Dopamine has a modulatory role in generalized absence epilepsy, a state
in which the EEG is characterized by spontaneous occurring spike-wave discharges.
It has been proposed that the relatively low dopaminergic activity in the
dorsal striatum and the high dopaminergic activity in the ventral striatum,
two features of the apomorphine-susceptible (APO-SUS) rat, contribute to
the high incidence of spike-wave discharges in these rats2. The results
of Buzsáki1 are in favour of this view: dopamine blockers (chlorpromazine
and acepromazine), injected directly into the dorsal striatum, enhanced
these discharges. Rats of the WAG/Rij rat strain show many of these spike-wave
discharges and this strain is considered as an adequate model of absence
epilepsy4. Since WAG/Rij rats abundantly show spike-wave discharges, it
was hypothesized that these rats should also have a low dopaminergic activity
in the dorsal striatum, comparible to the APO-SUS strain. The ACI and the
apomorphine-unsusceptible (APO-UNSUS) rat strain with only few discharges
per hour, were used as controls. The gnawing response to apomorphine, used
as a parameter for dopaminergic activity, was measured in a test box that
was almost identical to that described by Ljungberg and Ungerstedt3. A
high gnawing score indicates a low baseline dopaminergic activity in the
dorsal striatum. Dopamine agonists induce stereotypy and the gnawing response
itself is due to activation of dopamine receptors in the dorsal striatum.
As predicted the APO-SUS rats responded under apomorphine (1.5 mg/kg) with
high gnawing scores, while the reverse applied to the APO-UNSUS and ACI
rats. However, WAG/Rij rats injected with apomorphine showed low gnawing
responses, suggesting a high dopaminergic activity in the dorsal striatum.
It can be concluded that a low dopaminergic activity of the dorsal
striatum alone does not necessarily run in parallel with a high incidence
of spike-wave discharges. In particular the WAG/Rij rats, a model for human
absence epilepsy, are characterized by a high dopaminergic activity in
the dorsal striatum.
1 Buzsáki (1990) Neuroscience, 36, 1-14
2 Cools & Peeters (1992) Neurosci Lett, 134, 253-256
3 Ljungberg and Ungerstedt (1978) Pharmacol Biochem Be, 8, 483-489
4 van Luijtelaar & Coenen (1986) Neurosci Lett, 70, 393-397
PROTEIN METHYLTRANSFERASE III IN BOVINE ADRENAL
MEDULLA
H. De Busser(1), G. Van Dessel(1,2) and A. Lagrou(1), (1)Ruca-Research
Unit for Human Biochemistry and (2)UIA-Laboratory for Pathological Biochemistry,
University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
Hilde De Busser <hidebus@ruca.ua.ac.be>
Methylation of the carboxylate group of the C-terminal cysteine residue
terminates the post-translational isoprenylation of a family of small GTP-binding
proteins, suggested to be involved in a number of cellular processes including
cell growth and differentiation as well as in signal transduction mechanisms.
The membrane bound isoprenylated protein methyltransferase activity was
assayed using synthetic substrates e.g. acetylfarnesylcysteine(1) and (3H)methyl
SAM as methyl donor. From this membranous fraction 0.25% CHAPS could solubilize
53% of the enzymatic activity with a concommitant 39% extraction of total
protein (1.4 x enrichment). The Michaëlis-Menten kinetics of both
the membrane bound and solubilized enzyme were investigated as well as
the influence of cations, SH-reagents and detergents on the activity. Sinefungine,
homocysteine, farnesylthiosalicylate and farnesylthio-acetate inhibited
the methyltransferase activity. SDS-PAGE showed methylation of proteins
with a MM around 20 kD.
Adrenal medulla and chromaffin cells were homogenized and fractionated
by differential centrifugation(2,3) and by buyoant density gradient centrifugation.
The first approach showed the highest relative specific activity for methyltransferase
type III in the microsomal fraction. From the second approach a bi-phase
profile was observed: the first peak (d:1.13) coincided with the plasma
membranes, while the second peak (d:1.19) paralleled the distribution profile
of the chromaffin granules.
