Abstract: Mutations in the PARK2 gene encoding parkin cause autosomal recessive juvenile parkinsonism, but have also been found in patients diagnosed with certain tauopathies. Conversely, mutations in the MAPT gene encoding tau are present in some types of parkinsonism. In order to investigate the possible relationship between these two proteins, we generated a double mutant mouse that is deficient in PARK2 and that over-expresses the hTauVLW transgene, a mutant form of the tau protein present in FTDP-17. Independent deletion of PARK2 or over-expression of the hTauVLW transgene produces mild phenotypic alterations, while a substantial increase in parkin expression is observed in hTauVLW transgenic mice. However, double mutant mice present memory and exploratory deficits, and accumulation of PHF-1 and AT8 hyperphosphorylated tau epitopes in neurons. These phenomena are coupled with reactive astrocytosis, DNA fragmentation, and variable cerebral atrophy. Here, we show that cortical and hippocampal neurons of double mutant mice develop argyrophilic Gallyas-Braak aggregates of phosphorylated tau from 3 months of age. Their number decreases in old animals. Moreover, numerous phosphorylated tau aggregates were identified with the conformation-dependent Alz-50 antibody and the S-Thioflavin staining. Ventral motor nuclei of the spinal cord also present Alz-50, AT8, and PHF1 hyperphosphorylated tau aggregates when parkin is deleted in mice over-expressing the hTauVLW transgene, begining at early ages. Thus, the combination of PARK2 gene deletion with hTauVLW over-expression in mice produces abnormal hyperphosphorylated tau aggregates, similar to those observed in the brain of patients diagnosed with certain tauopathies. In the light of these changes, these mice may help to understand the molecular processes responsible for these diseases, and they may aid the development of new therapeutic strategies to treat neurodegenerative diseases related to tau and parkin proteins.

Abstract: While mutations in the Park-2 gene are the most frequent cause of autosomal-recessive juvenile parkinsonism (AR-JP), they are also present in several forms of tauopathies. Conversely, in some forms of parkinsonism, mutations in the tau gene have also been observed. Deletion of the Park-2 gene and over-expression of mutant tau independently produce mild brain alterations in mice. However, the presence of both mutations simultaneously causes a tau neuropathology, involving reactive astrocytosis, neuron loss in the cortex and hippocampus, and lesions in nigrostriatal and motor neurons. Furthermore, mutant tau over-expression in mice produces important memory impairment. When "parkin" function was abolished in young tau transgenic mice, the memory alterations were exaggerated. Moreover, additional exploratory and motor deficits were observed in older mice, causing the memory alterations to be underestimated. Thus, while memory deficits are more severe in young mice they were somehow attenuated by exploratory impairments in ageing mutants. This double mutant animal will serve as a useful experimental tool to investigate the abnormal processing of hyperphosphorylated tau and its relationship to the development of the cognitive deficits associated with certain neurodegenerative diseases.

Abstract: Mutations, haplotypes, and polymorphisms of tau and Park-2 genes constitute risk factors for developing tauopathies. In order to analyze the possible relationship between parkin and tau we generated a double-mutant mouse deficient for Park-2 expression and overexpressing a mutant tau protein (hTauVLW). Mice develop normally, although the median survival rate is considerably reduced with respect to wild type (45%). Aggregates of phosphorylated tau in neurons and reactive gliosis are quite abundant in cortex and hippocampus of these mice. Moreover, while in young transgenic mice the hTauVLW immunostained transgene product is observed in both cell bodies and dendrites, the hTauVLW mutant protein is only detected in the neuronal cell bodies when Park-2 gene is additionally deleted. Moreover, DNA fragmentation was detected by the TUNEL method, and cerebral atrophy is also present in these regions. The levels of phosphorylated tau and Hsp70 are increased in the double-mutant mice, while CHIP expression in hippocampus is lower when the Park-2 gene is deleted. Thus, the combination of Park-2 gene deletion with hTauVLW transgene overexpression in mice produces serious neuropathological effects, which reflect the existence of some relationship between both proteins.

