Differential insular cortex subregional vulnerability to α-synuclein pathology in Parkinson's disease and dementia with Lewy bodies.

AIM: The insular cortex consists of a heterogenous cytoarchitecture and diverse connections and is thought to integrate autonomic, cognitive, emotional and interoceptive functions to guide behaviour. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), it reveals α-synuclein pathology in advanced stages. The aim of this study is to assess the insular cortex cellular and subregional vulnerability to α-synuclein pathology in well-characterized PD and DLB subjects. METHODS: We analysed postmortem insular tissue from 24 donors with incidental Lewy body disease, PD, PD with dementia (PDD), DLB and age-matched controls. The load and distribution of α-synuclein pathology and tyrosine hydroxylase (TH) cells were studied throughout the insular subregions. The selective involvement of von Economo neurons (VENs) in the anterior insula and astroglia was assessed in all groups. RESULTS: A decreasing gradient of α-synuclein pathology load from the anterior periallocortical agranular towards the intermediate dysgranular and posterior isocortical granular insular subregions was found. Few VENs revealed α-synuclein inclusions while astroglial synucleinopathy was a predominant feature in PDD and DLB. TH neurons were predominant in the agranular and dysgranular subregions but did not reveal α-synuclein inclusions or significant reduction in density in patient groups. CONCLUSIONS: Our study highlights the vulnerability of the anterior agranular insula to α-synuclein pathology in PD, PDD and DLB. Whereas VENs and astrocytes were affected in advanced disease stages, insular TH neurons were spared. Owing to the anterior insula's affective, cognitive and autonomic functions, its greater vulnerability to pathology indicates a potential contribution to nonmotor deficits in PD and DLB.

Expression of amphetamine sensitization is associated with recruitment of a reactive neuronal population in the nucleus accumbens core.

RATIONALE: Repeated exposure to psychostimulant drugs causes a long-lasting increase in the psychomotor and reinforcing effects of these drugs and an array of neuroadaptations. One such alteration is a hypersensitivity of striatal activity such that a low dose of amphetamine in sensitized animals produces dorsal striatal activation patterns similar to acute treatment with a high dose of amphetamine. OBJECTIVES: To extend previous findings of striatal hypersensitivity with behavioral observations and with cellular activity in the nucleus accumbens and prefrontal cortex in sensitized animals. MATERIALS AND METHODS: Rats treated acutely with 0, 1, 2.5, or 5 mg/kg i.p. amphetamine and sensitized rats challenged with 1 mg/kg i.p. amphetamine were scored for stereotypy, rearing, and grooming, and locomotor activity recorded. c-fos positive nuclei were quantified in the nucleus accumbens and prefrontal cortex after expression of sensitization with 1 mg/kg i.p. amphetamine. RESULTS: Intense stereotypy was seen in animals treated acutely with 5 mg/kg amphetamine, but not in the sensitized group treated with 1 mg/kg amphetamine. The c-fos response to amphetamine in the accumbens core was augmented in amphetamine-pretreated animals with a shift in the distribution of optical density, while no effect of sensitization was seen in the nucleus accumbens shell or prefrontal cortex. CONCLUSIONS: A lack of stereotypy in the sensitized group indicates a dissociation of behavioral responses to amphetamine and striatal immediate-early gene activation patterns. The increase in c-fos positive nuclei and shift in the distribution of optical density observed in the nucleus accumbens core suggests recruitment of a new population of neurons during expression of sensitization.

C-fos activation patterns in rat prefrontal cortex during acquisition of a cued classical conditioning task.

