Focused ultrasound delivery of a selective TrkA agonist rescues cholinergic function in a mouse model of Alzheimer's disease.

The degeneration of cholinergic neurons is a prominent feature of Alzheimer's disease (AD). In animal models of injury and aging, nerve growth factor (NGF) enhances cholinergic cell survival and function, contributing to improved memory. In the presence of AD pathology, however, NGF-related therapeutics have yet to fulfill their regenerative potential. We propose that stimulating the TrkA receptor, without p75NTR activation, is key for therapeutic efficacy. Supporting this hypothesis, the selective TrkA agonist D3 rescued neurotrophin signaling in TgCRND8 mice, whereas NGF, interacting with both TrkA and p75NTR, did not. D3, delivered intravenously and noninvasively to the basal forebrain using MRI-guided focused ultrasound (MRIgFUS)-mediated blood-brain barrier (BBB) permeability activated TrkA-related signaling cascades and enhanced cholinergic neurotransmission. Recent clinical trials support the safety and feasibility of MRIgFUS BBB modulation in AD patients. Neuroprotective agents targeting TrkA, combined with MRIgFUS BBB modulation, represent a promising strategy to counter neurodegeneration in AD.

P53 regulates disruption of neuronal development in the adult hippocampus after irradiation.

Inhibition of hippocampal neurogenesis is implicated in neurocognitive dysfunction after cranial irradiation for brain tumors. How irradiation results in impaired neuronal development remains poorly understood. The Trp53 (p53) gene is known to regulate cellular DNA damage response after irradiation. Whether it has a role in disruption of late neuronal development remains unknown. Here we characterized the effects of p53 on neuronal development in adult mouse hippocampus after irradiation. Different bromodeoxyuridine incorporation paradigms and a transplantation study were used for cell fate mapping. Compared with wild-type mice, we observed profound inhibition of hippocampal neurogenesis after irradiation in mice deficient in p53 despite the absence of acute apoptosis of neuroblasts. The putative neural stem cells were apoptosis resistant after irradiation regardless of p53 genotype. Cell fate mapping using different bromodeoxyuridine incorporation paradigms revealed enhanced activation of neural stem cells and their consequential exhaustion in the absence of p53 after irradiation. Both p53-knockout and wild-type mice demonstrated similar extent of microglial activation in the hippocampus after irradiation. Impairment of neuronal differentiation of neural progenitors transplanted in irradiated hippocampus was not altered by p53 genotype of the recipient mice. We conclude that by inhibiting neural progenitor activation, p53 serves to mitigate disruption of neuronal development after irradiation independent of apoptosis and perturbation of the neural stem cell niche. These findings suggest for the first time that p53 may have a key role in late effects in brain after irradiation.

Gene delivery to the spinal cord using MRI-guided focused ultrasound.

Non-invasive gene delivery across the blood-spinal cord barrier (BSCB) remains a challenge for treatment of spinal cord injury and disease. Here, we demonstrate the use of magnetic resonance image-guided focused ultrasound (MRIgFUS) to mediate non-surgical gene delivery to the spinal cord using self-complementary adeno-associated virus serotype 9 (scAAV9). scAAV9 encoding green fluorescent protein (GFP) was injected intravenously in rats at three dosages: 4 × 10(8), 2 × 10(9) and 7 × 10(9) vector genomes per gram (VG g(-1)). MRIgFUS allowed for transient, targeted permeabilization of the BSCB through the interaction of focused ultrasound (FUS) with systemically injected Definity lipid-shelled microbubbles. Viral delivery at 2 × 10(9) and 7 × 10(9) VG g(-1) leads to robust GFP expression in FUS-targeted regions of the spinal cord. At a dose of 2 × 10(9) VG g(-1), GFP expression was found in 36% of oligodendrocytes, and in 87% of neurons in FUS-treated areas. FUS applications to the spinal cord could address a long-term goal of gene therapy: delivering vectors from the circulation to diseased areas in a non-invasive manner.

Proliferation, differentiation and amyloid-ß production in neural progenitor cells isolated from TgCRND8 mice.

