MPTP animal model of Parkinsonism: dopamine cell death or only tyrosine hydroxylase impairment? A study using PET imaging, autoradiography, and immunohistochemistry in the cat.

AIMS: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin widely used to produce experimental models of Parkinson's disease in laboratory animals. It is believed to cause a selective destruction of substantia nigra dopamine neurons, mainly based on a large reduction of tyrosine hydroxylase (TH), the catecholamine's synthesizing enzyme. Unlike Parkinson's disease in humans, however, all animal models are able to recover more or less rapidly from the MPTP induced Parkinsonian syndrome. This raises the question as whether MPTP causes a cell death with a decrease in dopamine transporter or a simple impairment of TH. METHODS: To respond to this question, we quantified in a cat model of Parkinson's disease (MPTP 5 mg/kg i.p. during 5 days) the dopamine transporter using positron emission tomography (PET) imaging and autoradiography of [(11) C]PE2I and compared the data with the TH-immunoreactivity. RESULTS: We found no changes in [(11) C]PE2I PET binding either 5 or 26 days after MPTP treatment when compared to baseline levels. Similarly, there were no significant changes in [(11) C]PE2I autoradiographic binding in the cat brain one week after MPTP treatment. In sharp contrast, MPTP treated cats exhibited severe Parkinson-like motor syndrome during the acute period with a marked decrease in TH-immunoreactivity in the striatum. CONCLUSION: These data suggest that MPTP toxicity impairs efficiently TH and that such an effect is not necessarily accompanied by significant reduction of dopamine transporter seen with in vitro or in vivo [(11) C]PE2I binding.

MicroPET imaging of 5-HT 1A receptors in rat brain: a test-retest [18F]MPPF study.

PURPOSE: Earlier studies have shown that positron emission tomography (PET) imaging with the radioligand [(18)F]MPPF allows for measuring the binding potential of serotonin 5-hydroxytryptamine(1A) (5-HT(1A)) receptors in different regions of animal and human brain, including that of 5-HT(1A) autoreceptors in the raphe nuclei. In the present study, we sought to determine if such data could be obtained in rat, with a microPET (R4, Concorde Microsystems). METHODS: Scans from isoflurane-anaesthetised rats (n = 18, including six test-retest) were co-registered with magnetic resonance imaging data, and binding potential, blood to plasma ratio and radiotracer efflux were estimated according to a simplified reference tissue model. RESULTS: Values of binding potential for hippocampus (1.2), entorhinal cortex (1.1), septum (1.1), medial prefrontal cortex (1.0), amygdala (0.8), raphe nuclei (0.6), paraventricular hypothalamic nucleus (0.5) and raphe obscurus (0.5) were comparable to those previously measured with PET in cats, non-human primates or humans. Test-retest variability was in the order of 10% in the larger brain regions (hippocampus, medial prefrontal and entorhinal cortex) and less than 20% in small nuclei such as the septum and the paraventricular hypothalamic, basolateral amygdaloid and raphe nuclei. CONCLUSIONS: MicroPET brain imaging of 5-HT(1A) receptors with [(18)F]MPPF thus represents a promising avenue for investigating 5-HT(1A) receptor function in rat.

Decreased [18F]MPPF binding potential in the dorsal raphe nucleus after a single oral dose of fluoxetine: a positron-emission tomography study in healthy volunteers.

BACKGROUND: Brain serotonin-1A (5-HT(1A)) autoreceptors internalize when activated by agonist or by their endogenous ligand, serotonin. This positron-emission tomography (PET) study tested the hypothesis that 5-HT(1A) autoreceptor internalization might be indexed in vivo by a decrease in the specific binding of the 5-HT(1A) radioligand, 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1 piperazinyl]ethyl-N-2-pyridinyl-benzamide ([(18)F]MPPF), in the dorsal raphe nucleus (DRN) of healthy adult men administered a single oral dose of the selective serotonin reuptake inhibitor, fluoxetine. METHODS: [(18)F]MPPF binding potential was measured in the DRN and other brain regions endowed with 5-HT(1A) receptors in eight healthy volunteers, 5 hours after the randomized, double-blind administration of fluoxetine (20 mg) or placebo. RESULTS: In every subject, [(18)F]MPPF binding potential was decreased in the DRN only (44% +/- 22 SD), in response to fluoxetine. CONCLUSIONS: Imaging the functional state of 5-HT(1A) autoreceptors (i.e., internalization) in the human brain, using [(18)F]MPPF/PET, may represent a promising avenue for investigating the neurobiology of serotonin-related disorders and notably of major depression.

[18F]MPPF as a tool for the in vivo imaging of 5-HT1A receptors in animal and human brain.

