Rosuvastatin enhances anti-inflammatory and inhibits pro-inflammatory functions in cultured microglial cells.

Microglial activation results in profound morphological, functional and gene expression changes that affect the pro- and anti-inflammatory mechanisms of these cells. Although statins have beneficial effects on inflammation, they have not been thoroughly investigated for their ability to affect microglial functions. Therefore the effects of rosuvastatin, one of the most commonly prescribed drugs in cardiovascular therapy, either alone or in combination with bacterial lipopolysaccharide (LPS), were profiled in pure microglial cultures derived from the forebrains of 18-day-old rat embryos. To reveal the effects of rosuvastatin on a number of pro- and anti-inflammatory mechanisms, we performed morphometric, functional and gene expression studies relating to cell adhesion and proliferation, phagocytosis, pro- and anti-inflammatory cytokine (IL-1ß, tumor necrosis factor α (TNF-α) and IL-10, respectively) production, and the expression of various inflammation-related genes, including those related to the above morphological parameters and cellular functions. We found that microglia could be an important therapeutic target of rosuvastatin. In unchallenged (control) microglia, rosuvastatin inhibited proliferation and cell adhesion, but promoted microspike formation and elevated the expression of certain anti-inflammatory genes (Cxcl1, Ccl5, Mbl2), while phagocytosis or pro- and anti-inflammatory cytokine production were unaffected. Moreover, rosuvastatin markedly inhibited microglial activation in LPS-challenged cells by affecting both their morphology and functions as it inhibited LPS-elicited phagocytosis and inhibited pro-inflammatory cytokine (IL-1ß, TNF-α) production, concomitantly increasing the level of IL-10, an anti-inflammatory cytokine. Finally, rosuvastatin beneficially and differentially affected the expression of a number of inflammation-related genes in LPS-challenged cells by inhibiting numerous pro-inflammatory and stimulating several anti-inflammatory genes. Since the microglia could elicit pro-inflammatory responses leading to neurodegeneration, it is important to attenuate such mechanisms and promote anti-inflammatory properties, and develop prophylactic therapies. By beneficially regulating both pro- and anti-inflammatory microglial functions, rosuvastatin may be considered as a prophylactic agent in the prevention of inflammation-related neurological disorders.

Development of the microglial phenotype in culture.

Selected morphological, molecular and functional aspects of various microglial cell populations were characterized in cell cultures established from the forebrains of E18 rat embryos. The mixed primary cortical cultures were maintained for up to 28days using routine culturing techniques when the microglial cells in the culture were not stimulated or immunologically challenged. During culturing, expansion of the microglial cell populations was observed, as evidenced by quantitative assessment of selected monocyte/macrophage/microglial cell-specific markers (human leukocyte antigen (HLA) DP, DQ, DR, CD11b/c and Iba1) via immunocyto- and histochemistry and Western blot analysis. The Iba1 immunoreactivity in Western blots steadily increased about 750-fold, and the number of Iba1-immunoreactive cells rose at least 67-fold between one day in vitro (DIV1) and DIV28. Morphometric analysis on binary (digital) silhouettes of the microglia revealed their evolving morphology during culturing. Microglial cells were mainly ameboid in the early stages of in vitro differentiation, while mixed populations of ameboid and ramified cell morphologies were characteristic of older cultures as the average transformation index (TI) increased from 1.96 (DIV1) to 15.17 (DIV28). Multiple immunofluorescence labeling of selected biomarkers revealed different microglial phenotypes during culturing. For example, while HLA DP, DQ, DR immunoreactivity was present exclusively in ameboid microglia (TI<3) between DIV1 and DIV10, CD11b/c- and Iba1-positive microglial cells were moderately (TI<13) and progressively (TI<81) more ramified, respectively, and always present throughout culturing. Regardless of the age of the cultures, proliferating microglia were Ki67-positive and characterized by low TI values (TI<3). The microglial function was assessed by an in vitro phagocytosis assay. Unstimulated microglia with low TI values were significantly more active in phagocytosing fluorescent microspheres than the ramified forms. In vitro studies on microglial population dynamics combined with phenotypic characterization can be of importance when different in vivo pathophysiological situations are modeled in vitro.

Disparate changes in the expression of transient receptor potential vanilloid type 1 receptor mRNA and protein in dorsal root ganglion neurons following local capsaicin treatment of the sciatic nerve in the rat.

In situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis were applied to study the changes in expression of the major nociceptive ion channel transient receptor potential vanilloid type 1 receptor (TRPV1) after the perineural application of capsaicin or nerve transection. In control rats, quantitative morphometric and statistical analyses of TRPV1 protein and mRNA expression in L5 dorsal root ganglion cells revealed distinct populations of small (type C) and small-to-medium (type B) neurons, which showed very high and moderate levels of TRPV1, whereas larger (type A) neurons mostly did not express this receptor. After either transection or capsaicin treatment of the sciatic nerve, immunohistochemistry and Western blotting demonstrated a massive (up to 80%) decrease in the proportion of TRPV1-immunoreactive neurons and TRPV1 protein at all postoperative survival times. In situ hybridization indicated marked decreases (up to 85%) in the proportion of neurons that expressed TRPV1 mRNA after sciatic nerve transection. In contrast, although perineural treatment with capsaicin resulted in similar substantial decreases in the proportions of type B and C neurons of the L5 dorsal root ganglia 3 days postoperatively, a clear-cut tendency to recovery was observed thereafter. Hence, the proportions of both type B and C neurons expressing TRPV1 mRNA reached up to 70% of the control levels at 30 days postoperatively. In accord with these findings, quantitative RT-PCR revealed a marked and significant recovery in TRPV1 mRNA after perineural capsaicin but not after nerve transection. These observations suggest the involvement of distinct cellular mechanisms in the regulation of the TRPV1 mRNA expression of damaged neurons, specifically triggered by the nature of the injury. The present findings imply that the antinociceptive and anti-inflammatory effects of perineurally applied capsaicin involve distinct changes in neuronal TRPV1 mRNA expression and long-lasting alterations in (post)translational regulation.

Ontogeny of calmodulin gene expression in rat brain.

Calmodulin (CaM), a multifunctional intracellular calcium receptor, is a key element in signaling mechanisms. It is encoded in vertebrates by multiple apparently redundant genes (CaM I, II, III). To investigate whether differential expression takes place in the developing rat brain, a quantitative in situ hybridization analysis was carried out involving 15 brain areas at six ages between embryonic day 19 and postnatal day 20 (PD20) with gene-specific [(35)S]cRNA probes. A widespread, developmental stage-specific and differential expression of the three CaM genes was observed. The characteristic changes in the CaM mRNA levels in the examined time frame allowed the brain regions to be classified into three categories. For the majority of the areas (e.g. the piriform cortex for CaM III), the signal intensities peaked at around PD10 and the expression profile was symmetric (type 1). Other regions (e.g. the cerebral cortex, layer 1 for CaM II) displayed their highest signal intensities at the earliest age measured, followed by a gradual decrease (type 2). The signal intensities in the regions in the third group (e.g. the hypothalamus for CaM III) fluctuated from age to age (type 3). Marked CaM mRNA levels were measured for each transcript corresponding to the three CaM genes in the molecular layers of the cerebral and cerebellar cortici and hippocampus, suggesting their dendritic translocation. The highest signal intensity was measured for CaM II mRNA, followed by those for CaM III and CaM I mRNAs on PD1. However, the CaM II and CaM III mRNAs subsequently decreased steeply, while the CaM I mRNAs were readily detected even on PD20. Our results suggest that during development (1) the transcription of the CaM genes is under differential, area-specific control, and (2) a large population of CaM mRNAs is targeted to the dendritic compartment in a gene-specific manner.

Branching-pattern analysis of the dendritic arborization in the thalamic nuclei of the rat brain.

We investigated the dendritic patterns of rapid Golgi-impregnated, highly similar multipolar neurons from two functionally different thalamic regions of the rat brain: two dorsal nuclei (the nucleus laterodorsalis thalami, pars dorsomedialis and the nucleus laterodorsalis thalami, pars ventrolateralis), and two ventral nuclei (the nucleus ventrolateralis thalami and the nucleus ventromedialis thalami). The analysis involved conventional morphometric parameters (height and size) and a new parameter derived from graph theory, the relative imbalance (RI), derived from the branching patterns of the dendrites, which permits quantitative characterization of the dendritic arborization of a neuron. On this basis, neurons can be grouped into three fundamentally different types: type A, or highly-polarized (imbalanced) neurons (RI values close to 1); type B, or medium-polarized neurons (RI values around 0.5); and type C, or balanced neurons with low polarization (RI values close to 0). The orientations of the dendritic arbor, and thus the receptive fields, of the dorsal and ventral thalamic neurons, were mutually perpendicular. The H and S values indicated that the neurons in the dorsal and ventral thalamic nuclei differed significantly. However, their RI values demonstrated that they were similar neurons of type B. Our data reveal that 1 ) the dendritic arbor cannot be reliably characterized purely on the basis of height and size, and 2) RI is a valuable morphometric parameter that identifies the true nature of the dendritic arborization.

