Dopamine receptor functions: lessons from knockout mice [corrected].

In the past few years, a number of laboratories have used gene targeting via homologous recombination to generate mice deficient for key molecules involved in dopaminergic (DAergic) transmission. This tremendous effort has resulted in the successful generation and characterization of mice deficient for the neurotransmitter DA, the main terminator of DAergic neurotransmission (the DA transporter), and all five subtypes of DA receptors. This review summarizes the results from studies of the various DA receptor knockout mice and of mice deficient in proteins that mediate DA receptor signaling. It focuses on a comparison of the locomotor phenotypes and responses to drugs of abuse (psychostimulants), and reviews the results of anatomic studies examining the morphological and neurochemical differentiation of the striatum in these mutants. Moreover, an overview of recently published results highlighting the physiological relevance of the interaction between different DA receptors and between DA receptors and other neurotransmitter receptors in the modulation of behavioral and molecular responses to DAergic stimulation is presented. Finally, in view of the recently discovered heteroligomeric assemblies of neurotransmitter receptors that involve DA receptor subtypes, the potential value of knockout mice as a tool for testing the in vivo significance of these heteroligomeric receptors is discussed.

Electrical stimulation of cerebellar fastigial nucleus protects rat brain, in vitro, from staurosporine-induced apoptosis.

Electrical stimulation of the cerebellar fastigial nucleus (FN) elicits a prolonged ( approximately 10 days) and substantial (50-80%) protection against ischemic and excitotoxic injuries. The mechanism(s) of protection are unknown. We investigated whether FN stimulation directly protects brain cells against apoptotic cell death in an in vitro rat brain slice culture model. Rats were electrically stimulated in FN or, as control, the cerebellar dentate nucleus (DN). Coronal slices through the forebrain were explanted, exposed to staurosporine, harvested, and analyzed for caspase-3 activity by a fluorescence assay. FN, but not DN, stimulation significantly reduced staurosporine-induced caspase-3 activity by 39 +/- 7% at 3 h, 31 +/- 3% at 6 h and 26 +/- 4% at 10 h of incubation. Immunocytochemistry revealed FN-specific reductions in activated caspase-3 mainly in glial-like cells throughout the forebrain. FN stimulation also results in a 56.5% reduction in cytochrome c release upon staurosporine incubation. We conclude that neuroprotection elicited from FN stimulation can directly modify the sensitivity of brain cells to apoptotic stimuli and thereby suppress staurosporine induced apoptosis in adult rat brain slices. This model indicates that neuroprotection can be studied in vitro and provides new insight into the potential role of glial cells in ischemic protection of neurons induced by FN stimulation.

Stimulation of the subthalamic vasodilator area and fastigial nucleus independently protects the brain against focal ischemia.

We investigated whether stimulation of the functionally discrete subthalamic region, subthalamic cerebrovasodilator area (SVA), which increases cerebral blood flow (CBF) when excited, would, like stimulation of cerebellar fastigial nucleus (FN), produce central neurogenic neuroprotection. A 1-h electrical stimulation of SVA or FN reduced infarctions triggered by permanent occlusion of middle cerebral artery (MCA) by 48-55% in Sprague-Dawley rats and by 59% in Fisher rats. The salvaging effect of SVA stimulation, similar to FN, was long lasting and reduced the volume of infarctions placed 72 h or 10 days later by 58 and 26%, respectively, in Fisher rats. Bilateral lesioning of FN neurons by the microinjection of ibotenic acid 5 days before SVA stimulation did not affect SVA-evoked neuroprotection. Bilateral lesions of SVA neurons administered 5 days before FN stimulation had no effect on FN-induced neuroprotection but reversed the stimulus-locked increase in CBF accompanying FN stimulation. This study demonstrates that (1) excitation of neurons and/or fibers projecting through the SVA reduces ischemic infarctions as substantially as excitation of FN neurons; (2) the effects are long-lasting and not attributable to increases in cerebral blood flow, changes in blood gases or brain temperature, or rat strain; (3) the neuroprotective effects of SVA and FN stimulation are mutually independent and (4) FN-evoked cerebrovasodilation is mediated by SVA neurons. The SVA and FN are part of a neuronal system in CNS, which is distributed and, when excited, acts to protect the brain from ischemic injury.