The amyloid precursor protein (APP) and amyloid-ß (Aß) peptide play central roles in the pathology and etiology of Alzheimer's disease. Amyloid-induced impairments in neurogenesis have been investigated in several transgenic mouse models but the mechanism of action remains to be conclusively demonstrated. The changes in neurogenesis during this transition of increasing Aß levels and plaque formation were investigated in the present study. We found that the proliferation of newborn cell in the dentate gyrus was enhanced prior to elevations in soluble Aß production as well as amyloid deposition in 5-week-old TgCRND8 mice, which are well-established Alzheimer's disease models, compared to non-transgenic (Non-Tg) mice. The number of BrdU-positive cells remained higher in TgCRND8 vs Non-Tg mice for a period of 8weeks. The numbers of BrdU/NeuN-positive cells were not significantly different in TgCRND8 compared to Non-Tg mice. A significant decrease in BrdU/GFAP but not in BrdU/S100ß was found in Tg vs Non-Tg at 6-weeks of age. In addition, a unique observation was made using isolated neuroprogenitor cells from TgCRND8 mice which were found to be less viable in culture and produced substantial amounts of secreted Aß peptides. This suggests that the proliferation of neural progenitors in vivo may be modulated by high levels of APP expression and the resulting Aß generated directly by the progenitor cells. These findings indicate that cell proliferation is increased prior to Aß deposition and that cell viability is decreased in TgCRND8 mice over time.

Endothelial cells regulate p53-dependent apoptosis of neural progenitors after irradiation.

Endothelial cells represent an important component of the neurogenic niche and may regulate self-renewal and differentiation of neural progenitor cells (NPCs). Whether they have a role in determining the apoptotic fate of NPCs after stress or injury is unclear. NPCs are known to undergo p53-dependent apoptosis after ionizing radiation, whereas endothelial cell apoptosis after irradiation is dependent on membrane acid sphingomyelinase (ASMase) and is abrogated in sphingomyelin phosphodiesterase 1 (smpd1)- (gene that encodes ASMase) deficient mice. Here we found that p53-dependent apoptosis of NPCs in vivo after irradiation was inhibited in smpd1-deficient mice. NPCs cultured from mice, wild type (+/+) or knockout (-/-), of the smpd1 gene, however, demonstrated no difference in apoptosis radiosensitivity. NPCs transplanted into the hippocampus of smpd1-/- mice were protected against apoptosis after irradiation compared with those transplanted into smpd1+/+ mice. Intravenous administration of basic fibroblast growth factor, which does not cross the blood-brain barrier, known to protect endothelial cells against apoptosis after irradiation also attenuated the apoptotic response of NPCs. These findings provide evidence that endothelial cells may regulate p53-dependent apoptosis of NPCs after genotoxic stress and add support to an important role of endothelial cells in regulating apoptosis of NPCs after injury or in disease.

Overexpression of the vesicular acetylcholine transporter increased acetylcholine release in the hippocampus.

Cholinergic neurotransmission in the hippocampus is involved in cognitive functions, including learning and memory. Strategies to enhance septohippocampal cholinergic neurotransmission may therefore be of therapeutic value to limit cognitive decline during cholinergic dysfunction. In addition to current strategies being developed, such as the use of acetylcholinesterase inhibitors, enhancing acetylcholine (ACh) release may be critical for optimal cholinergic neurotransmission. Vesicular acetylcholine transporter (VAChT) activity limits the rate of formation of the readily releasable ACh pool. As such, we sought to determine the influence of increased VAChT expression on the septohippocampal cholinergic system. To do this, we used the B6.eGFPChAT congenic mouse, which we show contains multiple gene copies of VAChT. In this transgenic mouse, the increased VAChT gene copy number led to an increase in VAChT gene expression in the septum and a corresponding enhancement of VAChT protein in the hippocampal formation. VAChT overexpression enhanced the release of ACh from ex vivo hippocampal slices. From these findings, we conclude that VAChT overexpression is sufficient to enhance ACh release in the hippocampal formation. It remains to be established whether, in cases of cholinergic deficits, increasing VAChT expression would re-establish adequate levels of cholinergic neurotransmission, thereby providing a valid therapeutic target.

Pathogenesis of levodopa-induced dyskinesia: focus on D1 and D3 dopamine receptors.

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa therapy for Parkinson's disease. Taking advantage of a monkey brain bank constituted to study the pathophysiology of levodopa-induced dyskinesia, we here report the changes affecting D1, D2 and D3 dopamine receptors within the striatum of four experimental groups of non-human primates: normal, parkinsonian, parkinsonian treated with levodopa without or with dyskinesia. We also report the possible role of arrestin and G protein-coupled receptor kinases.