Abstract: Independent deletion of the PARK2 gene and hTauVLW over-expression in mice produce mild alterations in the brain. However, the presence of both mutations in a parkin-deficient and hTauVLW double mutant mouse causes a tau neuropathology, reactive astrocytosis, and neuronal loss in the cortex and hippocampus, as well as lesions in nigrostriatal and motor neurons. Moreover, these mutants display some memory and exploratory defects that reflect a functional link between parkin and tau proteins. We have tested the motor activity and coordination of these double mutant mice to determine the effects of parkin deletion in mice over-expressing the hTauVLW transgene. While the loss of parkin alone produces increased exploration and alterations in gait and motor coordination, in hTauVLW transgenic mice the absence of parkin causes less prominent motor impairments. These effects suggest the existence of some compensatory mechanisms that are activated when the hTauVLW transgene is over-expressed in the absence of parkin. This mouse model will hopefully help to study the causes of the motor deficits associated with certain neuropathologies related to the tau and parkin proteins, and to find appropriate treatments.

Abstract: Abnormal deposition of protein tau takes place in the brain of patients with several neurodegenerative diseases. Few of these patients present frontotemporal dementia with parkinsonism and amyotrophy (FTDPA-17), an autosomal dominant tauopathy related to mutations of the gene that codes for protein tau, localized in chromosome 17. The great majority of patients with tauopathies such as Alzheimer's disease, sporadic frontotemporal dementia or progressive supranuclear palsy do not show a Mendelian pattern of inheritance. We have occasionally seen tauopathies in patients with parkin mutations and, therefore, hypothesized that the protein tau interacts with parkin. We have tested that hypothesis in mice with combined genetic modifications of tau (over-expression of human tau with three mutations known to produce FTDPA-17) and parkin (deleted) proteins. Homozygote parkin null or over-expressing mutated-human tau mice have subtle behavioral and molecular abnormalities but do not express a clinical phenotype of neurodegenerative disease. Mice with combined homozygous mutations of these two genes show progressively abnormal walking already noticeable at 3 months of age, loss of dopamine and dopamine markers in striatum, nuclear tau immunoreactive deposits in motor neurons of the spinal cord, abnormal expression of glial markers and enhanced levels of pro-apoptotic proteins; findings that were absent or less pronounced in homozygote animals with deletions of parkin or over-expression of tau. The double transgenic mice do not express normal mechanisms of adaptation to stress such as increased levels of GSH and Hsp-70. In addition, they have reduced levels of CHIP-Hsc70, a complex known to attenuate aggregation of tau and to enhance ubiquitination of phosphorylated tau. We have found high levels of phosphorylated tau in parkin-/-+tau(VLW) mice and a relative decrease of the inactivated pSer9 to total GSK-3 levels. Our data reveal that there are interactions between tau and parkin that could be relevant for the pathogenesis and treatment of tauopathies. Similarly, we hope that the double transgenic parkin-/-+tau(VLW) mice could be useful for testing of compounds with putative therapeutic value in human tauopathies.

Abstract: Cinnarizine, a calcium antagonist that produces parkinsonism in humans, induces behavioural changes such as alopecia, buco-lingual dyskinesia and reduction of motor activity in female parkin knock out (PK-KO) mice but not in wild-type (WT) controls. PK-KO mice have high striatal dopamine levels and increased dopamine metabolism in spite of low reduced tyrosine hydroxylase protein. Cinnarizine, which blocks dopamine receptors and increases dopamine release, further increased dopamine metabolism. PK-KO mice increased GSH levels as a compensatory mechanism against enhanced free radical production related to acceleration of dopamine turnover. Neuronal markers, such as beta-tubulin slightly increased in PK-KO and furthermore with cinnarizine. Astroglial markers were decreased in PK-KO mice, and this effect was potentiated by cinnarizine, suggesting abnormal glia in these animals. Microglia was hyperactivated in PK-KO midbrain, suggesting inflammation in these animals. Proapoptotic proteins were increased by cinnarizine and, to a lesser extent, in PK-KO mice. Our data indicate that mutation of parkin is a risk factor for drug-induced parkinsonism.