Catecholamine monoamine oxidase a metabolite in adrenergic neurons is cytotoxic in vivo.

3,4-Dihydroxyphenylglycolaldehyde is the monoamine oxidase-A metabolite of two catecholamine neurotransmitters, epinephrine and norepinephrine. This aldehyde metabolite and its synthesizing enzymes increase in cell bodies of catecholamine neurons in Alzheimer's disease. To test the hypothesis that 3,4-dihydroxyphenylglycolaldehyde, but not epinephrine or its major metabolite 4-hydroxy-3-methoxyphenylglycol, is a neurotoxin, we injected 3,4-dihydroxyphenylglycolaldehyde onto adrenergic neurons in the rostral ventrolateral medulla. Injections of epinephrine or 4-hydroxy-3-methoxyphenylglycol were made into the same area of controls. A dose response and time study were performed. Adrenergic neurons were identified by their content of the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase, immunohistochemically. Apoptosis was evaluated by in situ terminal deoxynucleotidyl-transferase mediated dUTP nick end label staining. Injection of 3,4-dihydroxyphenylglycolaldehyde in amounts as low as 50 ng results in loss of adrenergic neurons and apoptosis after 18 h. The degree of neurotoxicity is dose and time dependent. Doses of 3,4-dihydroxyphenylglycolaldehyde 10-fold higher produce necrosis. Neither epinephrine nor 4-hydroxy-3-methoxyphenylglycol are toxic. A 2.5 microg injection of 3,4-dihydroxyphenylglycolaldehyde is toxic to cortical neurons but not glia. Active uptake of the catecholamine-derived aldehyde into differentiated PC-12 cells is demonstrated. Implications of these findings for catecholamine neuron death in neurodegenerative diseases are discussed.

Intrinsic neurons of fastigial nucleus mediate neurogenic neuroprotection against excitotoxic and ischemic neuronal injury in rat.

Electrical stimulation of the cerebellar fastigial nucleus (FN) elevates regional cerebral blood flow (rCBF) and arterial pressure (AP) and provides long-lasting protection against focal and global ischemic infarctions. We investigated which neuronal element in FN, perikarya or axons, mediates this central neurogenic neuroprotection and whether it also protects against excitotoxicity. In anesthetized rats, the FN was stimulated for 1 hr, and ibotenic acid (IBO) was microinjected unilaterally into the striatum. In unstimulated controls, the excitotoxic lesions averaged approximately 40 mm3. Stimulation of FN, but not dentate nucleus (DN), significantly reduced lesion volumes up to 80% when IBO was injected 15 min, 72 hr, or 10 d, but not 30 d, thereafter. In other rats, intrinsic neurons of FN or DN were destroyed by pretreatment with IBO. Five days later, the FN was stimulated, and 72 hr later, IBO was microinjected into the striatum. Lesions of FN, but not DN, abolished neuroprotection but not the elevations of rCBF and AP elicited from FN stimulation. Excitotoxic lesions of FN, but not DN, also abolished the 37% reduction in focal ischemic infarctions produced by middle cerebral artery occlusion. Excitation of intrinsic FN neurons provides long-lasting, substantial, and reversible protection of central neurons from excitotoxicity, as well as focal ischemia, whereas axons in the nucleus, probably collaterals of ramified brainstem neurons, mediate the elevations in rCBF, which do not contribute to neuroprotection. Long-lived protection against a range of injuries is an unrecognized function of FN neurons transmitted over pathways distinct from those regulating rCBF.

Stimulation of cerebellar fastigial nucleus inhibits interleukin-1beta-induced cerebrovascular inflammation.

