In vivo imaging of synaptic SV2A protein density in healthy and striatal-lesioned rats with [11C]UCB-J PET.

The number of functionally active synapses provides a measure of neural integrity, with reductions observed in neurodegenerative disorders. [11C]UCB-J binds to synaptic vesicle 2A (SV2A) transmembrane protein located in secretory vesicles. We aimed to assess [11C]UCB-J PET as an in vivo biomarker of regional cerebral synaptic SV2A density in rat lesion models of neurodegeneration. Healthy anesthetized rats had [11C]UCB-J PET and arterial blood sampling. We compared different models describing [11C]UCB-J brain uptake kinetics to determine its regional distribution. Blocking studies were performed with levetiracetam (LEV), an antiepileptic SV2A antagonist. Tracer binding was measured in rodent unilateral acute lesion models of Parkinsonism and Huntington's disease, induced with 6-hydroxydopamine (6-OHDA) and quinolinic acid (QA), respectively. [3H]UCB-J autoradiography was performed in postmortem tissue. Rat brain showed high and fast [11C]UCB-J uptake and washout with up to 80% blockade by LEV. [11C]UCB-J PET showed a 6.2% decrease in ipsilateral striatal SV2A binding after 6-OHDA and 39.3% and 55.1% decreases after moderate and high dose QA confirmed by autoradiography. In conclusion, [11C]UCB-J PET provides a good in vivo marker of synaptic SV2A density which can potentially be followed longitudinally along with synaptic responses to putative neuroprotective agents in models of neurodegeneration.

Transport of 5-hydroxydopamine and horseradish peroxidase through the perilymph-endolymph barrier.

The distribution of 5-hydroxydopamine (5-OHDA) and horseradish peroxidase (HRP) in the cochlea was examined by light and electron microscopy after perilymphatic perfusion. Reaction product of 5-OHDA was observed along the endolymphatic surface of the cochlear duct in both 10-minute and 30-minute perfusions, while that of HRP was observed only in the 30-minute perfusion. Both tracers were found in pinocytotic vesicles of Reissner's membrane, Claudius' cells, and Hensen's cells. No labeling was seen in tight junctions of the epithelial cells lining the scala media. The epithelial cells of the spiral prominence showed various degrees of degeneration in the 5-OHDA experiment. These morphologic findings suggest that 5-OHDA and HRP can enter the endolymphatic space from the perilymphatic space by pinocytosis and that the degree of transport depends on the size of the particles.

The release of catecholamines by an endogenous factor that inhibits neuronal Na+,K(+)-ATPase.

It is known that the inhibition of Na+,K(+)-ATPase induces neurotransmitter release in several experimental models. In this laboratory it was previously observed that a brain soluble fraction separated by Sephadex G-50 (peak II) is able to inhibit Na+,K(+)-ATPase but not other membrane-bound enzymes. The object of the present study was to test the effect of brain peak II fraction on neurotransmitter content of the pineal nerves synaptic vesicles. Uninjected rats and rats injected 30 min before with 5-hydroxydopamine (30 mg per kg,i.p.) were used. 5-hydroxydopamine produces a false neurotransmitter whose presence in the synaptic vesicles is visualized after fixation with glutaraldehyde-osmium as an electron dense material with the electron microscope which fills totally or partially the vesicles. In uninjected rats the osmiophilia and the chromaffin reaction of the electron dense core were studied. The pineal glands were incubated in Tyrode solution without calcium in the presence and absence of peak II at room temperature and processed for electron microscopy. When the glands from rats pretreated with 5-hydroxydopamine were incubated with peak II a significant decrease in the number of vesicles totally stained was observed. This indicates a reduction in false neurotransmitter content, specially in the matrix of the synaptic vesicles. In the presence of an aged peak II, which does not inhibit Na+,K(+)-ATPase, this effect on the synaptic vesicles was not observed. When the glands from uninjected rats were incubated with peak II no changes in the osmiophilia and the chromaffin reaction of the synaptic vesicles were found. The osmiophilia and the chromaffin reaction of the cores marks the monoamines storage site (catechol and indoleamines in the pineal nerves). These results are coherent with the idea of a relationship between the inhibition of Na+,K(+)-ATPase activity and the release of a pool of neurotransmitter stored in the nerve endings.

The release of catecholamines by an endogenous factor that inhibits neuronal Na+, K(+)-ATPase