Abstract: BACKGROUND: Progressive supranuclear palsy (PSP) is a clinicopathological syndrome related to tau deposits and in linkage disequilibrium with tau polymorphisms. Some rare familial PSP cases have been related to tau gene mutations. OBJECTIVE: To present the clinical, pathological, and molecular data of one family with early-onset autosomal dominant PSP. DESIGN: We performed clinical examinations, quantitative neurological tests, positron emission tomographic scans with fluorodopa F 18 and raclopride C 11, analysis of tau mutations, neuropathological examinations, and protein analyses on brain specimens. RESULTS: Three family members had PSP confirmed by pathological features in the proband. A novel mutation of tau, G303V, was found in the proband and other family members. tau Isoforms with 4 microtubule-binding repeats were overexpressed in the proband brain. CONCLUSIONS: The G303V mutation of tau is associated with autosomal dominant PSP. Expression of 4 microtubule-binding repeat tau isoforms is increased in the proband.

Abstract: Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism (EO-AR). Here we show that inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection.

Abstract: Neurotrophic factors (NFs) are proteins that enhance neuronal survival, differentiation, neurotransmitter function and resistance to neurotoxins and lesions. For these reasons the NFs are considered as a new potential therapeutic tool for the treatment of neurodegenerative disorders, a group of diseases that produce the most important cause for disability in the Western world. Some NFs prevent or even reverse the behavioral, biochemical, pharmacological and histological abnormalities observed in several in vitro and in vivo models of neurodegenerative disorders, namely Parkinson's disease. Several NFs have been investigated in primate models of neurological disorders and some of them have been used for patients with these diseases. The results so far obtained in humans have been disappointing for several reasons, including technical problems for delivery, unbearable side effects or lack of efficacy. Future approaches for the use of NFs in humans should include the following: (1) Investigation of the putative compounds in animal models more related to the pathophysiology of each disease, such as in genetic models of neurodegenerative diseases; (2) New methods of delivery including genetic engineering by viral vectors and administration through implantable devices; (3) More precise methods of continuous response evaluation, including the novel neuroimaging techniques; (4) Investigation of the effects of behavioral stimulation and conventional pharmacotherapy on the metabolism of NFs.

Abstract: Steele-Richardson-Olszewski syndrome (SROS) is a neurodegenerative disorder of unknown aetiology, most frequently sporadic. Familial cases of SROS have been described. An intronic polymorphism of the tau gene is associated with sporadic SROS and mutations of the tau gene are present in atypical cases of SROS. The role of tau has been excluded in other families with pathology proven SROS, suggesting that this syndrome may have multiple causes. An 82-year-old patient, father of 3 children with autosomal recessive juvenile parkinsonism due to combined heterozygous mutations of the parkin gene, developed clinical features of SROS 2 years before death. The diagnosis was confirmed by pathology. He carried the C212Y mutation of the parkin gene and was homozygous for the A0 polymorphism and for the H1 haplotype. The role of parkin in the processing of tau is discussed.

Abstract: Autosomal recessive-juvenile parkinsonism (AR-JP) is one of the most common forms of familial Parkinson's disease (PD) and is related to mutations in the Park-2 gene, encoding for a protein ligase of ubiquitin, parkin. Different mutations located along the parkin gene have been observed in different AR-JP affected families, possibly interfering with the normal function of parkin and the proteasome system. Two cases of patients with AR-JP have been recently described presenting different homo- and heterozygous parkin mutations and limited tau pathology. We report here the case of a patient with clinical and pathological findings compatible with progressive supranuclear palsy (PSP), carrier of a single, heterozygous mutation of the parkin gene, and homozygous for the H1/H1 haplotype in the tau gene. Abnormal tau hyperphosphorylation has been observed in our patient brain samples, suggesting that a partial deficit of parkin, a protein with ubiquitin-ligase function, may trigger tau pathology in individuals with molecular genetic risk factors.