Electrical stimulation of the cerebellar fastigial nucleus (FN) in rat protects the brain against ischemia. We studied whether FN could reduce the cerebrovascular inflammation as a mechanism of protection. FN or dentate nucleus (sham controls) was electrically stimulated for 1 h, and 72 h later rats were either injected with interleukin (IL)-1beta into the striata or processed to analyze inflammatory responses in isolated brain microvessels. In striata, IL-1beta induced a recruitment of leukocytes that was reduced by 50% by FN stimulation. In isolated microvessels, IL-1beta induced the transient and dose-dependent upregulation of the mRNAs encoding for the inducible nitric oxide synthase (NOS-2), intercellular adhesion molecule 1 (ICAM-1), and inhibitory kappaB-alpha (IkappaB-alpha), an inhibitor of nuclear factor-kappaB. FN stimulation decreased the upregulation of NOS-2 and ICAM-1 mRNAs, whereas it increased IkappaB-alpha mRNA expression. Dentate nucleus stimulation did not mimic the FN actions. These findings suggest that FN stimulation may render brain microvessels refractory to IL-1beta by overproduction of IkappaB-alpha and support the hypothesis that alteration of microvascular inflammation may contribute to the central neurogenic neuroprotection elicited from the FN.

Visceral afferent pathways to the thalamus and olfactory tubercle: behavioral implications.

The goal of this study was to support the hypothesis that visceral signals may integrate and influence behavior by way of direct pathways from the nucleus tractus solitarii (NTS) to the olfactory tubercle and the midline/intralaminar thalamus. An anterograde tracer, biotinylated dextran amine (BDA) was iontophoresed bilaterally into the caudal NTS to optimize terminal labeling. NTS-cortical projections traversed both limbs of the diagonal bands providing heavy innervation, and terminated lightly within layer 3 of the olfactory tubercle. NTS-thalamic projections terminated within anterior and, as previously shown, posterior divisions of nucleus paraventricularis thalami and avoided the adjoining mediodorsal thalamic nucleus. Heretofore unrecognized projections were traced to the parafascicular and reuniens thalamic nuclei, and the peripeduncular nucleus. Control experiments identified the nucleus gracilis as the principal source of ascending projections to ventroposterior lateral, posterior and intralaminar thalamic nuclei. Our data corroborate the supposition that olfactory signals may integrate with visceral stimuli in the striatal compartment of olfactory tubercle. NTS projections encompass thalamic nuclei that project topographically to the prefrontal cortex, hippocampus and ventral (limbic) striatum, regions activated by visceral stimulation. Structural data support the idea that compartments of the non-discriminative thalamus may contribute to perception and behavioral responses to visceral stimulation.

Cerebellar stimulation reduces inducible nitric oxide synthase expression and protects brain from ischemia.

A focal infarction produced by occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats induced expression of inducible nitric oxide synthase (iNOS) mRNA, measured by competitive reverse transcription-polymerase chain reaction. The mRNA appeared simultaneously in the ischemic core and penumbra at 8 h, peaked between 14 and 24 h, and disappeared by 48 h. At 24 h, inducible nitric oxide synthase (iNOS)-like immunoreactivity was present in the endothelium of cerebral microvessels and in scattered cells, probably representing leukocytes or activated microglia. Electrical stimulation of the cerebellar fastigial nucleus (FN) for 1 h, 48 h before MCAO, reduced infarct volumes by 45% by decreasing cellular death in the ischemic penumbra. It also reduced by >90% the expression of iNOS mRNA and protein in the penumbra, but not core, and decreased by 44% the iNOS enzyme activity. We conclude that excitation of neuronal networks represented in the cerebellum elicits a conditioned central neurogenic neuroprotection associated with the downregulation of iNOS mRNA and protein. This neuroimmune interaction may, by blocking the expression of iNOS, contribute to neuroprotection.