Joachim Bormann

Effects of the 1-amino-adamantanes at the MK-801-binding site of the NMDA-receptor-gated ion channel: a human postmortem brain study

Recent studies from our laboratory have provided evidence that the 1-amino-adamantane derivative memantine (1-amino-3,5-dimethyl-adamantane) binds to the MK-801-binding site of the N-methyl-D-aspartate (NMDA)-receptor-gated ion channel. This action has been suggested to account for the antiparkinsonian and antispastic activity of the drug. In the present investigation we have extended our work by testing a series of 1-amino-adamantanes, including amantadine (1-amino-adamantane) and memantine, for their ability to compete with [3H]MK-801 binding in membrane homogenates of postmortem human frontal cortex. The most potent substance (1-amino-3,5-diethyl-adamantane) had a Ki-value of 0.19 +/- 0.06 microM while the weakest substance (1-N-methyl-amino-adamantane) had a Ki-value of 21.72 +/- 1.63 microM. The Ki-value of amantadine was 10.50 +/- 6.10 microM. In agreement with our earlier investigation, the Ki-value of memantine was 0.54 +/- 0.23 microM. The results indicate that 1-amino-adamantanes, in general, may produce their pharmacological effects through an interaction with the NMDA-receptor-gated ion channel. The displacement of [3H]MK-801 binding thus may provide the basis to predict the antiparkinsonian and antispastic activity of novel substituted 1-amino-adamantanes and possibly of other drugs.

Memantine displaces [3H]MK-801 at therapeutic concentrations in postmortem human frontal cortex

It has recently been shown that MK-801 ((+ )5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a NMDA antagonist, has therapeutic activity in an animal model of Parkinsons disease. MK-801 reversed the reduced locomotor activity in the reserpineand amethyl-paratyrosine-pretreated mouse, indicating a mechanism independent of dopamine or noradrenaline release (Carlsson and Carlsson, 1989). This suggests that NMDA antagonists might be useful in the treatment of Parkinsons disease . Conversely, antiparkinson drugs might have NMDA antagonistic activity (Olney et al ., 1987) . The present investigation demonstrates that the long known antiparkinson and antipastic substance, memantine (1-amino-3,5-dimethyladamantane), inhibits the binding of [ 3H]MK-801 to postmortem human brain homogenates at therapeutic concentrations . Tissue from the frontal cortex was taken at autopsy from 3 subjects with no apparent history of neurological or psychiatric disorders . There were 2 males, aged 36 and 60, and 1 female aged 85 years. The postmortem interval was between 24 and 48 h. All details of the experimental procedures were identical to those previously described (Kornhuber et al., 1989) . MK-801 and memantine were investigated within the same assay and all

gp120 of HIV-1 induces apoptosis in rat cortical cell cultures: prevention by memantine

After incubation of rat cortical cell cultures with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 12 h, cells showed fragmentation of DNA at internucleosomal linkers, the characteristic feature of apoptosis. In a quantitative approach, it was determined that the percentage of DNA fragmentation increased from 7%, in the absence of gp120, to 62% following incubation with 24 ng/ml of gp120. Simultaneously, the percentage of viable cells decreased from 94% to 33%. Memantine (1-amino-3,5-dimethyladamantane), a drug currently used in the therapy of spasticity and Parkinsons disease as well as the NMDA antagonist MK-801 both prevented the effects of gp120 at micromolar concentrations. In human cultured astrocytes, gp120 was ineffective with respect to DNA fragmentation and cell toxicity. From these data, we conclude that the gp120-induced apoptosis may contribute to the neurological complications frequently associated with the immunodeficiency syndrome. The cytoprotective effect of memantine in cortical cell cultures may qualify the drug for the treatment of AIDS-related dementia.

Expression of GABA Receptor ρ1 and ρ2 Subunits in the Retina and Brain of the Rat

We have investigated the distribution of GABA receptor ρ1 and ρ2 subunits in the rat central nervous system. Cloning of rat ρ1 and ρ2 cDNA fragments revealed similarities to the corresponding human sequences of 99% (ρ1) and 88% (ρ2) at the protein level. Whereas the human ρ2 subunit has no consensus sequence for phosphorylation by protein kinase C, the cytoplasmic loop of the rat sequence contains two such sites. Use of the polymerase chain reaction with reverse‐transcribed total RNA (RT‐PCR) from different brain tissues revealed that transcript for the ρ1 subunit was present in the retina only. The ρ2 mRNA was detected in all brain regions, with the highest level of expression in the retina. In situ hybridization of retinal sections revealed that ρ1 and ρ2 transcripts are present in the inner nuclear layer. RT‐PCR and in situ hybridization of isolated retinal cells showed that both ρ subunits are present in rod bipolar cells. Since these cells express bicuculline‐insensitive GABA receptors, our results further support the idea that ρ subunits are part of the GABAC receptor.

Synaptic clustering of GABAC receptor ρ-subunits in the rat retina

Polyclonal antibodies which recognize the ρ‐subunits of the GABAC receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABAC receptors at synaptic sites. During postnatal development diffuse ρ‐immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the ρ‐immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the ρ‐antiserum were performed. The experiments showed that ρ‐immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABAC receptor ρ‐subunits coassemble with GABAA receptor subunits, double‐labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABAC receptor expressing puncta did not coincide with GABAA receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABAA and GABAC receptor subunits are coassembled. Similar double‐labelling experiments were also performed to find out whether GABAC receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABAC receptors consist of ρ‐subunits and are not coassembled with GABAA‐ or glycine‐receptor subunits.