Abstract: INTRODUCTION: Microtubules are the essential components of the cytoskeleton, they are responsible for the formation and maintenance of the neuronal morphology and their specific connections. The microtubule associated proteins (MAPs) contribute to regulate the dynamism and stability of the microtubules, and therefore they are essential to maintain the correct function of the microtubules. Among them, tau is a protein that seems to be crucial in stabilizing the neuronal polarity. DEVELOPMENT: In this paper, factors affecting the affinity of tau to bind microtubules are reviewed, giving special attention to the processes that take place in the neurodegenerative diseases that present neurofibrillary tangles (NFTs), aggregates composed of modified tau in form of paired helical filaments (PHFs). One of the most important tau modification in this aberrant aggregates is the hyperphosphorylation. Thus, kinases and phosphatases responsible for tau modification could be altered in certain pathologies, leading to a decrease in the affinity of tau to bind microtubules and carrying out its self assembling and aberrant aggregation in the neurons of the affected nervous system regions. Those pathologies presenting a tau disfunction are known as tauopathies.

Abstract: Microtubule-associated protein 1B (MAP1B) has been implicated in axogenesis in cultured cells. To gain insight into the functions that MAP1B plays in vivo, we analyzed a strain of Map1B mutant mice generated by a gene trapping approach. Homozygous mice die on the first day after birth, probably due to a severe abnormal development of the nervous system. They present alterations in the structure of several brain regions. The normal Map1B gene yields different protein isoforms from alternatively spliced transcripts. The smaller isoforms were present in wild type, hetero-, and homozygous mice, but their expression was higher in the mutants than in the wild-type. Moreover, trace amounts of MAP1B protein were also observed in Map1B homozygous mutants, indicating an alternative splicing around the gene trap insertion. Thus, the Map1B gene trapped mutation reported in this work did not generated a null mutant, but a mouse with a drastic deficiency in MAP1B expression. Analyses of these mice indicate the presence of several neural defects and suggest the participation of MAP1B in neuronal migration.

Abstract: The Eph family of receptors, the largest subgroup within the tyrosine protein kinase receptor family, are comprised of at least thirteen members, many of which are predominantly expressed in the developing and adult nervous system. In this study, we have isolated a full-length cDNA, encoding the mouse homologue of a previous partially characterized Eek protein, a member of Eph receptor tyrosine kinase family. In a comparison of the amino acid sequences of various Eph family members, Eek is most similar to Ehk-3/MDK1, Sek/Cek8, Ehk-2, Hek/Mek4/Cek4, and Bsk/Ehk1/Rek7/Cek7, which are predominantly expressed in the nervous system. Additionally, we have used a low-stringency PCR cloning technique to identify ligands, related to B61, that may interact with Eek. Three different GPI-linked ligands, namely Elf-1/Cek7-L, Ehk1-L/Efl-2/Lerk3 and AL-1/RAGS, were isolated from mouse brain. To study the functional interactions between these ligands and the Eek receptors, we have constructed chimeric ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. These chimeric ligands bound to, and activated both the Eek receptors and the Eek-TrkB chimeric receptors expressed in NIH3T3 cells. These findings suggest that Eek receptor can be activated by at least three different GPI-linked ligands.

Abstract: Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for dopaminergic neurons and motor neurons in culture. It also protects these neurons from degeneration in vitro, and improves symptoms like Parkinson's disease induced pharmacologically in rodents and monkeys. Thus GDNF might have beneficial effects in the treatment of Parkinson's disease and amyotrophic lateral sclerosis. To examine the physiological role of GDNF in the development of the mammalian nervous system, we have generated mice defective in GDNF expression by using homologous recombination in embryonic stem cells to delete each of its two coding exons. GDNF-null mice, regardless of their targeted mutation, display complete renal agencies owing to lack of induction of the ureteric bud, an early step in kidney development. These mice also have no enteric neurons, which probably explains the observed pyloric stenosis and dilation of their duodenum. However, ablation of the GDNF gene does not affect the differentiation and survival of dopaminergic neurons, at least during embryonic development.