Somato-dendritic synapses in the nucleus reticularis thalami of the rat.

In the reticular nucleus of the rat thalamus, about 30% of the synapses are brought about by the perikarya of parvalbumin-immunopositive neurons, which establish somato-dendritic synapses with large dendrites of nerve cells of specific thalamic nuclei. Although the parvalbumin-immunopositive presynaptic structures bear resemblance to goblet-like or calyciform axonal endings, electron microscopic immunocytochemistry and in situ hybridization revealed that these structures are parts of the perikaryal cytoplasm studded with synaptic vesicles. In about 15% of the somato-dendritic synapses, axons are seen to be in synaptic contact with the parvalbumin-immunoreactive perikaryon. Double immunohistochemical staining revealed that the parvalbumin immunoreactive presynaptic perikarya and dendrites contained GABA. It is assumed that the peculiar somato-dendritic synaptic complexes subserve the goal of filtration of impulses arriving at the reticular nucleus from various thalamic nuclei, thus processing them for further sampling.

Methods for quantification of in situ hybridization signals obtained by film autoradiography and phosphorimaging applied for estimation of regional levels of calmodulin mRNA classes in the rat brain.

Comparative analysis of the regional abundances of the various mRNAs in neural tissues requires the quantitation of target nucleic acid sequences while their tissue distribution is preserved. A quantitative in situ hybridization protocol is presented for the assessment of regional levels of calmodulin (CaM) I, II and III mRNAs in the rat brain. Coronal brain cryostat sections were hybridized with multiple CaM [35S]cRNA probes and co-exposed to an autoradiographic film or storage phosphor screen, together with a membrane-based radioactive standard scale. The membrane scale was calibrated against a brain paste standard scale. Regression analyses of the sensitometric graphs of standard scales corresponding to the autoradiographic film and to the storage phosphor screen were performed by means of exponential (ROD=p(1)(1-exp[-p(2)x])) and linear (LI=ax) functions, respectively (ROD is relative optical density, LI is labeling intensity, and x is radioactivity). The ROD/LI values for the hybridized brain regions were converted into cRNA probe copy numbers (estimations of mRNA copy numbers) through use of the above standard scales. This method was applied to compare the regional abundances of multiple CaM mRNAs in the brains of control, dehydrated, chronic ethanol-treated and ethanol withdrawal-treated animals.

Postischemic calmodulin gene expression in the rat hippocampus.

The hippocampal calmodulin (CaM) gene expression was determined in the rat after transient forebrain ischemia. Ischemia was induced by the 4-vessel occlusion model, and the hybridized mRNA copy numbers corresponding to the 3 CaM genes detected by phosphorimaging were determined by quantitative in situ hybridization 24 h post-insult. A small, but significant upregulation (by 8.8%) of the CaM I gene was observed in the CA1 pyramidal cell layer, whereas other regions exhibited a maintained or slightly decreased CaM gene expression. The CaM mRNA levels decreased most markedly (by 10-15%) in the hippocampal molecular layers. No consistent correlation was found between the ischemic vulnerability and the CaM gene expression pattern. The results indicate that the induction of delayed neuronal death is not incidental to the transcriptional activation of the CaM genes in the ischemic rat hippocampus in vivo. As the calcium-bound CaM content increases during the ischemic insult, downregulation of the CaM gene expression could be a homeostatic response aimed at maintaining the intracellular level of the active CaM pool.

Multiple calmodulin genes exhibit systematically differential responses to chronic ethanol treatment and withdrawal in several regions of the rat brain.

