Christer Köhler

II. Excitotoxic models for neurodegenerative disorders

Abstract In recent years, considerable interest has been shown in the neurotoxic properties of excitatory amino acids and their possible relevance for the study of human neurodegenerative disorders. The term “excitotoxin” has been coined for a family of acidic amino acids which are neuroexcitants and produce a characteristic type of “axon-sparing” neuronal lesion. Intracerebral infusions of kainic and ibotenic acids, the two most commonly used excitotoxins, result in a morphological and biochemical picture in experimental animals which resembles that observed in the brains of Huntingtons disease and epilepsy victims. The emergence of such animal models for neurodegenerative disorders has led to the hypothesis that endogenous excitotoxins may exist which are linked to the pathogenesis of human diseases. The most promising candidate discovered so far is quinolinic acid, a hepatic tryptophan metabolite which has recently also been found to occur in brain tissue. The particular excitotoxic properties of quinolinic acid warrant a thorough investigation of its metabolic and synaptic disposition in normal and abnormal brain function. While little is known about the mechanisms by which excitotoxins cause selective neuronal death, most current speculations propose the participation of specific synaptic receptors for acidic amino acids. The recent development of selective antagonists of such receptors has aided in the elucidation of excitotoxic mechanisms. Although a biochemical link between endogenous excitotoxins and human neurodegenerative disorders remains elusive at present, pharmacological blockade of excitotoxicity may constitute a novel therapeutic strategy for the treatment of these disease states.

Specific in vitro and in vivo binding of 3H-raclopride. A potent substituted benzamide drug with high affinity for dopamine D-2 receptors in the rat brain.

The substituted benzamide drug raclopride, [((-)-(S)-3,5-dichloro-N-((1-ethyl-2-pyrrolidinyl) methyl)-6-methoxy-salicylamide tartrate; FLA 870(-); A40664] was shown to be a potent and selective antagonist of dopamine D-2 receptors by its high affinity for striatal 3H-spiperone binding sites and low potency to block dopamine stimulated adenylate cyclase in vitro. In vitro studies showed that 3H-raclopride binds with a high affinity (KD = 1.2 nM) and a low proportion of non-specific binding to rat striatal homogenates. The binding of 3H-raclopride is saturable (Bmax = 23.5 pmoles/g wet wt) and reversible (dissociation half-time = 30 min) with a regional distribution of the specifically bound drug showing the following rank-order: striatum greater than nucleus accumbens greater than olfactory tubercle greater than septum greater than hypothalamus greater than hippocampus greater than frontal cortex. After in vivo administration, 3H-raclopride accumulates preferentially in dopamine rich brain areas with approximately 10 times higher levels in the striatum than in the cerebellum, when examined 30 min after injection. The in vivo binding of 3H-raclopride was saturable, reversible and showed a low component of non-specific binding. More than 90% of the drug reached the brain in a non-metabolized form as judged by thin-layer chromatography. Pharmacological analysis of 3H-raclopride binding showed that it could be displaced by dopamine agonists and antagonists but not by serotoninergic or noradrenergic drugs. Taken together, the results suggest that 3H-raclopride labels dopamine D-2 receptors with high specificity in the rat brain both in vitro and in vivo, and thus, that it should be a useful tool in studies of central dopamine D-2 receptors.

Septal neurons containing glutamic acid decarboxylase immunoreactivity project to the hippocampal region in the rat brain

SummaryInjections of the fluorescent dyes Fast Blue or Granular Blue into either the hippocampus (volume approximately 50 nl) or the entorhinal area (100–150 nl) resulted in labeling by retrograde axonal transport of cells in the diagonal band of Broca (dbB) and the medial septum (MS). A large number (approximately 30%) of these cells contained glutamic acid decarboxylase (GAD)-like immunoreactivity, as determined by combined retrograde fluorescent tracing and GAD-immunohistochemistry. Not all GAD positive cells in the dbB and MS were labeled by fluorochromes in a single experiment. The GAD-stained and fluorochrome-containing cells were present at all rostro-caudal levels of the septum and appeared not to belong to any single morphological class of cells. Double staining experiments showed that the GAD-positive cells did not contain acetylcholinesterase reaction product. These findings provide evidence that a significant portion of the septohippocampal projection may utilize gamma-aminobutyric acid as a neurotransmitter.

Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain

We have studied the localization of serotonin- and non-serotonin-containing cell bodies in the midbrain raphe nuclei that project to the entorhinal area and the hippocampal formation in the rat brain, using the technique of combined retrograde fluorescent tracing and immunohistochemistry on the same tissue section. The branching properties of these neurons were studied by retrograde double labelling using two fluorochromes which emit fluorescence with different spectral characteristics. After injections of granular blue or propidium iodide into the medial entorhinal area, retrogradely-labelled cells were found situated bilaterally in the caudal half of the dorsal raphe nucleus, the medial part of the median raphe and throughout the rostrocaudal extension of the nucleus reticularis tegmentipontis. Injections placed successively more laterally in the entorhinal area labelled progressively less cells contralaterally in the dorsal raphe and the reticular tegmental nucleus of the pons. After fluorochrome injections into the dorsal part of the hippocampal formation, retrogradely-labelled cells were found in the caudal part of the dorsal raphe, in the peripheral part of the median raphe and to a minor extent in the medial part of this nucleus, but not in the nucleus reticularis tegmentipontis. The experiments with double retrograde fluorescent tracing showed that the raphe nuclei do not send bilateral projections to the entorhinal area in spite of the fact that many of these cells are located contralateral to the injected hemisphere in single labelling experiments. Injections of the fluorochromes into the entorhinal area and hippocampal formation showed that at least 10% of the raphe cells project to both areas simultaneously. Analysis of sections incubated with antiserum to serotonin showed that a majority of the retrogradely-labelled versus serotonin-immunoreactive cells was found to vary within different parts of the individual raphe nuclei: the ventromedial part of the dorsal, the medial part of the median and the nucleus reticularis tegmentipontis being the highest. The findings indicate that both serotonin- and non-serotonin-containing neurons in the raphe innervate the hippocampal region, that these projections may be crossed but not bilateral, and that the same neuron in the raphe may influence the neural activity in the entorhinal area and the hippocampus simultaneously.

Comparison of ibotenate and kainate neurotoxicity in rat brain: A histological study

The neurotoxic properties of ibotenate and kainate after intracerebral application were compared in several regions of the rat brain. Ibotenate, being 5-10 times less toxic than kainate, caused lesions which were generally found to extend spherically from the tip of the injection cannula. In contrast, kainate injections often resulted in neuronal degeneration distant from the site of infusion, thus severely limiting its use as a tool for causing lesions in neurobiological studies. In some of the brain regions examined (hippocampus, septum), neurons appeared differentially susceptible to kainate but uniformly vulnerable to ibotenate. Some cell groups, such as those in the medial septum and the locus coeruleus, proved highly resistant to kainate but could be selectively ablated by ibotenate. These findings, together with differences between the two toxins in the evolution of neuronal degeneration (exemplified here in the hippocampal formation), appear to support previous suggestions that ibotenate and kainate exert their excitotoxic actions via different mechanisms. On the other hand, neuropathological changes caused in the cerebellum did not differ, since both ibotenate and kainate preferentially destroyed granule cells. Two nuclei, the arcuate nucleus of the hypothalamus and the nucleus of the fifth nerve, were found to be extremely resistant to either neurotoxin.

The selective dopamine D2 receptor antagonist raclopride discriminates between dopamine-mediated motor functions.

The actions on central dopamine (DA) mechanisms of raclopride, a new substituted benzamide, were studied by means of behavioural and biochemical methods in the rat. Raclopride blocked the in vitro binding of the dopamine D2 antagonist 3H-spiperone (IC50=32 nM), but not of the unselective D1 antagonist 3H-flupenthixol (IC50>100,000 nM) in rat striatum, and failed to inhibit striatal DA-sensitive adenylate cyclase in vitro (IC50>100,000 nM). Raclopride caused a dose-dependent increase in the DA metabolites HVA and DOPAC in the striatum and olfactory tubercle. Behavioural studies showed that raclopride discriminates between the motor behaviours induced by the DA agonist apomorphine. Thus, unlike haloperidol, raclopride blocked apomorphine-induced hyperactivity at considerably lower doses than those inhibiting oral stereotypies. Moreover, raclopride showed a high separation between the doses for blockade of apomorphine-induced hyperactivity and those inducing catalepsy in rats. Raclopride caused a dose-dependent blockade of the specific binding of 3H-spiperone and 3H-N-n-propylnorapomorphine (3H-NPA) in vivo at doses similar to those blocking the behavioural effects of apomorphine. The maximal blockade of 3H-spiperone binding in vivo was lower for raclopride than for haloperidol. Raclopride caused a greater inhibition of 3H-NPA than of 3H-spiperone in vivo binding in the striatum. It is suggested that the ability of raclopride to discriminate between different DA-mediated functions may be attributed to a preferential blockade of a subclass of functionally coupled dopamine D2 receptors in striatal as well as in extrastriatal brain regions in the rat.

