animals.

Stress enhancement of craving during sobriety: a risk for relapse.

This report of the proceedings of a symposium presented at the 2004 Research Society on Alcoholism Meeting provides evidence linking stress during sobriety to craving that increases the risk for relapse. The initial presentation by Rajita Sinha summarized clinical evidence for the hypothesis that there is an increased sensitivity to stress-induced craving in alcoholics. During early abstinence, alcoholics who were confronted with stressful circumstances showed increased susceptibility for relapse. George Breese presented data demonstrating that stress could substitute for repeated withdrawals from chronic ethanol to induce anxiety-like behavior. This persistent adaptive change induced by multiple withdrawals allowed stress to induce an anxiety-like response that was absent in animals that were not previously exposed to chronic ethanol. Subsequently, Amanda Roberts reviewed evidence that increased drinking induced by stress was dependent on corticotropin-releasing factor (CRF). In addition, rats that were stressed during protracted abstinence exhibited anxiety-like behavior that was also dependent on CRF. Christopher Dayas indicated that stress increases the reinstatement of an alcohol-related cue. Moreover, this effect was enhanced by previous alcohol dependence. These interactive effects between stress and alcohol-related environmental stimuli depended on concurrent activation of endogenous opioid and CRF systems. A.D. Lê covered information that indicated that stress facilitated reinstatement to alcohol responding and summarized the influence of multiple deprivations on this interaction. David Overstreet provided evidence that restraint stress during repeated alcohol deprivations increases voluntary drinking in alcohol-preferring (P) rats that results in withdrawal-induced anxiety that is not observed in the absence of stress. Testing of drugs on the stress-induced voluntary drinking implicated serotonin and CRF involvement in the sensitized response. Collectively, the presentations provided convincing support for an involvement of stress in the cause of relapse and continuing alcohol abuse and suggested novel pharmacological approaches for treating relapse induced by stress.