The immunophenotype of peripheral blood lymphocytes in clinically healthy dogs and dogs with lymphoma in remission.

Lymphoma is a common cancer of dogs that frequently is treated with chemotherapy or radiation therapy. Response to therapy is variable and currently available diagnostic tests do not reliably predict response to therapy. Treatment for lymphoma often results in lymphopenia, but it is unknown whether the changes in circulating lymphocytes result from generalized or specific reduction of lymphocytes. In this study, blood lymphocytes from 12 clinically healthy dogs, 10 dogs in remission because of treatment for B-cell lymphoma, and 8 dogs in remission from T-cell lymphoma were analyzed by flow cytometry by using a panel of 20 antibodies reactive with canine leukocyte antigens. Results identified similar lymphocyte parameters in treated dogs regardless of the type of lymphoma. Treated dogs had >50% reduction in blood lymphocyte concentration, and an absolute decrease in most subsets of lymphocytes. Both groups of treated dogs had relative increases in the proportion of CD3+, T-cell receptor (TCR)alphabeta+, and CD90+ lymphocytes, and a decreased proportion of CD45RA+ cells. In addition, dogs with T-cell lymphoma in remission had a significant increase in the proportion of CD49d+ lymphocytes. These findings were interpreted as representing likely suppression of lymphocyte regeneration by chemotherapy, with a relative increase in the proportion of memory over naive lymphocytes. Lack of correlation with the T- or B-cell origin of the initial lymphoma suggested that, by using flow cytometric methods, residual circulating neoplastic cells could not be detected. However, the changes in the lymphocyte profile of dogs treated with chemotherapy may have relevance to their immunocompetence.

Flow cytometric immunophenotype of canine lymph node aspirates.

Increasing availability of reagents able to distinguish subtypes of lymphocytes and other leukocytes has enabled greater understanding of lymphocyte biology and pathology in the dog. Lymphocytes in circulation most commonly are subjected to immunophenotypic assessment by flow cytometry, but needle aspirates of lymph nodes can be similarly suitable for immunophenotypic examination. In this investigation, the feasibility of immunophenotyping samples obtained by needle aspiration of lymph nodes from 32 dogs with no physical abnormalities and 6 dogs with lymphoma was determined. In addition, samples from 6 dogs were stored overnight at 4 degrees C and reanalyzed 24 hours later. For each sample, stained smear preparations were examined microscopically for lymphocyte morphology, neoplasia, and the presence of inflammatory cells. Expression of antigens on a corresponding sample of aspirated cells was determined by flow cytometric detection of antibody binding on a minimum of 10,000 events. The distribution of data was determined with Anderson-Darling tests, and reference intervals incorporating the central 95% of values were established. Adequate samples were obtained from 30 of 32 clinically normal dogs. Immunophenotypic results after 24 hours of storage were consistent with those obtained immediately after sampling. Reference intervals for lymphocyte subsets from normal dog lymph nodes were similar to the proportions of CD3+, CD4+, CD8+, and CD21+ lymphocytes found in blood. Aspirates of enlarged lymph nodes from dogs with lymphoma were readily classified by this technique. Aspiration of lymph nodes from dogs for comprehensive analysis by flow cytometry is feasible and applicable to immunophenotyping of lymphoma.

Mapping, characterization, and expression analysis of the SM-20 human homologue, c1orf12, and identification of a novel related gene, SCAND2.

Major psychosis was shown to segregate with a balanced translocation (1q42.1; 11q14.3) in a multigenerational family. This study describes the identification of a human SM-20 homologue gene that lies at about 400 kb on the centromeric side of the 1q42.1 breakpoint. The full-length cDNA sequence and gene structure were determined. Expression analysis was performed, showing high expression levels in skeletal and cardiac muscles; in the central nervous system, expression was restricted to dopaminergic neurons and spinal motoneurons. A second gene displaying high sequence similarity with SM-20 was also identified by BLAST. This gene, located on chromosome 15, is likely to have evolved by retroposition of SM-20 mRNA and an exon-shuffling mechanism. It encodes a 306-amino-acid protein harboring strong homology with an N-terminal motif found in some zinc-finger proteins. This gene was named SCAND2 (SCAN domain-containing 2).

Toward a primate model of L-dopa-unresponsive parkinsonism mimicking striatonigral degeneration.