The prefrontal cortex (PFC) is known to be involved in associative learning; however, its specific role in acquisition of cued classical conditioning has not yet been determined. Furthermore, the role of regional differences within the PFC in the acquisition of cued conditioning is not well described. These issues were addressed by exposing rats to either one or four sessions of a cued classical conditioning task, and subsequently examining c-fos immunoreactivity in various areas of the PFC. Differences in patterns of c-fos immunopositive nuclei were found when comparing the PFC areas examined. No significant differences were found between rats presented with a temporally contingent conditioned stimulus (CS) light and food (paired groups) and those presented with the same stimuli temporally non-contingently (unpaired groups). In lateral and orbital PFC, both the paired and unpaired groups showed more c-fos immunopositive nuclei than control groups exposed only to the behavioral setup (context exposed groups), and all groups showed a drop in c-fos immunopositive nuclei from session 1 to session 4. In dorsal medial PFC, no differences were seen between the paired, unpaired and context exposed groups. These groups did, however, differ from naive animals, an effect that was not seen in the ventral medial PFC. The results of this study do not support a role for the PFC in the acquisition of a cued classical conditioning task. The differences seen between paired, unpaired and context exposed groups in orbital and lateral PFC could be due to contextual conditioning or reward-related effects.

Delayed occurrence of enhanced striatal preprodynorphin gene expression in behaviorally sensitized rats: differential long-term effects of intermittent and chronic morphine administration.

Protracted changes in basal "steady-state" opioid peptide gene expression in the brain may represent adaptations underlying the behavioral effects of drugs of abuse, observed long after drug exposure. Here, we have studied the long-term effects of two distinct regimens of morphine administration ("intermittent" vs "chronic" morphine treatment) on behavioral sensitization and "steady-state" striatal preprodynorphin and preproenkephalin gene expression in rats. Opioid peptide gene expression was investigated using in situ hybridization at three rostrocaudal levels (rostral, intermediate and caudal) of the caudate-putamen and the nucleus accumbens. Behavioral studies showed that the intermittent morphine treatment resulted in a significantly greater enhancement of morphine-induced locomotion than the chronic morphine treatment three weeks after cessation of opiate exposure. The intermittent morphine treatment resulted in an initial decrease of preprodynorphin gene expression of about 5-10% in the caudate-putamen and the nucleus accumbens at the rostral and intermediate levels one day after the last morphine administration. In contrast, a protracted increase of preprodynorphin gene expression of about 20% throughout the caudate-putamen and of about 6% in intermediate sections of the nucleus accumbens was observed 21 days after cessation of intermittent morphine treatment. Although the chronic morphine treatment induced a decrease of preprodynorphin messenger RNA levels one day after the last administration, no significant changes were observed three weeks after cessation of chronic morphine treatment. No long-term changes were observed in preproenkephalin gene expression after either morphine treatment. Since the intermittent morphine administration induced long-term behavioral sensitization much more effectively than the chronic morphine treatment, we tentatively suggest that the protracted increase of preprodynorphin gene expression may play a facilitative role in the long-term character of opiate-induced behavioral sensitization.

Morphine-induced increase in D-1 receptor regulated signal transduction in rat striatal neurons and its facilitation by glucocorticoid receptor activation: possible role in behavioral sensitization.

One month (but not 1-3 days) after intermittent morphine administration, the hyperresponsiveness of rats toward the locomotor effects of morphine and amphetamine was associated with an increase in dopamine (DA) D-1 receptor-stimulated adenylyl cyclase activity and enhanced steady state levels of preprodynorphin gene expression in slices of the caudate/putamen and nucleus accumbens. Such an enduring increase in postsynaptic D-1 receptor efficacy also occurred in cultured gamma-aminobutyric acid (GABA) neurons of the striatum obtained from rats prenatally treated with morphine. Interestingly, in vitro glucocorticoid receptor activation in these cultured striatal neurons by corticosterone potentiated this neuroadaptive effect of prior in vivo morphine exposure. Since activation of glucocorticoid receptors by corticosterone did not affect D-1 receptor functioning in cultured neurons of saline-pretreated rats, prior intermittent exposure to morphine (somehow) appears to induce a long-lasting state of corticosterone hyperresponsiveness in striatal neurons. Therefore, DA-sensitive striatal GABA neurons may represent common neuronal substrates acted upon by morphine and corticosterone. We hypothesize that the delayed occurrence of these long-lasting morphine-induced neuroadaptive effects in GABA/dynorphin neurons of the striatum is involved in the enduring nature of behavioral sensitization to drugs of abuse and cross-sensitization to stressors.