Ethanol induces profound alterations in the neuronal signaling systems, including the calcium (Ca(2+)) signaling. Prolonged exposure to ethanol evokes adaptive changes in the affected systems as they strive to restore the normal neuronal function. We investigated the involvement of calmodulin (CaM) genes, coding for the major mediator protein of intracellular Ca(2+) signals, in these adaptive processes at the mRNA level. The changes induced in the regional abundances of the CaM I, II, and III mRNA classes by chronic ethanol treatment and withdrawal were examined by means of quantitative in situ hybridization, employing gene-specific [35S]cRNA probes on rat brain cryostat sections. Regional analysis of the resulting changes in mRNA levels highlighted brain areas that belong in neuronal systems known to be especially sensitive to the action of ethanol. The results revealed systematically differential regulation for the three mRNA classes: the CaM I and CaM III mRNA levels displayed increases, and CaM II levels decreases in the affected brain regions, in both chronic ethanol- and withdrawal-treated animals. As regards the numbers of brain regions undergoing significant alterations in mRNA content, the CaM I mRNA levels exhibited changes in most brain areas, the CaM II levels did so in a lower number of brain regions, and the CaM III levels changed in only a few brain areas. These results suggest a differential regulation for the CaM genes in the rat brain and may help towards elucidation of the functional significance of the multiple CaM genes in the mammalian genome.

Calmodulin gene expression in an immortalized striatal gabaergic cell line.

We have demonstrated the presence of the mRNAs transcribed from the three calmodulin (CaM) genes in the GABAergic cell line M26-1F derived from embryonic rat striatal cells and immortalized by oncogene transduction. Similarly as in the rat striatum in vivo, these clonal cells express CaM I, CaM II and CaM III mRNAs differently, the CaM I mRNA population being the most abundant, followed in sequence by the CaM II and CaM III mRNA populations. The proportions of these transcripts resemble those in the adult striatum. The possibility of deriving immortalized cell lines from primary neuronal tissue which exhibit characteristics similar to those of the tissue of origin could provide an important tool in many types of in vitro studies.

Calculation of maximal hybridization capacity (Hmax) for quantitative in situ hybridization: a case study for multiple calmodulin mRNAs.

In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CaM) I, II, and III [(35)S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [(35)S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm(2) units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.

Differential regulation of vasopressin gene expression in the hypothalamus of endotoxin-treated 14-day-old rat.

The E. coli endotoxin 0111 B4, a lipopolysaccharide (LPS), in a dose of 200 ng/kg body weight/50 microl artificial cerebrospinal fluid (CSF) was given intracisternally to 14-day-old rats. Four hours later CSF, blood and urine were sampled, and consecutive brain sections from the hypothalamic area of the brain were prepared for in situ hybridization. The LPS treatment resulted in a significant (p<0.001) pleocytosis and an elevation of the protein content of the CSF. There were no changes observed in the chemical parameters of the CSF, plasma, blood or urine, i.e. vasopressin (VP) levels, osmolality, Na+ and K+ concentrations, glucose level, pH, bicarbonate or PaCO2, PaO2 values. LPS injection, however, resulted in a significantly (p<0.01) increased VP mRNA level (121% of the control value) in the supraoptic nuclei (SON), but not in the paraventricular nuclei (PVN), as compared to controls. Our findings suggest an early effect of LPS on VP gene expression selectively in the SON of 14-days-old rats. This animal model might be suitable for studying the regulation of VP gene expression and the role of this peptide in the pathogenesis of bacterial meningitis in pediatric patients.

Differential distribution and intracellular targeting of mRNAs corresponding to the three calmodulin genes in rat brain. A quantitative in situ hybridization study.

To investigate the pattern of expression of the three calmodulin (CaM) genes by in situ hybridization, gene-specific [35S]-cRNA probes complementary to the multiple CaM mRNAs were hybridized in rat brain sections and subsequently detected by quantitative film or high-resolution nuclear emulsion autoradiography. A widespread and differential area-specific distribution of the CaM mRNAs was detected. The expression patterns corresponding to the three CaM genes differed most considerably in the olfactory bulb, the cerebral and cerebellar cortices, the diagonal band, the suprachiasmatic and medial habenular nuclei, and the hippocampus. Moreover, the significantly higher CaM I and CaM III mRNA copy numbers than that of CaM II in the molecular layers of certain brain areas revealed a differential dendritic targeting of these mRNAs. The results indicate a differential pattern of distribution of the multiple CaM mRNAs at two levels of cellular organization in the brain: (a) region-specific expression and (b) specific intracellular targeting. A precise and gene-specific regulation of synthesis and distribution of CaM mRNAs therefore exists under physiological conditions in the rat brain.

beta-Amyloid[1-40]-induced early hyperpolarization in M26-1F cells, an immortalized rat striatal cell line.