We developed a primate model of striatonigral degeneration (SND), the neuropathology underlying levodopa-unresponsive parkinsonism associated with multiple systemic atrophy (MSA-P), by sequential systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 3-nitropropionic acid (3NP) in a Macaca fascicularis monkey. L-Dopa-responsive parkinsonian features emerged after MPTP injections. Subsequent chronic 3NP administration aggravated the motor symptoms and abolished the L-dopa response. In vivo magnetic resonance imaging revealed bilateral striatal lesions. Histopathologically, there was severe dopaminergic cell loss in the substantia nigra pars compacta compared with the control monkey. Furthermore, we observed circumscribed areas of severe neuronal degeneration in the motor striatum. These changes were absent in the control monkey, and they were associated with diffuse metabolic failure as demonstrated by cytochrome oxidase histochemistry. The striatal pathology predominantly involved output pre-pro-enkephalin A- and substance P-containing cells, whereas somatostatin (NADPH-diaphorase)-containing interneurons were relatively spared. Our model therefore reproduced levodopa-unresponsive parkinsonism and SND-like pathologic changes characteristic of MSA-P. The double-lesion primate model of SND may serve as a preclinical test-bed for the evaluation of novel therapeutic strategies in MSA-P.

Phenotypical characterization of the neurons expressing the D1 and D2 dopamine receptors in the monkey striatum.

The striatum is regulated by dopaminergic inputs from the substantia nigra. Several anatomical studies using in situ hybridization have demonstrated that in rodents, dopamine D1 and D2 receptors are segregated into distinct striatal efferent populations: dopamine D1 receptor into gamma-aminobutyric acid (GABA)/substance P striatonigral neurons, and dopamine D2 receptor into GABA/enkephalin striatopallidal neurons. The existence of such a segregation has not been investigated in primates. Therefore, to quantify the efferent striatal GABAergic neurons in the adult Cynomolgus monkey, we detected GAD67 mRNA expression while considering that only a minority of the GABAergic population is composed of interneurons. To characterize the peptidergic phenotype of the neurons expressing dopamine D1 or D2 receptors, we examined the mRNA coding for these receptors in the striatum at the cellular level using single- and double in situ hybridization with digoxigenin and 35S ribonucleotide probes. Double in situ hybridization demonstrated a high coexpression of dopamine D1 receptor and substance P mRNAs (91-99%) as well as dopamine D2 receptor and preproenkephalin A mRNAs (96-99%) in medium-sized neurons throughout the nucleus caudatus, putamen, and nucleus accumbens. Only a small subpopulation (2-5%) of the neurons that contained dopamine D1 receptor mRNA also expressed dopamine D2 receptor mRNA in all regions. Large-sized neurons known to be cholinergic expressed D2R mRNA. However, within the nucleus basalis of Meynert, the large cholinergic neurons expressed D2R mRNA, but the neurons producing enkephalin expressed neither D1R nor D2R mRNA. These results demonstrate that the striatal organizational pattern of D1 and D2 receptor segregation in distinct neuronal populations described in rodent also exists in primate.

Expression of L1 and PSA during sprouting and regeneration in the adult hippocampal formation.

The objective of the present study was to evaluate the expression of polysialic acid (PSA) and the cell adhesion molecule L1 during axonal regeneration and sprouting after injury to the adult rat brain. All animals received a complete lesion of the fimbria-fornix (FF). Grafts of nerve growth factor (NGF)- or beta-galactosidase (betaGal)-producing fibroblasts were placed in the FF lesion cavity and induced septohippocampal cholinergic regeneration or sympathetic tyrosine hydroxylase (TH)-positive sprouting, respectively. Cholinergic regeneration was evaluated from 2 to 8 weeks following grafting of NGF-producing fibroblasts in the FF lesion cavity. In the graft area, choline acetyltransferase (ChAT)-positive fibers expressed L1 and PSA. Once cholinergic axons reached the hippocampal formation (HF), they no longer expressed L1 or PSA. Eight weeks after a lesion of the FF and transplantation of betaGal-producing fibroblasts, TH-positive fibers sprouted in the denervated HF and expressed L1 but not PSA. At the zone of reactive gliosis, PSA but not L1 expression was increased following a lesion of the FF and transplantation of NGF- or betaGal-producing fibroblasts. In animals that received a graft of NGF-producing fibroblasts in the FF lesion cavity, numerous ChAT-positive axons were observed along these areas rich in PSA and reactive astrocytes. Taken together, these results suggest that the expression of PSA and L1 is upregulated on regenerating cholinergic axons during axonal elongation and downregulated upon target innervation. In contrast, TH-positive fibers that sprout in the denervated HF express and maintain their expression of L1. Finally, the expression of PSA in the area of reactive gliosis may contribute to a permissive environment for axonal regrowth.