Differential localization of mRNAs encoding dopamine D1 or D2 receptors in cholinergic neurons in the core and shell of the rat nucleus accumbens.

By combining immunocytochemistry for ChAT and in situ hybridization for dopamine D1 or D2 receptor mRNA in the striatum, it was found that (1) the percentage of ChAT/D2 mRNA co-localization is higher in the caudate-putamen than in the shell and core of the nucleus accumbens, (2) in the shell the degree of ChAT/D2 mRNA co-localization is higher rostrally than caudally, and 3) no significant regional differences exist in the degree of co-localization of ChAT and D1 mRNA.

Secondary structure of the single-stranded DNA binding protein encoded by filamentous phage Pf3 as determined by NMR.

Nuclear magnetic resonance spectroscopy was employed to study the single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3. The protein is 78 amino acids long and occurs in solution predominantly as a homodimer with a molecular mass of 18 kDa. Sequence-specific 1H and 15N resonance assignments have been obtained using homo- and heteronuclear two- and three-dimensional experiments. The secondary structure of the protein monomer was determined from a qualitative interpretation of nuclear Overhauser enhancement spectra and amide exchange data. It consists of a five-stranded antiparallel beta-sheet and three beta-hairpins. Problems caused by the protein's tendency to aggregate at concentrations needed for NMR spectroscopy were largely overcome by designing a mutant (Phe36-->His) which exhibits significantly improved solubility characteristics over the wild-type protein. It is shown that this mutation only locally affects the structure of the protein; the chemical shifts of the wild-type and mutant species differ only for a few residues near the site of the mutation, and the secondary structures of the proteins are identical. The secondary structure of the Pf3 single-stranded DNA binding protein is compared to that of the Ff gene V protein, the only single-stranded DNA binding protein for which the complete three-dimensional structure is known to date [Folkers, P. J. M., Nilges, M., Folmer, R. H. A., Konings, R. N. H. & Hilbers, C. W. (1994) J. Mol. Biol. 236, 229-246; Skinner, M. M., Zhang, H., Leschnitzer, D. H., Guan, Y., Bellamy, H., Sweet, R. M., Gray, C. W., Konings, R. N. H., Wang, A. H.-J. & Terwilliger, T. C. (1994) Proc. Natl Acad. Sci. USA 91, 2071-2075]. It is found that the secondary structures of the two proteins are very similar which supports the hypothesis that a five-stranded antiparallel beta-sheet with protruding beta-hairpins is a common motif in a certain class of single-stranded DNA binding proteins. In addition, the sequence and folding predicted earlier for the DNA binding wing in the single-stranded DNA binding protein of phage Pf3 [de Jong, E. A. M., van Duynhoven, J. P. M., Harmsen, B. J. M., Tesser, G. I., Konings, R. N. H. & Hilbers, C. W. (1989) J. Mol. Biol. 206, 133-156] is borne out by the present study. It closely resembles that in the single-stranded DNA binding protein of phage Ff, which may indicate that such a wing is a recurrent motif as well.

Differential effects of dopamine depletion on the binding and mRNA levels of dopamine receptors in the shell and core of the rat nucleus accumbens.

In the present study, using quantitative receptor autoradiography and in situ hybridization histochemistry the effects of unilateral 6-hydroxydopamine lesions on the binding density levels of dopamine D1 and D2 receptors and the levels of mRNA encoding D1 and D2 receptors were investigated in the core and shell territories of the nucleus accumbens (Acb) and in the caudate-putamen (CP). The lesions induced contrasting effects on the D1 binding and D1 mRNA in the Acb and CP, i.e. an increase in binding and a decrease in the mRNA levels. For the D2 receptor an increase in both the binding density and mRNA levels was observed. The lesion-induced effects displayed regional differences. For D1 mRNA and D1 and D2 binding, the lesion effect was more pronounced in the core than in the shell of the Acb. For the D2 mRNA levels an increase was observed in the CP but not in the two territories of the Acb. Furthermore, the decrease in D1 mRNA was greater in the rostral than in the caudal parts of the core and shell of the Acb. These results indicate that the core and shell of the Acb and the CP respond differentially to dopamine depletion.