Remoxipride, a new potential antipsychotic compound with selective anti-dopaminergic actions in the rat brain

The novel substituted benzamide, remoxipride, preferentially blocked apomorphine-induced hyperactivity with weak effects on stereotypies. The potency of remoxipride was about 50 times higher than that of sulpiride. Remoxipride caused a weak, atypical form of catalepsy and showed a high separation between the ED50 for blockade of apomorphine-induced hyperactivity and the ED50 for induction of catalepsy (ratio 24). Remoxipride was shown to be a selective dopamine D2 receptor antagonist since it displaced [3H]spiperone (IC50 = 1570 nM) but not [3H]flupentixol (IC50 greater than 100 000 nM) in rat striatum, and did not inhibit striatal DA-sensitive adenylate cyclase in vitro (IC50 greater than 100 000 nM). Remoxipride is a potent antagonist of D2 receptors showing a dose-dependent blockade of [3H]spiperone and [3H]n-propylnorapomorphine in vivo binding with a potency equal to that of chlorpromazine. In contrast to haloperidol, remoxipride caused a preferential blockade of in vivo [3H]spierone binding in the mesolimbic DA rich areas and the substantia nigra with much less effect in the striatum. In addition, remoxipride produced a preferential increase of DA utilization following synthesis inhibition in the olfactory tubercle. Only minor changes in NA and 5-HT metabolism were observed while HVA and DOPAC levels were markedly elevated. Taken together, these results indicate that remoxipride is a potent, selective D2 receptor blocking agent with a preferential action in mesolimbic and extrastriatal dopamine-containing neurons.

Differential vulnerability of central neurons of the rat to quinolinic acid

Infusion of 120 nmol quinolinic acid into several regions of the rats brain revealed differences in vulnerability to its neurotoxic effects, as judged by light microscopical analysis. The striatum, the pallidal formation and the hippocampus were the most susceptive brain areas whereas the cerebellum, substantia nigra, amygdala, medial septum and hypothalamus proved more resistant.

Evidence for separate projections of hippocampal pyramidal and non-pyramidal neurons to different parts of the septum in the rat brain

Large (200 nl) intraseptal injections of horseradish peroxidase (HRP) resulted in retrograde axonal labeling of both pyramidal and non-pyramidal neurons throughout all septo-temporal levels of the hippocampal formation in the rat brain. Small (50 nl) injections of HRP into the medial septum labeled cells of non-pyramidal shape in the stratum oriens and the stratum radiatum of regio inferior, stratum oriens of regio superior and the hilus of the area dentata. Small (50 nl) injections of HRP restricted to the lateral septum resulted in retrograde labeling of pyramidal cells in regio inferior and regio superior without labeling of non-pyramidal cells. These results suggest a new efferent projection system from the hippocampus consisting of non-pyramidal neurons which innervate the medial septum/diagonal band complex in the rat brain.

Regional Distribution of Cytochrome P‐450 in the Rat Brain: Spectral Quantitation and Contribution of P‐450b,e and P‐450c,d

Abstract: The cytochrome P‐450 (P‐450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P‐450b,e and P‐450c,d to brain P‐450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P‐450b and P‐450c (anti‐P‐450b and anti‐P‐450c, respectively). P‐450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p‐chloroamphetamine‐coupled Sepharose. The yield of P‐450 from whole brain was 90 ± 19 pmol/g of tissue, which is ∼ 1% of the level in liver microsomes from control rats. The amount of P‐450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P‐450 content, with yields of up to 400 pmol/g, whereas the substantia nigra yielded only 16–20 pmol/g. Immunohistochemical studies with anti‐P‐450b and anti‐P‐450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti‐P‐450c. In the cerebellum, both anti‐P‐450b and anti‐P‐450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti‐P‐450b gave weak staining of certain astroglia, but with anti‐P‐450c, there was intense staining of neuronal somata. Western immunoblots with P‐450 isolated from different brain regions confirmed the distribution of P‐450b,e and P‐450c,d observed with immunohistochemistry. Of all the brain regions examined, P‐450b,e was detected only in P‐450 obtained from the cerebellum and P‐450c only in the cerebellum and thalamus. However, quantitation of the P‐450b,e and P‐450c bands on the immunoblots by 125I‐labeled protein A revealed that these forms of P‐450 account for <1% of the P‐450 in the cerebellum and thalamus. This low content of P‐450b and P‐450c was also reflected in a low level of ethoxycoumarin O‐deethylase activity in the cerebellum and thalamus. From these studies, it is concluded that there are multiple forms of P‐450 in the brain and these different forms of P‐450 are highly selectively localized to certain cells. Furthermore, most of the P‐450 in the brain remains uncharacterized.