Cellular distribution of adenosine A2A receptor mRNA in the primate striatum.

The cellular expression of adenosine A2A receptor mRNA in the adult monkey and human striatum was examined by using single and double in situ hybridization with ribonucleotide probes. Analysis on adjacent sections demonstrated a homogeneous overlapping expression of adenosine A2A receptor and preproenkephalin A mRNAs throughout nucleus caudatus, putamen, and nucleus accumbens. By contrast, high expression of preproenkephalin A mRNA but no expression of adenosine A2A receptor mRNA was found in the nucleus basalis of Meynert. Double in situ hybridization demonstrated an extensive colocalization of adenosine A2A receptor and preproenkephalin A mRNAs in approximately 50% of the medium-sized spiny neurons of the monkey nucleus caudatus, putamen, and nucleus accumbens. A small number of neurons (4-12%) that contained adenosine A2A receptor mRNA but not preproenkephalin A mRNA was found along the ventral borders of the striatum. Virtually all adenosine A2A receptor mRNA-containing neurons co-expressed dopamine D2 receptor mRNA, whereas only very few adenosine A2A receptor mRNA containing neurons co-expressed dopamine D1 receptor or substance P mRNAs. In addition, a sub-population of adenosine A2A receptor mRNA-expressing neurons that also contained preproenkephalin A mRNA was found in the septum in monkeys. These results demonstrate that there is a high expression of adenosine A2A receptor mRNA in the primate striatum that is extensively co-localized with dopamine D2 receptor and preproenkephalin A mRNAs. It is concluded that adenosine A2A receptors are likely to be important for the parallel organization of primate striatal neurotransmission and that these receptors could be a target for drug therapy in Parkinson's disease.

Hippocampal grafts of acetylcholine-producing cells are sufficient to improve behavioural performance following a unilateral fimbria-fornix lesion.

Lesions of the septohippocampal pathway produce cognitive deficits that are partially attenuated by grafts of cholinergic-rich tissue into denervated target regions or by systemic administration of cholinomimetic drugs. In the present study, fibroblasts engineered to produce acetylcholine were used to test the hypothesis that restoration of hippocampal acetylcholine in rats with septohippocampal lesions is sufficient to improve cognitive processing post-damage. Rats received unilateral grafts of acetylcholine-producing or control fibroblasts into the hippocampus immediately prior to an aspirative lesion of the ipsilateral fimbria-fornix. Some rats with fimbria-fornix lesions were implanted with acetylcholine-producing or control fibroblasts into the neocortex, another major target of the basal forebrain cholinergic system, to determine if the site of acetylcholine delivery to the damaged brain is critical for functional recovery. Rats were tested in a hidden platform water maze task, a cued water maze task and activity chambers between one and three weeks post-grafting. Compared to unoperated controls, rats with fimbria fornix lesions only were significantly impaired in hidden platform water maze performance. Hippocampal grafts of acetylcholine-producing cells reduced lesion-induced deficits in the water maze, whereas hippocampal control grafts and cortical grafts of either cell type were without effect. Locomotor activity and cued water maze performance were unaffected by the lesion or the implants. Taken together, these data indicate that water maze deficits produced by fimbria fornix lesions, which disrupt a number of hippocampal neurotransmitter systems, can be attenuated by target specific replacement of acetylcholine in the hippocampus and that this recovery occurs in the absence of circuitry repair.

Ontogeny of the striatal neurons expressing the D2 dopamine receptor in humans: an in situ hybridization and receptor-binding study.