Adaptive changes in rat striatal preproenkephalin expression and dopamine-opioid interactions upon chronic haloperidol treatment during different developmental stages.

This study was designed to evaluate the effects of a chronic treatment with the classical neuroleptic drug haloperidol on the preproenkephalin (ppEnk) mRNA synthesis and its consequences for opioid and dopamine (DA) receptor-regulated adenylate cyclase in the developing and adult rat striatum. Prenatal exposure to haloperidol (2 mg/kg, 14 days) caused a 40% reduction of striatal ppEnk mRNA levels, but had no consequences for DA-stimulated or Met-enkephalin-inhibited adenylate cyclase activity in striatal slices from embryonic day 21 (E21) foetal brain. Postnatal treatment of rat pups from day 10 (P10) until P23 and adult rats resulted in significant increases of mRNA levels of 8 and 41%, respectively, a clear reduction of D1 DA receptor-stimulated cAMP production and a profound desensitization of delta-opioid receptors inhibitory coupled to adenylate cyclase. Since striatal D2 receptor-mediated inhibition of adenylate cyclase activity, in contrast to its activation through D1 receptors, is not present in the prenatal period, this study indicates that the tonic inhibitory effect of DA on striatal ppEnk mRNA synthesis is dependent on the presence of adenylate cyclase-coupled D2 receptors which gradually develops postnatally and further supports the idea that striatal D1 and D2 DA receptors have bidirectional effects on enkephalin synthesis in this brain area. The adaptive changes in D1 DA and delta receptor-regulated adenylate cyclase activity are discussed in relation to the well-known increase in the locomotor and reinforcing effects of mu-opioid receptor agonists upon chronic neuroleptic treatment.

Rostrocaudal subregional differences in the response of enkephalin, dynorphin and substance P synthesis in rat nucleus accumbens to dopamine depletion.

Quantitative in situ hybridization histochemistry was used to examine the effects of unilateral 6-hydroxydopamine lesions of the ascending dopaminergic fibres on levels of mRNA encoding the neuropeptides enkephalin, dynorphin and substance P in subregions of the nucleus accumbens. The nucleus accumbens was divided into quadrants and changes in mRNA were measured along the rostrocaudal extent of the nucleus. Two weeks after the lesion an increase was found in enkephalin mRNA in the lesioned side compared to the non-lesioned side, whereas a decrease was observed for dynorphin and substance P mRNA. The changes in mRNA levels differed from quadrant to quadrant and were not uniformly distributed along the rostrocaudal axis. Both types of changes, i.e. increase and decrease, were much higher in rostral parts of the nucleus than in caudal parts, indicating regional differences in the effects of blockade of the dopaminergic neurotransmission. The lesion-induced increases and decreases in mRNA levels occurred in both the shell and the core subregions of the nucleus accumbens and were not specifically related to either of these areas. Factors are discussed that may contribute to the rostrocaudal gradient in the changes of enkephalin, substance P and dynorphin mRNA levels. On the basis of their afferent and efferent connections, the rostral and caudal parts of the nucleus accumbens are considered to be involved in different functions. The present results suggest that dopamine depletion may affect these functions in a differential manner.

Sequence-specific 1H-NMR assignment and secondary structure of the Tyr41----His mutant of the single-stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13.