(1) Tan, E.W., Perez-Sala, D., Canada F.J. and Rando, R.R. (1991) J.
Biol. Chem. 266: 10719-10722.
(2) Darchen, F., Zahraoui, A., Monteils, M-P., Travitian, A. and Scherman,
D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87: 5692-5696.
(3) Wolff, J. and Jones, A.B. (1971) J. Biol. Chem. 246: 3939-3947.
FUNCTIONAL RECOVERY AND RESTORATION OF AXONAL
PROJECTIONS OF AN IDENTIFIED MOLLUSCAN NEURON FOLLOWING AXOTOMY
C.J. Koert, A.B. Smit, R.E. van Kesteren, and W.P.M. Geraerts. Department
of Molecular and Cellular Neurobiology, Research Institute Neurosciences
Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, koert@bio.vu.nl
(niels koert)
The two cerebral giant cells (CGCs) in the CNS of the freshwater snail
Lymnaea stagnalis form an electrical couple of neurons that is involved
in the modulation of feeding behavior. The CGCs have a major axon projecting
from the cerebral ganglia into the buccal ganglia, that is capable of regeneration
after injury. The CGC synthesize serotonin and myomodulin neuropeptides,
as inferred from immunocytochemistry. Using mass spectrometry (MALDI-MS),
HPLC, and cDNA cloning we confirmed the expression of myomodulin in the
CGC, and in addition, characterized two novel CGC neuropeptides. We then
optimized the surgical experimental procedures and identified the different
phases of CGC regeneration , using electrophysiological techniques, whole-mount
immunocytochemistry, and a behavioral read-out system. In situ hybridization
and immunocytochemistry, were then employed to quantify the CGC neurotransmitters
and/or their transcript levels during the process of axonal regeneration.
Whether the transmitters of the CGC are functionally implicated in neuronal
outgrowth of the cell after injury is currently under investigation.
Ca2+-DEPENDENT REDISTRIBUTION OF SYNAPTIC VESICLES
IN SYNAPTOSOMES INDUCED BY SHORT DEPOLARIZATIONS
A.G.M. Leenders, F.H. Lopes da Silva and W.E.J.M. Ghijsen, Institute
of Neurobiology, Faculty of Biology, University of Amsterdam, Kruislaan
320, 1098 SM Amsterdam.The Netherlands.
In nerve terminals two morphologically distinct types of vesicles are
found : small clear-cored vesicles (SCCV) and large dense-cored vesicles
(LDCV). The SCCV release the fast acting classical neurotransmitters at
the active zone and the LDCV release slower acting modulatory neuropeptides
outside the active zone. Using a rapid mixing device we have shown that
there is a difference in kinetics of transmitter release from these two
vesicle types in rat cortex synaptosomes. There is a fast (t < 50msec)
release of glutamate and GABA from SCCV, whereas cholecystokinin release
from LDCV requires seconds.
In the present study we investigated the changes in distribution of
these different vesicle types in synaptosomes upon depolarization at the
ultrastructural level using electron microscopy. Synaptosomes were fixated
(2% paraformaldehyde and 2.5% glutaraldehyde) before or immediatly after
a short K-induced depolarization. Control synaptosomal sections have an
average diameter of 0.55µM and contain about 55 SCCV. Short depolarizations
did not change the total number of SCCV per synaptosome section. However,
the distribution of the SCCV within the synaptosomes was changed. 100msec
depolarization resulted in increased clustering and docking of SCCV at
their release site, the active zone. The amount of LDCV in synaptosomes
did not change during a 100msec depolarization, but was reduced to 50%
upon a 1sec depolarization. Both the redistribution of the SCCV and the
reduction of LDCV were not seen when depolarization was induced without
extracellular Ca2+ or in the presence of P-,Q- and N-type Ca2+-channel
toxins. In conclusion, these morphological data showed a depolarization
induced and Ca2+-channel dependent fast recruitement of SCCV towards the
active zone and a slow release of LDCV at remote sites.