Abstract: Tyrosine protein kinases are likely to play an important role in the maintenance and/or development of the nervous system. In this study we have used the PCR cloning technique to isolate sequences derived from tyrosine kinase genes expressed in cultured hippocampal neurons obtained from 17.5-day-old rat embryos. Nucleotide sequence analysis of 209 independent clones revealed sequences derived from 25 tyrosine kinases, of which two corresponded to previously unreported genes. One of the PCR clones, ptk-2, belongs to the Jak family of cytoplasmic tyrosine kinases. The second clone, ptk-3, was derived from a gene encoding an additional class of tyrosine kinase receptors whose extracellular domains contain regions of homology with coagulation factors V and VIII and complement component C1. Transcripts encoding the Ptk-3 receptor are present in a variety of embryonic and adult tissues with highest levels observed in brain. During development, ptk-3 transcripts are most abundant in the proliferative neuroepithelial cells of the ventricular zone, raising the possibility that this receptor may play an important role in the generation of the mammalian nervous system.

Abstract: The distribution of calbindin immunoreactivity was studied in the developing rat dorsal thalamus at embryonic days 14, 16, 18 and 20. At early stages (days 14-16), calbindin is expressed throughout the dorsal thalamic cell mass. Most intense labeling occurs in cells adjacent to the ventricular surface, in a spatial gradient reflecting the well-known outside-in generation pattern. Between days 16 and 20, calbindin-positive periventricular cells are redistributed in the dorsal thalamus according to two different patterns. They first become oriented tangentially within the periventricular layer, and diminish in number at the central locus where midline thalamic fusion occurs at 18 days. Periventricular calbindin immunoreactivity becomes restricted to a ring of late-born cells surrounding the gray commissure. Recognizable portions of this ring-shaped primordium will mature forming n.paratenialis, n.reuniens, n.paraventricularis, and n.subparafascicularis magnocellularis. Simultaneously, a massive contingent of radially-oriented, fusiform, calbindin-positive young neurons extends from the periventricular ring-shaped aggregate to the lateral brain surface at the caudoventral pole of the dorsal thalamus at embryonic days 17/18. These cells surround the primordium of the medial geniculate body, participating in the constitution of its marginal zone, and invade the lateral posterior nucleus, accumulating within its caudomedial part. Other portions of this stream form the parvocellular subparafascicular nucleus and the peripeduncular nucleus. The observed patterns of calbindin expression suggest that dorsal thalamic postmitotic neurons transiently express the marker during initial phases of axogenesis, whereas a specific, late-born population expresses calbindin continuously into adulthood. This late subpopulation displays migratory behavior, and finally subdivides into several nuclei of the mature midline, superficial and posterior thalamus.

Abstract: Immunocytochemical techniques were used to analyze the distribution of the calcium-binding proteins calbindin and parvalbumin during the pre- and postnatal development of the rat somatosensory cortex. Calbindin occurs in most early differentiated neurons that form the primordial plexiform layer at embryonic day 14. This expression in transient; during the perinatal period, calbindin becomes immunologically undetectable within the structures derived from the primordial plexiform layer, i.e., the prospective layers I and VIb. Immunoreactive neurons are also absent from adult layers I and VIb. Calbindin is also detected in a second population of neurons which, from embryonic day 18 onwards, distributes diffusely within the cortical plate. Some neurons of this population show morphological traits of immaturity, while others show complete dendritic arborization. The definitive pattern of distribution of calbindin-immunoreactive neurons is achieved by postnatal day 22. Infragranular layers contain intensely-immunoreactive cells whose numerical density decreases during postnatal development, whereas in supragranular layers similar neurons are interspersed among numerous faintly-stained neurons. Parvalbumin is detected for the first time at postnatal day 6, within a small group of neurons located in cortical layer V, and extends afterwards through the whole thickness of the cerebral cortex. At this same postnatal stage, groups of immunoreactive puncta are also found in layer IV of the somatosensory cortex; these puncta increase in density progressively and, at embryonic day 13, immunoreactive cells appear also grouped at this level. At this postnatal age, parvalbumin immunostaining delineates the somatosensory map in cortical layer IV. From this stage to adulthood, the number of immunoreactive neurons increases in the whole thickness of the somatosensory cortex. Barrels in layer IV become less distinct as immunoreactive cells and processes invade the septa. Layer IV in the adult somatosensory cortex appears more densely populated by parvalbumin immunoreactive neurons and puncta than in the surrounding areas.