Sequence-specific 1H-NMR assignments are reported for the Tyr41----His (Y41H) mutant of the single-stranded DNA binding protein, encoded by gene V of the filamentous bacteriophage M13 (GVP). The mutant protein was chosen for this purpose because it exhibits significantly improved solubility characteristics over wild-type GVP [Folkers et al. (1991) Eur. J. Biochem. 200, 139-148]. The secondary structure elements present in the protein are deduced from a qualitative interpretation of the nuclear Overhauser enhancement spectra and amide exchange data. The protein is entirely composed of antiparallel beta-structure. It is shown that identical structural elements are present in wild-type GVP. Previously, we have demonstrated that the secondary structure of the beta-loop, encompassing residues 13-31 which is present in GVP in solution, deviates from that proposed for the same amino acid sequence on the basis of X-ray diffraction data [van Duynhoven et al. (1990) FEBS Lett. 261, 1-4]. Now that we have arrived at a complete description of the secondary structure of the protein in solution, other deviations with respect to the crystallographically determined structure became apparent as well. The N-terminal part of the protein is, in solution, part of a triple-stranded beta-sheet while, in the crystal, it is an extended strand pointing away from the bulk of the protein dimer. One of the antiparallel beta-sheets in the protein which had been designated earlier as the complex loop has, in the solution structure, a different pairwise arrangement of the residues in its respective beta-ladders. Residues 30 and 48 are opposite to one another in the solution structure while in the crystal structure residues 32 and 48 are paired. A similar observation is made for the so-called dyad domain of the protein of which the beta-sheet in the solution structure is shifted by one residue with respect to that of the crystal structure.

Alpha-MPT does not affect dopamine levels in the periventricular organ of lizards.

Recent studies indicate that the liquor (CSF)-contacting cell bodies in the periventricular organ of non-mammalian vertebrates accumulates rather than synthesizes dopamine. We therefore gave the lizard Gekko gecko injections of the dopamine synthesis inhibitor alpha-methylparatyrosine (alpha-MPT). Brains were fixed 105-240 min after injection. Subsequent staining with dopamine antiserum revealed almost no changes in staining in the periventricular organ but there was a dramatic decrease in the other dopaminergic cell groups of the brain. The data support the notion that the cells in the periventricular organ accumulate dopamine and that the CSF plays an important role in dopamine neurotransmission in non-mammalian vertebrates.

Comparative analysis of vasotocin-like immunoreactivity in the brain of the turtle Pseudemys scripta elegans and the snake Python regius.

The distribution of vasotocin in the brains of the turtle Pseudemys scripta elegans and the snake Python regius was studied with immunohistochemical methods. In both species, vasotocin-immunoreactive (VTi) cells were found in the supraoptic nucleus, the paraventricular nucleus and the bed nucleus of the stria terminalis. No VTi cell bodies were seen in the brainstem. Vasotocinergic fibers were found in all major brain divisions. Intrahypothalamic VTi fibers were observed between the supraoptic and the paraventricular nuclei and in the median eminence. An extensive network of extrahypothalamic VTi fibers extends from the olfactory bulb to the spinal cord. Limbic structures, such as the nucleus accumbens, the septal area and the ventral amygdaloid nucleus, contain a moderate to dense VTi plexus. Other areas with a substantial number of VTi fibers are the lateral habenular nucleus, the ventral tegmental area, the substantia nigra, the locus coeruleus and the nucleus of the solitary tract. Sex-related differences in the density of the VTi fibers were observed in the lateral septal nucleus, the mid-brain periaqueductal gray and, to a lesser extent, in the ventral amygdaloid nucleus, the lateral habenular nucleus, the ventral tegmental area and the substantia nigra. In these areas, the density of VTi fibers is higher in males than in females. The distribution of vasotocin-like immunoreactivity in the brains of Pseudemys and Python resembles the pattern previously observed in the lizard Gekko gecko. However, among the three species several differences exist, the most remarkable one being the variation in number of liquor-contacting VTi cells in the paraventricular nucleus.

Distribution of phenylethanolamine-N-methyltransferase-immunoreactive perikarya and fibers in the brain of the lizard Gekko gecko.