CORTICAL SPREADING DEPRESSION ABOLISHES SPIKE-WAVE
DISCHARGES IN THE WAG/RIJ RAT
H.K.M. Meeren, N. Ates, A.M.L. Coenen, E.L.J.M. Van Luijtelaar, and.
W.H.I.M. Drinkenburg, Dept. Comparative & Physiological Psychology,
Nijmegen Institute for Cognition and Information, University of Nijmegen,
P.O. Box 9104, 6500 HE Nijmegen, The Netherlands.
WAG/Rij rats spontaneously show generalized spike-wave discharges (SWDs)
in their neocortical EEG, and constitute a validated genetic model of absence
epilepsy. It has been established that these SWDs reflect highly synchronized
oscillations in the thalamocortical circuit. It is generally assumed that
the thalamus is the central pacemaker for these oscillations. The role
of the cortex, however, is less clear. To assess whether the cortex is
a prerequisite for SWDs, the cortex was reversibly eliminated by the technique
of cortical spreading depression (CSD). CSD was induced by bilateral application
of 3 M KCl solution to the surface of the dura in freely moving rats, while
cortical and thalamic EEG was recorded. Repetitive waves of spreading depression
(SD) were reliably elicited for two hours, as monitored by DC potentials.
During this period SWDs were completely abolished in both cortical and
thalamic EEG. SWDs did not not return until 10 hours after the last SD
wave, and reappeared simultaneously in cortex and thalamus. CSD also completely
abolished Hypnorm facilitated SWDs, in both cortex and thalamus. These
results clearly show that the cortex is a prerequisite for the occurrence
of SWDs. Although the thalamus is thought to contain the pacemaker for
oscillations, the intrathalamic circuitry in itself is not sufficient to
generate SWDs. In the absence of corticothalamic input, the thalamus probably
fails to recruite and/or synchronize enough neurons in order to generate
highly synchronized spike-wave discharges.
ELECTRICALLY STIMULATED GABA RELEASE IN RAT HIPPOCAMPUS
CA1 REGION IS ENHANCED AFTER KINDLING EPILEPTOGENESIS
M. Zuiderwijk, F.H. Lopes da Silva and W.E.J.M. Ghijsen. Graduate School
for the Neurosciences, Institute for Neurobiology, University of Amsterdam,
The Netherlands.
It is still unclear how the release of GABA and its presynaptic regulation is altered in kindling epileptogenesis. In the present study we investigated GABA release upon locally applied high frequency electrical stimulations in rat hippocampal slices of kindled and control animals. Male Wistar rats were kindled via implanted electrodes in area CA1 of the hippocampus by stimulating twice a day until 7 generalised tonic/clonic seizures occurred. Hippocampal slices were isolated and transferred to a submerged recording chamber. Stimulation electrodes were positioned in the Schaffer-collaterals and glass electrodes were placed in stratum radiatum to monitor field potentials. GABA release was measured by collecting 1-min fractions of extracellular fluid via a small glass cannula positioned just above the dendritic layers of CA1 pyramidal neurones and analysed by HPLC. In order to measure GABA release, the presence of the GABA uptake carrier blocker SK&F 89976-A (SK&F) was required. In control slices, GABA release was transiently increased upon 50Hz electrical stimulation (3s trains of 50Hz stimuli, repeated every 20s during a 4 min period). Both low frequency (0.07Hz) and 50Hz stimulated GABA release were significantly enhanced (P<0.05) in kindled compared to control slices. To test whether this effect depended on GABA-B autoreceptors, the GABA-B receptor antagonist saclofen was added. Application of saclofen significantly further increased the 50Hz stimulated GABA release in control slices (P<0.03), but had no effect in kindled slices. In conclusion, high frequency electrically stimulated GABA release is regulated by presynaptic GABA-B autoreceptors. This regulation is absent when animals have reached a fully epileptic state. (Supported by grant 903-42-091 of the Netherlands Organization for Scientific Research).