Abstract: The distribution of the GABAA receptor/benzodiazepine receptor/chloride channel complex was investigated in the thalamus of the rat by means of immunohistochemistry in adulthood, as well as during embryonic and postnatal development, using a monoclonal antibody. In adults, the immunoreactivity for the GABAA receptor complex was intensely expressed by neuronal processes throughout the dorsal thalamus. Neuronal perikaryal membranes were frequently outlined by punctate immunostaining; cell bodies, intrathalamic fibre bundles and the internal capsule did not display immunoreactivity for the GABAA receptor. Regional differences in the expression of the receptor were consistently observed: the immunostaining was much lighter in the thalamic reticular nucleus than in the dorsal thalamic nuclei and, among the latter, the anteroventral nucleus and the ventral nuclear complex displayed the most intense immunopositivity. Immunostaining for the GABAA receptor was already expressed in embryos at E14, and was homogeneously distributed throughout the neuropil of the dorsal and ventral thalamic primordia. During the first two postnatal weeks, a regional differentiation of the immunopositivity was appreciable in the thalamus, with a progressive reduction in the reticular nucleus and a parallel increase in the dorsal thalamic structures. Immunoreactive neuronal perikarya were not observed in the thalamus at any developmental stage. The expression of the GABAA receptor complex appeared to have reached a mature configuration by the end of the third postnatal week. These findings indicate that in adults the GABAA receptor is differentially expressed by thalamic nuclear structures, including the reticular nucleus. Furthermore, the maturation of the receptor in the thalamus undergoes a rearrangement during the first postnatal weeks that results in a considerable regression within the reticular nucleus.

Abstract: A monoclonal antibody against benzodiazepines (21-7F9) was used to study the distribution of benzodiazepine-like immunoreactivity in the rat brain. Immunodensitometry in combination with image analysis were used for quantification. The results showed a ubiquitous distribution of benzodiazepine-like immunoreactivity throughout the brain. Very high levels of benzodiazepine-like immunoreactivity were found in the Purkinje cell layer of the cerebellum, in the primary olfactory cortex, in the stratum pyramidale of the hippocampus and in the mitral cell layer of the olfactory bulb. High densities of benzodiazepine-like immunoreactivity were found in the granule cell layer of the cerebellum, the pyramidal cell layer of the olfactory tubercle, the granule layer of the dentate gyrus, the arcuate nucleus of the hypothalamus, the mammillary bodies, the interstitial nucleus of Cajal and superficial grey layer of superior colliculus. The substantia nigra pars compacta, the islands of Calleja and layers II, III, V and VI of the cerebral cortex had moderate levels of benzodiazepine-like immunoreactivity. Lower densities were found in the internal granular layer and the external plexiform layer of the olfactory bulb, in the molecular layer of the dentate gyrus, in layers I and IV of the cerebral cortex, in the nucleus caudate-putamen and most of the thalamic nuclei. The lowest density of immunoreactivity was found in the globus pallidus, and the strata radiatum, oriens and lacunosum-moleculare of the hippocampus. The distribution of endogenous benzodiazepine-like immunoreactivity was compared with the distribution of the GABA/benzodiazepine receptor by using both immunocytochemistry and receptor autoradiography. Our studies have shown a clear mismatch between the localization of the benzodiazepine-like immunoreactivity and the GABA/benzodiazepine receptors.

Abstract: The maturation of the calcium binding proteins calbindin-D28k (CB) and parvalbumin (PV) during the first 3 postnatal weeks was studied in the rat thalamus using immunohistochemistry. These two proteins display a non-homogeneous distribution in the adult thalamus. In the rat, CB is mainly localized in the neurons and neuropil of the thalamic midline, intralaminar, and ventromedial nuclei, as well as in the posterior complex. At birth, CB-immunoreactive cell bodies were evident in thalamic midline structures, and especially in the nucleus reuniens. The number of thalamic CB-positive cell bodies, as well as the intensity of the neuropil immunostaining, increased progressively in the first postnatal weeks. This quantitative increase was first apparent in the midline structures and then in the other thalamic territories which are CB-positive in adulthood, and followed a mediolateral gradient. The mature pattern was achieved by the end of the third postnatal week. In the adult rat thalamus the neurons of the reticular nucleus display PV-immunostaining and PV-positive fibers densely innervate most of the dorsal thalamic domains. PV-immunoreactivity was clearly evident at birth in the cell bodies of the reticular nucleus. The density of PV-containing fibers increased progressively after birth in the dorsal thalamus, with a lateromedial gradient. At the end of the third postnatal week the ventroposterior (VP) complex appeared heavily innervated by PV-positive fibers, whose density in more medial structures was still lower than in the adult thalamus. A transient hyperinnervation of PV-immunoreactive fibers, displaying a dishomogenous organization in distinct segments, was observed in VP, and especially in the ventroposteromedial nucleus, during the second postnatal week. Altogether these findings indicate that the maturation of CB and PV requires postnatally a relatively prolonged period of time. The possible involvement of these proteins in different functional aspects of thalamic neuronal maturation is discussed.