The short-term (20-minute) action of beta[1-40]-amyloid on the resting transmembrane potential was investigated by means of flow-cytofluorimetric studies in M26-1F cells, an immortalized rat striatal cell line, using the potential-sensitive fluorescent probe bis-oxonol. The distribution of the individual cell-associated probe fluorescence was found to be shifted to lower levels in cells treated with beta-amyloid[1-40] for 20 minutes as compared with that of their untreated counterparts. A change in the same direction was caused by valinomycin, a hyperpolarizing ionophore, whereas gramicidin, a depolarizing ionophore, induced a shift to higher fluorescence intensities. These findings, together with the reported behaviour of this particular fluorescent probe at different transmembrane potential levels, indicate that beta-amyloid[1-40] is capable of inducing early hyperpolarization in M26-1F cells. This is one of the earliest cell physiological effect of beta-amyloid peptides that has been reported so far. Moreover, our findings indicate an ionophore-like action of amyloid peptides.

Water deprivation upregulates the three calmodulin genes in exclusively the supraoptic nucleus of the rat brain.

Calmodulin (CaM), the ubiquitous intracellular calcium-binding protein, is coded by three bona fide CaM genes (CaM I, CaM II and CaM III) in mammals. They code for the same protein and are transcribed at particularly high levels in the brain, where CaM plays an essential role in basic neuronal functions. In this study, the expression of the three CaM genes in response to osmotic stimuli by water deprivation was investigated in the rat brain, with particular interest as concerns the hypothalamic magnocellular nuclei. CaM mRNA levels were determined by quantitative in situ hybridization autoradiography with gene-specific [35S]cRNA probes. In response to osmotic challenge, it was found that upregulation of the three CaM genes participates in the activation of the hypothalamo-hypophyseal system in the supraoptic nucleus (SON) (126% to 169%), but not in the magnocellular part of the paraventricular hypothalamic nucleus (PVN) (-10%). CaM mRNA levels decreased by 10%-15% in the suprachiasmatic nucleus (SCh) and many other extrahypothalamic brain areas. The opposite responses of the CaM gene expression in the SON and the magnocellular part of the PVN suggest a functional difference between them. Moreover, the significantly different magnitudes of the changes in the CaM mRNA levels in the SON nucleus (138%, 126% and 169% for CaM I, CaM II and CaM III, respectively) exemplify the precise differential control of the CaM gene expression in the brain.

Slide-binding characterization and autoradiographic localization of delta opioid receptors in rat and mouse brains with the tetrapeptide antagonist [3H]TIPP.

Slide-binding and autoradiographic studies were performed on cryostat sections from brains of adult Sprague-Dawley rats and BALB C mice to describe the binding characteristics of the tetrapeptide [3H]TIPP, an antagonist with high specificity and affinity for the delta opioid receptors. Steady-state binding of [3H]TIPP to cryostat sections of brain paste was reached in 120-180 min of incubation. Specific [3H]TIPP binding resulted in maximal numbers of binding sites (Bmax) of 15.59 and 23.91 fmol/mg protein, and dissociation constants (Kd) of 0.46 and 0.85 nM for rat and mouse brain paste sections, respectively. TIPP displayed the highest affinity for delta opioid receptors in inhibiting specific [3H]TIPP binding, with IC50 values of 0.82 nM and 0.14 nM in rat and mouse brain sections, respectively. While DPDPE was also effective in displacing the specific binding of [3H]TIPP (IC50 = 3.18 +/- 0.53 nM and 0.63 +/- 0.42 nM in rat and mouse brain paste sections, respectively), other subclass-selective or nonopioid ligands were much less effective, or ineffective. Autoradiographic localization of [3H]TIPP binding revealed the characteristic distribution of delta opioid receptors in both species. In consequence of its antagonistic nature, and of its unnatural amino acid residue, which makes this ligand more resistant to biodegradation, [3H]TIPP is a superior ligand for evaluation of the binding characteristics and autoradiogaphic distribution of the delta opioid receptors.