Se ha demostrado que la inhibición de la Na+, K+-ATPasa produce liberación de neurotransmisor en distintos modelos experimentales. En este laboratorio se observó previamente que una fracción soluble separada mediante Sephadex G-50 (pico II) es capaz de inhibir la actividad de Na+, K+-ATPasa pero no de otras enzimas asociadas a membranas. El objetivo del presente trabajo fue probar el efecto de la fracción pico II de cerebro sobre el contenido de neurotransmisor de las vesículas sinápticas de los nervios pineales. Se usaron ratas no inyectadas y ratas inyectadas 30 min antes con 5-hidroxidopamina (30 mg per Kg, i.p.). La 5-hidroxidopamina produce un falso neurotransmisor cuya presencia en las vesículas sinápticas se visualiza luego de la fijación con glutaraldehído-osmio como un material electrón denso que llena total o parcialmente las vesículas. En ratas no inyectadas se estudió la osmiofilia y la reacción cromafín del nucleoide electron denso. Las glándulas pineales se incubaron en solución Tyrode sin calcio en presencia y ausencia de pico II a temperatura ambiente y se estudiaron al microscopio electrónico. Cuando las glándulas de las ratas pretratadas con 5-hidroxidopamina se incubaron con pico II se observó una disminución siginificativa en el número de vesículas totalmente llenas de material electrón denso. Esto indica una reducción en el contenido de falso neurotransmisor contenido en la matriz de las vesículas sinápticas. Este efecto sobre las vesículas sinápticas no se observó en presencia de pico II invejecido, que no inhibe la Na+, K+-ATPasa. Cuando las gládulas de ratas no inyectadas se incubaron con pico II no se observaron cambios ni en la osmiofilia ni en la reacción cromafin de las vesículas sinápticas. La osmiofilia y la reacción cromafin del nucleoide electrón denso marca el sitio de acumulación de monoaminas (catecol e indolaminas en los nervios pineales). Estos resultados son coherentes con la idea de una relación entre la inhibición de la actividad de Na+, K+-ATPasa y la liberación de una fracción de neurotransmisor acumulado en los terminales nerviosos

Cardiovascular functions of central noradrenergic neurons in rabbits.

1. The cardiovascular actions of central noradrenergic (NA) neurons was examined using the acute neurotransmitter releasing actions of intracisternal 6-hydroxydopamine in conscious rabbits. 2. The predominant actions of NA pathways in the brain-stem are to inhibit vasomotor and cardiac sympathetic activity and to facilitate cardiac vagal responses. 3. NA projections to the spinal cord tonically inhibit vasoconstrictor tone and form part of a high gain control system for responding to changes in specific afferent information. 4. The forebrain NA projections influence heart rate through modulation of the baroreflex gain of the cardiac vagus. 5. Central antihypertensive drugs such as clonidine and alpha-methyldopa mimic most of the brainstem actions of the central NA neurons. They also utilize the ascending NA pathways influencing the baroreflex vagal gain to minimize the baroreflex effects of reducing mean arterial pressure. 6. Thus, NA neurons influence the circulatory system through actions at all levels of the central nervous system and provide a comprehensive framework for integrating cardiovascular information from multiple inputs. This provides a number of targets for future development of useful pharmacological agents.

Uptake of horseradish peroxidase by the middle ear mucosa in experimentally induced otitis media.

Electron microscopy was used to study the characteristics of uptake of the protein tracer, horseradish peroxidase (HRP), by the middle ear mucosa of chinchillas. Two types of experimental otitis media were used: serous otitis media (SOM) produced by eustachian tube obstruction and purulent otitis media (POM) produced by inoculation of Streptococcus pneumoniae type 7F. HRP was placed in each tympanic bulla for either 1 min or 10 min. In the 1-min installation of HRP, the degree of HRP uptake was higher in SOM and POM animals than in control animals. Following the 10-min installation of HRP, the degree of HRP uptake was higher in SOM and POM animals than in control animals. Following the 10-min installation of HRP, the degree of tracer uptake was almost the same as among the control, SOM and POM animals. The mechanism of uptake was pinocytosis. In another experiment, 5-hydroxydopamine (mol. wt. 256) was used as a small tracer placed into the bulla to examine the permeability of epithelial tight junctions. No labelling of reaction product was observed in the tight junctions of the control, SOM or POM animals. These findings suggest that otitis media increases the uptake of HRP by pinocytosis and that epithelial tight junctions remain intact in inflammation of the middle ear cavity.

Further observation on the intraepithelial nerve fibers of rabbit ocular ciliary epithelium.

The intraepithelial nerve fibers of rabbit ciliary epithelium were observed under the electron microscope after administration of 5-hydroxydopamine. All the fibers so far observed contained small and large cored vesicles, thus indicating their noradrenergic nature.

In vivo voltammetric study of 6-hydroxydopamine-induced neuronal degradation.

Freely moving rats were injected intraventricularly with 200 micrograms of 6-hydroxydopamine (6-OHDA) and examined for transport of this substance to the striatum, degradation of dopamine (DA) neurons, and functional recovery through microcomputer-controlled in vivo voltammetry. Approximately 6 min after the injection, 6-OHDA began to appear in the extracellular fluid of the contralateral striatum. It increased linearly and began to decrease exponentially with the termination of the injection. Two hours after the injection with 6-OHDA, a peak began to appear at the same potential as the peak of DA in the differential pulse voltammogram. It persisted for approximately 3 days. When the rats were injected intraperitoneally with L-3,4-dihydroxyphenylalanine (L-Dopa), the conversion of L-Dopa to DA was not found 1 week after the 6-OHDA injection, but was clearly recognized 5 months after the injection. When the rats were examined for behavioral changes arising from the L-Dopa injection, they were found to be clearly less hyperactive 5 months after the 6-OHDA injection than 1 week after.