Abstract: The ontogenesis of cells showing GABA-like immunoreactivity, and the distribution of the immunoreactivity for the GABAA receptor were studied immunocytochemically in the prenatal rat brain. By embryonic day 14, a few GABA-like immunoreactive (GABA-positive) cells scattered at the subpial limit of the marginal zone (primordial plexiform layer) in the lateral part of the developing cortex. GABA-positive cells appeared progressively within the dorsal and medial sectors of the primordial plexiform layer, occupying deeper positions within the layer. The immunoreactivity for the GABAA receptor covered the whole thickness of the primordial plexiform layer. By embryonic day 16, most GABA-positive cells populated three distinct laminar compartments of the developing cortex: the prospective lamina I, the subplate, and the lower part of the intermediate zone. The GABA-positive cells of the lower intermediate zone appeared to be typical of the developing cerebral cortex of the rat: their neuronal nature was assessed immunocytochemically, using monoclonal antibodies against microtubule-associated protein 2, mainly expressed in neuronal somata and dendrites, and against intermediate filament protein vimentin, expressed in glia. The lower intermediate zone contained cells immunoreactive for microtubule-associated protein 2, although the immunostaining was less intense than in the prospective lamina I and the subplate. Preliminary results showed no vimentin-positive cells in the lower intermediate zone. At embryonic day 16, immunoreactivity for the GABAA receptor was present within the prospective lamina 1 and the subplate. Preliminary results showed no vimentin-positive cell in the lower intermediate zone. At embryonic day 16, immunoreactivity for the GABAA receptor was present within the prospective lamina 1 and the subplate, but not in the lower intermediate zone. From embryonic day 18 onwards, the immunostaining for the GABAA receptor labelled, unambiguously, the subplate as a lamina clearly separated from the suprajacent cortical plate. At embryonic day 18, the GABAA receptor started to be expressed within the lower, differentiating part of the cortical plate. Within the cortical plate, the expression of GABA in neural cell perikarya, and the immunostaining for the GABAA receptor, followed a similar spatio-temporal ("inside-out") gradient during pre- and early postnatal stages. Most GABA-positive cells of the lower intermediate zone started to disappear (or stopped the expression of GABA) by embryonic day 20, but some remained until adulthood. A similar time-course was observed for the microtubule-associated protein 2-immunoreactive cell population located at the same level.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: The distribution of substance P receptors was examined by autoradiography at all levels of the human postmortem spinal cord using the ligand [125I]Bolton-Hunter substance P. Adjacent sections were used to localize substance P-like immunoreactivity by a radioimmunohistochemical technique. In the control spinal cord substance P-like immunoreactivity was found to be highly concentrated in the superficial layers of the dorsal horn, intermediolateral cell columns and lamina X, while lower levels of immunoreactivity were observed in other areas of the grey matter of the spinal cord. In contrast, high densities of substance P binding sites were localized not only to the substantia gelatinosa of the dorsal horn but also to other regions of the grey matter of the spinal cord, particularly in the area of the preganglionic sympathetic neurons in the intermediolateral cell column and in the region of the somatic motor neurons of the ventral horn. In 5 cases of amyotrophic lateral sclerosis we found a marked reduction of substance P binding, especially in the ventral horn associated with the loss of motor neurons. These results suggest a postsynaptic localization of substance P receptors to the motor neurons of the ventral horn in the human spinal cord and a role for substance P in the function of motor neurons.