Differential pharmacology of GABAA and GABAC receptors on rat retinal bipolar cells

GABAA and GABAC receptors were studied on cultured or freshly isolated rat retinal bipolar cells. The cells displayed GABA-induced whole-cell currents, which were only partially blocked by high concentrations (100 microM) of the GABAA receptor antagonist bicuculline. The bicuculline-resistant (GABAC) component was insensitive to the GABAA receptor modulators flunitrazepam (1 microM) and pentobarbital (50 microM). The bicuculline-sensitive portion of the current was strongly augmented by both drugs, indicating that it was mediated by conventional GABAA receptors. The GABAC and GABAA receptor subtypes displayed a 7-fold difference in their binding affinity for GABA, the EC50 values being 4.2 microM and 27.1 microM, respectively. The Hill coefficient was approximately 2 for both receptors. The bicuculline-insensitive GABAC receptors were markedly blocked by 100 microM picrotoxinin, 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide (SR-95531) and gamma-hexachlorocyclohexane, drugs known to be antagonists of GABAA receptors. Examination of single-channel currents indicated main-state conductances of 7.9 pS and 29.6 pS for GABAC and GABAA receptors, respectively. The pore diameter of open GABAC receptor channels was 5.1 A, i.e. close to the value of 5.6 A reported for the GABAA receptor. These results demonstrate that rod bipolar cells possess two populations of pharmacologically distinct GABA receptors, GABAA and novel-type GABAC receptors, which might subserve different physiological functions in controlling visual transduction in the retina.

Immunocytochemical localization of the GABAC receptor ? subunits in the cat, goldfish, and chicken retina

Polyclonal antibodies against the N‐terminus of the rat ρ 1 subunit were used to study the distribution of γ‐aminobutyric acid C (GABAC) receptors in the cat, goldfish, and chicken retina. Strong punctate immunoreactivity was present in the inner plexiform layer (IPL) of all three species. The punctate labelling suggests a clustering of the GABAC receptors at synaptic sites. Weak label was also found in the outer plexiform layer (OPL) and over the cell bodies of bipolar cells. Double immunostaining of vertical sections with an antibody against protein kinase C (PKC) showed the punctate immunofluorescence to colocalize with bipolar cell axon terminals. In the goldfish retina, the axon terminals of Mb1 bipolar cells were enclosed by ρ‐immunoreactive puncta. In the chicken retina, several distinct strata within the IPL showed a high density of ρ‐immunoreactive puncta. The results suggest a high degree of sequence homology between the ρ subunits of different vertebrate species, and they show that the retinal localization of GABAC receptors is similar across different species. J. Comp. Neurol. 380:520–532, 1997.

GABA-gated Cl− Channels in the rat retina

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian retina, and its physiological action is well established. GABA receptors have been localized immunocytochemically in the retina of different mammalian species, and all major retinal cell types have been found to express GABAA receptor subunits. Recently, a new type of GABA receptor with pharmacological and electrophysiological properties different from the known GABAA and GABAB receptors, has been described. These GABAC receptors are found predominantly in the vertebrate retina. This review concentrates on the electrophysiological characterization of GABA receptors expressed by amacrine and bipolar cells of the rat retina. We recorded GABA-induced currents from cultured neonatal amacrine and bipolar cells as well as from isolated bipolar cells of adult animals. While amacrine cells contain a homogeneous population of GABAA receptors, bipolar cells exhibit both GABAA and GABAC responses. Although both receptors gate chloride-selective ion channels, their biophysical and pharmacological properties differ markedly. These functional differences and the cellular distribution of GABAA and GABAC receptors suggest that they have different inhibitory functions in the rat retina.

Functional Heterogeneity of Hippocampal GABAA Receptors

γ‐Aminobutyric acid type A (GABAA) receptors were studied in cultured neurons taken from rat hippocampus at early postnatal stages. GABA‐induced whole‐cell currents showed a broad range of peak amplitudes and time‐courses of desensitization. Dose – response curves of rapidly and slowly desensitizing cells revealed EC50 values of 8.5 and 37.3 μM GABA, respectively, with the Hill coefficient being greater than unity. The main‐state conductance of GABAA receptor channels was 28 – 31 pS in all cells. GABA responses of low‐affinity cells were more strongly affected by benzodiazepine receptor agonists (e.g. flunitrazepam, clonazepam) and inverse agonists (e.g. methyl‐6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate), as compared to cells exhibiting high‐affinity GABA responses. Currents were also potentiated by zolpidem, but were little affected by Ro 15‐4513 and Zn2+. These data suggest the presence of physiologically and pharmacologically distinct GABAA receptor isoforms in neurons of the early postnatal hippocampus, which may subserve different inhibitory control mechanisms in this brain region.

Organotypic slice culture of the mammalian retina

Vertical slices of 6-day postnatal (P6) rat retina were cut at a thickness of 100 microns and cultured using the roller-tube technique. After 14-21 days in vitro there was significant distortion of normal retinal architecture, but localized areas of the slices showed the typical pattern of layering of mature retina. The following immunocytochemical markers were used to characterize the different retinal cell types: antibodies against protein kinase C (PKC), calcium binding protein (CabP 28kD), neurofilaments (NF), glia-specific antibodies (GFAP, vimentin), and transmitter-specific antibodies (GABA, TH). The expression of these markers was compared in P6 retina, adult retina, and slice culture. To further characterize the cultured cells, patch-clamp recordings were performed in combination with intracellular injection of Lucifer Yellow (LY). Transmitter- and voltage-gated membrane currents were recorded from morphologically identified neurons. The experiments show that a mammalian slice culture can be used to study differentiation and function of retinal cell types.