The distribution of phenylethanolamine N-methyltransferase (PNMT)-immunoreactive (PNMTi) cell bodies and fibers in the brain of the lizard Gekko gecko was studied by antibodies raised in rabbits against purified bovine adrenal PNMT. The PNMTi cell bodies were observed in the ventrolateral rhombencephalic tegmentum at the level of the obex. No immunoreactive perikarya were found in the nucleus of the solitary tract, the medial longitudinal fascicle or the hypothalamus. An extensive network of PNMTi fibers is present throughout the brain, extending rostrally as far as the olfactory peduncle. In the telecenphalon, moderate to dense plexuses of PNMTi fibers were observed in the medial part of the nucleus accumbens, the medial septal nucleus, the nucleus of the diagonal band, the caudoventral septal region and the central amygdaloid nucleus. In the diencephalon, the periventricular and lateral zones of the preoptic and hypothalamic areas, the medial forebrain bundle and the dorsomedial thalamic nucleus contain many PNMTi fibers. Brainstem structures innervated by PNMTi fibers are the ventral tegmental area, the substantia nigra, the periaqueductal gray, the locus coeruleus, the parabrachial region, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus and the ventrolateral region of the caudal brainstem. Although the brain of Gekko appears to lack PNMTi cells in areas comparable to the C2 and C3 cell groups in rats, the distribution of PNMTi fibers is nevertheless strikingly similar in both groups.

Efferent connections of the striatum and the nucleus accumbens in the lizard Gekko gecko.

The efferent connections of the striatum and the nucleus accumbens of the lizard Gekko gecko were studied with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). These structures were found to have segregated output systems. The striatum shows a major projection to the globus pallidus. Striatal fibers which are more caudally directed run through the lateral forebrain bundle and can be traced as far caudally as the pars reticularis of the substantia nigra where they exhibit many varicosities. Along its course, the lateral forebrain bundle issues fibers with varicosities to the anterior and posterior entopeduncular nuclei. The major recipient structure of the nucleus accumbens is the ventral pallidum. The nucleus accumbens, in addition, projects to the portion of the lateral hypothalamus in the path of the medial forebrain bundle and to the ventral tegmental area, which is its most caudal target. Subsequently, the same technique was used in an attempt to study the efferents of the globus pallidus and the ventral pallidum, the major recipient structures of the striatum and the nucleus accumbens. The globus pallidus was found to project to the rostral part of the suprapeduncular nucleus in the ventral thalamus and, in addition, may distribute fibers to the same structures as does the striatum. The ventral pallidum distributes fibers to the ventromedial thalamic nucleus. It probably also projects diffusely to the hypothalamus, the habenula, and the mesencephalic tegmentum.

Distribution of dopamine in the forebrain and midbrain of the red-eared turtle, Pseudemys scripta elegans, reinvestigated using antibodies against dopamine.

The distribution of dopamine (DA) immunoreactivity in the forebrain and midbrain of the red-eared turtle, Pseudemys scripta elegans, was studied using recently developed antibodies against DA. DA-containing cells were found around the glomeruli of the olfactory bulb but not in the telencephalon proper. In the diencephalon DA cells were observed in the preoptic region, several parts of the periventricular hypothalamic nucleus, the periventricular organ, the ependymal wall of the infundibular recess, the lateral hypothalamic area and the pretectal posterodorsal nucleus. In the midbrain DA cells were found in the ventral tegmental area, the substantia nigra and the presumed reptilian homologue of the mammalian A8 cell group. Dopaminergic fibers and terminals were observed throughout the whole brain, particularly in the telencephalon and diencephalon. The olfactory tubercle, the striatum and the nucleus accumbens appear to have the most dense innervation, but the anterior olfactory nucleus and the septal area also show numerous DA fibers and terminals. Cortical areas are in general not densely innervated by DA fibers. Compared to the results obtained for a lizard, Gekko gecko, with the same antibodies, the results of the present study are very similar as regards the distribution of DA neurons, fibers and terminals. In having better developed DA cell groups in the midbrain and a stronger innervation of the striatum, Pseudemys resembles mammals more than does Gekko. In contrast, the many cerebrospinal fluid-contacting DA neurons in the hypothalamus of Pseudemys are a primitive feature of the diencephalon. The previous immunohistochemical study of Gekko, a lizard, and the present account of Pseudemys, a turtle, indicate that at least two different lines of evolution exist within the reptiles with regard to the DA innervation of the dorsal ventricular ridge. One, including turtles and, probably, crocodilians with a weak DA innervation; and another, represented by lizards, with a strong DA immunoreactivity.