Serotonin (5-hydroxytryptamine, 5-HT) and its various receptors are involved in numerous CNS functions and psychiatric disorders. 5-HT(1A), the best-characterized subtype of currently known 5-HT receptors, is tightly implicated in the pathogenesis of depression, anxiety, epilepsy and eating disorders. It thus represents an important target for drug therapy. Specific radioligands and positron emission tomography (PET) allow for a quantitative imaging of brain 5-HT(1A) receptor distribution in living animals and humans. Recently, the selective 5-HT(1A) receptor antagonist, MPPF, has been successfully labeled with [(18)F]fluorine ([(18)F]MPPF), and an increasing number of academic and industry centres have used this radiotracer in preclinical and clinical studies. After a brief account of some of the structural, distributional and electrophysiological characteristics of brain 5-HT(1A) receptors, this review focuses on studies conducted with [(18)F]MPPF, with emphasis on preclinical results illustrating the actual and potential value of this PET radioligand for clinical research and drug development.

A PET imaging study of 5-HT(1A) receptors in cat brain after acute and chronic fluoxetine treatment.

Immuno-electron microscopic and beta-microprobe studies have demonstrated that the internalization of serotonin 5-HT(1A) autoreceptors, after acute treatment with the selective 5-HT(1A) receptor agonist 8-OH-DPAT or with the specific serotonin reuptake inhibitor (SSRI) fluoxetine, is associated with a marked decrease in the in vivo binding of [(18)F]MPPF in the nucleus raphe dorsalis (NRD) of rat. To determine whether this event might be amenable to brain imaging, the present [(18)F]MPPF positron emission tomographic (PET) study was carried out in anesthetized cats given or not a single dose (5 mg/kg, i.v.) or chronically treated with fluoxetine (5 mg/kg, s.c. for 21 days). Compared to control, [(18)F]MPPF binding potential was considerably (and visibly) decreased in the cat NRD after acute fluoxetine treatment, while it remained unchanged in other brain regions. Unexpectedly, after chronic fluoxetine treatment, [(18)F]MPPF binding potential was not affected in any brain region. In parallel immuno-electron microscopic experiments carried out in rat, the density of 5-HT(1A) autoreceptors on the plasma membrane of NRD dendrites was comparable to control after chronic fluoxetine treatment. If the decrease in [(18)F]MPPF binding at the onset of SSRI treatment was detectable by PET imaging, it could potentially serve as a biological index of efficacy.

A comparison of in vivo and in vitro neuroimaging of 5-HT 1A receptor binding sites in the cat brain.

To validate the cat as a suitable model for positron emission tomography imaging (PET) and to gain further knowledge on the anatomical distribution of the serotonin-1A receptor (5-HT 1A) in the feline brain, we used PET with [18F]MPPF and in vitro autoradiography with [3H]MPPF, [3H]8-OH-DPAT and [3H]paroxetine. PET radioactivity curves with [18F]MPPF were very reproducible in anaesthetized cats, with the highest radioactivity uptakes recorded in the hippocampus, cingulate cortex, septum, infralimbic cortex and raphe nucleus, whereas the lowest were found in the cerebellum. [3H]8-OH-DPAT binding displayed a comparable, albeit lower, regional distribution than with [3H]MPPF. Autoradiography also revealed the presence of 5-HT 1A receptor binding sites in the cortex and in the interpeduncular nucleus, due to its greater sensitivity and spatial resolution compared with PET imaging. The cat constitutes an interesting experimental model for PET imaging, as many physiological concepts have been well established with this animal. Our study also shows the advantages of combining complementary neuroimaging techniques such as in vivo PET imaging and in vitro autoradiography to visualize the distribution of the 5-HT 1A receptors.

Postnatal development of the cholinergic innervation in the dorsal hippocampus of rat: Quantitative light and electron microscopic immunocytochemical study.

Choline acetyltransferase (ChAT) immunocytochemistry was used to examine the distribution and ultrastructural features of the acetylcholine (ACh) innervation in the dorsal hippocampus of postnatal rat. The length of ChAT-immunostained axons was measured and the number of ChAT-immunostained varicosities counted, in each layer of CA1, CA3, and dentate gyrus, at postnatal ages P8, P16, and P32. At P8, an elaborate network of varicose ChAT-immunostained axons was already visible. At P16, the laminar distribution of this network resembled that in the adult, but adult densities were reached only by P32. Between P8 and P32, the mean densities for the three regions increased from 8.4 to 14 meters of axons and 2.3 to 5.7 million varicosities per cubic millimeter of tissue. At the three postnatal ages, the ultrastructural features of ChAT-immunostained axon varicosities from the strata pyramidale and radiatum of CA1 were similar between layers and comparable to those in adult, except for an increasing frequency of mitochondria (up to 41% at P32). The proportion of these profiles displaying a synaptic junction was equally low at all ages, indicating an average synaptic incidence of 7% for whole varicosities, as previously found in adult. The observed junctions were small, usually symmetrical, and made mostly with dendritic branches. These results demonstrate the precocious and rapid maturation of the hippocampal cholinergic innervation and reveal its largely asynaptic nature as soon as it is formed. They emphasize the remarkable growth capacities of individual ACh neurons and substantiate a role for diffuse transmission by ACh during hippocampal development.