A new quantitative film autoradiographic method of quantifying mRNA transcripts for in situ hybridization.

We developed and tested a novel quantitative method for the quantification of film autoradiographs, involving a mathematical model and a dot-blot-based membrane standard scale. The exponential model introduced here, ROD = p1(1 - exp[p2x]), appropriately (r2>0. 999), describes the relation between relative optical density (ROD) and radioactivity (x) in the range between 0 and 240 gray scale values (using a 256-gray scale level digitizer). By means of this model, standard curves with distinct quenching properties can be exactly interconverted, permitting the tissue-equivalent calibration of different standard scales. The membrane standard scale employed here has several advantages, including the flexible radioactivity range, the facile and rapid preparation technique, and the compact size. The feasibility of the quantification procedure is exemplified by the comparative quantification of multiple calmodulin mRNAs in the rat brain by in situ hybridization with [35S]-cRNA probes. The procedure for quantification provides a significant improvement in that the direct and exact comparison of radiolabeled species, even from different experiments, can be reliably performed. Further, the procedure can be adapted to the quantification of autoradiographs produced by other methods.

Changing the site of initiation of plus-strand DNA synthesis inhibits the subsequent template switch during replication of a hepadnavirus.

Unique to hepadnavirus reverse transcription is the process of primer translocation, in which the RNA primer for the initiation of plus-strand DNA synthesis is generated at one site on its template, DR1, and is moved to a new site, DR2. For duck hepatitis B virus (DHBV), DR2 is located within 50 nucleotides of the 5' end of the minus-strand DNA template. When the synthesis of plus-strand DNA proceeds to the 5' terminus of the minus strand, the 3' end of the minus strand becomes the template for DNA synthesis. This switch in templates circularizes the nascent genome and is required for the genesis of the relaxed circular form of the DNA and the mature capsid. Maturation of the capsid is a prerequisite for virus egress. We have analyzed a series of DHBV variants in which plus-strand DNA synthesis was initiated from a new position relative to the 5' end of the template. For these variants, the subsequent circularization was inhibited. We found that when the number of nucleotides between the site of initiation of plus-strand DNA synthesis and the 5' end of its template was restored to 54 nucleotides, circularization was substantially restored. These results mean that the process of circularization is influenced by the earlier steps in DNA replication. This sensitivity is consistent with the notion that this region of the nascent genome is in a dynamic structure that is crucial for successful DNA replication.

Beta-amyloid(Phe(SO3H)24)25-35 in rat nucleus basalis induces behavioral dysfunctions, impairs learning and memory and disrupts cortical cholinergic innervation.

Long-term behavioral effects, changes in learning and memory functions and aberrations of cholinergic fibers projecting to the parietal cortex were investigated after bilateral injections of beta-amyloid(Phe(SO3H)24)25-35 peptide in rat nucleus basalis magnocellularis (nbm). The beta-amyloid peptide used in these experiments contained the original beta-amyloid 25-35 sequence which was coupled to a phenylalanine-sulphonate group at position 24. This additional residue serves as a protective cap on the molecule without influencing its neurotoxic properties and results in water-solubility, stability and low rates of peptide metabolism. In this paper, home cage, locomotor and open-field activities, passive shock-avoidance and 'Morris' water maze learning abilities were assessed throughout a 35-day survival period. Subsequently, acetylcholinesterase (AChE) histochemistry was used to visualize alterations of parietal cortical cholinergic innervation. In response to the neurotoxic action of beta-amyloid(Phe(SO3H)24)25-35, a progressive hyperactivity developed in the rats in their home cages which were maintained throughout the 5-week post-injection period. This was accompanied by a significant hypoactivity in the novel environment of a locomotor arena. Beta-amyloid(Phe(SO3H)24)25-35-treated animals showed greatly impaired cortical memory functions in the step-through passive shock-avoidance paradigm, while spatial learning processes remained unaffected. Moreover, beta-amyloid(Phe(SO3H)24)25-35 injections in the nucleus basalis suppressed explorative behavior in rats and inhibited conditioned stress responses 28 days after surgery. Reductions of cortical cholinergic (AChE-positive) projections provided anatomical substrate for the behavioral changes. This indicated extensive, long-lasting neurodegenerative processes as a result of beta-amyloid(Phe(SO3H)24)25-35 infusion.