Fine structural features of the acetylcholine innervation in the developing neostriatum of rat.

The acetylcholine (ACh) innervation in the developing neostriatum of rat was examined by means of light and electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT). ChAT-immunoreactive cell bodies and their emerging processes, located at birth in the lateral part of neostriatum, progressively pervaded the whole region, to give rise to an extremely dense axonal network. As visualized and measured in single thin sections, at postnatal (P) ages P8, P16, and P32, the intrinsic and relational features of ChAT-immunostained profiles of axon varicosities in the lateral and medial parts of neostriatum were similar to those previously described in the adult. At the three postnatal ages, the immunoreactive profiles were comparable in shape, size, and vesicular content, and displayed one or more mitochondria with increasing frequency (from 10% at P8 to 29% at P32). The proportion which showed a synaptic junction was low at the three ages (8-16%), indicating an average synaptic incidence of 22% for whole varicosities after stereological extrapolation. The observed junctions were relatively small, mostly symmetrical, and made with dendritic spines or branches. The frequency of synapses on spines versus branches increased with age, from 20% at P8 to almost 60% at P32. Thus, the relational features of the neostriatal ACh innervation were similar to adult as soon as it appeared, as previously observed to be the case in the developing cerebral cortex. The diffuse mode of transmission may therefore be characteristic of both ACh interneurons (neostriatum) and projection neurons (cerebral cortex) in the CNS, and could be determining their functional properties during development as well as at maturity.

Pentylenetetrazol-induced seizures in immature rats provoke long-term changes in adult hippocampal cholinergic excitability.

PURPOSE: We previously demonstrated that the anticholinesterase eserine provokes interictal-like discharges in the CA3 area of hippocampal slices from rats in which generalized seizures had been induced by pentylenetetrazol (PTZ) when immature. In this study, we investigated several factors as the possible mechanism for this effect, including age at convulsions. METHODS: Rats were injected with PTZ on postnatal day (P) 18-20 or >P60, and neuronal activity was recorded intra- and extracellularly from CA3 5-10 or >40 days later. In additional experiments, convulsions were triggered by kainate or were blocked by pentobarbital. Hippocampal (a) acetylcholine (ACh) innervation density was measured by immunocytochemistry, and ACh and gamma-aminobutyric acid (GABA) contents were determined by high-performance liquid chromatography (HPLC)-electrospray ionization. RESULTS: The excitatory effect of eserine was the most consistent in slices from rats PTZ-treated when immature and after the long interval, whereas the reverse was true in rats treated as adults. This effect was dependent on the occurrence of a seizure and was less prevalent when the seizure had been provoked by kainate. Adult animals PTZ-treated at P20 did not differ from control in (a) poly- or monosynaptic GABAA and GABAB CA3 inhibitory postsynaptic potentials (IPSPs); (b) density of ACh innervation; or (c) tissue content of ACh and GABA. CONCLUSIONS: A PTZ-induced generalized seizure in immature rat provokes endogenous ACh-induced interictal-like discharges in adult hippocampal CA3. This effect is only transiently observed if the seizure was induced in adult. It does not appear to be related to a change in GABAergic inhibition, in density of ACh innervation, or in ACh or GABA content.

Comparative analysis of cholinergic innervation in the dorsal hippocampus of adult mouse and rat: a quantitative immunocytochemical study.

To obtain quantitative data on the distribution of the acetylcholine (ACh) innervation in the dorsal hippocampus of adult mouse (C57/B6) and rat (Sprague-Dawley), a semicomputerized method was used to measure the length of immunostained axons in hippocampal sections processed for light microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT). The results could be expressed in density of axons (meters per mm3) for the different layers and regions of dorsal hippocampus (CA1, CA3, DG), and also in density of axon varicosities (millions per mm3), after having determined the average number of varicosities per unit length of ChAT-immunostained axon (4 varicosities/10 microm). In mouse, the mean regional densities of ACh innervation were thus measured at 13.9, 16.1, and 15.8 m of axons, for 5.6, 6.4, and 6.3 million varicosities per mm3 of tissue, in CA1, CA3, and DG, respectively. The values were comparable in rat, except for CA1, in which the densities were lower than in mouse by 40% in the stratum lacunosum, and 20% in the stratum radiatum. Otherwise, the laminar patterns of innervation were similar in the two species, the highest densities being found in the stratum lacunosum moleculare of CA3, pyramidale of both CA1 and CA3, and moleculare of DG. These quantitative data will be of particular interest to evaluate changes in mutant mice, or mice and rats subjected to experimental conditions affecting the cholinergic phenotype.