D2 dopamine receptor (D2R) gene expression was analyzed by in situ hybridization and D2R ligand autoradiography in the human striatum during ontogeny. D2R mRNA and ([3H]YM-09151-2)-binding sites were detected in the striatum from week 12 of fetal life. At this time, D2R mRNA and binding sites were predominant in the putamen and occurred in a pattern of clusters. D2R-binding sites displayed a similar pattern. The signal in the caudate nucleus was weak from weeks 12 to 16. From week 20 of fetal life, D2R mRNA and D2R-binding sites signals became intense in the ventral striatum. At birth, D2R mRNA became homogeneously distributed while D2R-binding sites kept an heterogeneously distribution. Comparative topological and temporal analysis of the D2R, enkephalin and D1 dopamine receptor (D1R) mRNAs showed a distinct developmental pattern for each mRNA. Before birth, the neurons expressing enkephalin and D1R mRNAs were preferentially distributed in the matrix and in the striosomes, respectively, while the neurons expressing D2R mRNA did not display a preferential localization. At birth, high levels of enkephalin mRNA were restricted to the matrix; D1R mRNA level was homogeneous throughout the striatum. D2R mRNA was heterogeneously distributed in the whole striatum with high signals located both in the striosomes and the matrix. These results demonstrate that functional D2R are expressed as early as week 12 in the striatum with a heterogeneous distribution. Our findings also demonstrate that, in contrast to what was expected from similar studies in rodents, D2R mRNA and enkephalin mRNA do not display identical, overlapping expression patterns in striatal neurons during human ontogeny.

Molecular anatomy of the development of the human substantia nigra.

A series of 15 fetal and perinatal human brains (from week 12 of fetal life to day 2 after birth) was studied in order to describe the anatomical and molecular correlates of the substantia nigra ontogeny. In situ hybridization, immunohistochemistry and binding studies were used to detect D2 dopamine receptor (D2R) mRNA, D2R binding sites, dopamine membrane transporter (DAT) mRNA, tyrosine hydroxylase (TH) protein D1 dopamine receptor (D1R) protein and D1R binding sites. Dopaminergic (DA) neurons of the substantia nigra were detected through TH immunoreactivity from week 12. At week 16, the substantia nigra was clearly delineated as a compact group of intermingled neurons and fibers. From week 19, groups of DA neurons were segregated from the pars reticulata. These groups have been divided into the substantia nigra pars compacta, the ventral tegmental area and the retrorubral area. The DA neurons exhibited a gradual increase in size and branching development until birth. From week 12 onward they expressed several other markers of dopamine transmission, i.e., D2R mRNA, D2R binding sites and DAT mRNA. The ventral tegmental area expressed lower levels of mRNA for DAT and D2R than the pars compacta. From week 12, D1R immunoreactivity and D1R binding sites were also present in the substantia nigra pars reticulata. This suggests that projecting striatonigral neurons, known to express the D1R gene, have developed pathways connecting with the substantia nigra by week 12. Our results demonstrate that the developing substantia nigra in human displays early transcriptional and translational activity for the main constituents of dopaminergic transmission from week 12 and receives at this time dopaminoceptive inputs bearing D1 receptors from the striatum.

Developmental profiles of various cholinergic markers in the rat main olfactory bulb using quantitative autoradiography.

The existence of possible relationships among the developmental profile of various cholinergic markers in the main olfactory bulb (OB) was assessed by using in vitro quantitative autoradiography. Muscarinic receptors were visualized with [3H]pirenzepine (muscarinic M1-like sites) and [3H]AF-DX 384 (muscarinic M2-like sites); nicotinic receptors by using [3H]cytisine (nicotinic 42-like subtype) and [125I] alpha-bungarotoxin (nicotinic 7-like subtype); cholinergic nerve terminals by using [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites). These various cholinergic markers exhibited their lowest levels at birth and reached adult values by the end of the 4-5 postnatal weeks. However, the density of presynaptic cholinergic markers and nicotinic receptors at postnatal day 2 represented a large proportion of the levels observed in adulthood, and displays a transient overexpression around postnatal day 20. In contrast, the postnatal development of cholinergic muscarinic M1-like and M2-like receptors is apparently regulated independently of the presynaptic cholinergic markers and nicotinic receptors. Two neurochemically and anatomically separate olfactory glomeruli subsets were observed in the posterior OB of the developing rat. These atypical glomeruli expressed large amounts of [3H]vesamicol-and [3H]hemicholinium binding sites without significant amounts of muscarinic M1, M2, or nicotinic alpha 4 beta 2 receptor binding sites. A significant density of [125I] alpha-bungarotoxin binding sites could be detected only at early postnatal ages. A few olfactory glomeruli specifically restricted to the dorsal posterior OB expressed a high density of [3H]cytisine binding sites but lacked significant binding of the two presynaptic cholinergic markers used here, suggesting their noncholinergic but cholinoceptive nature.

Ontogeny of the striatal neurons expressing the D1 dopamine receptor in humans.

We studied D1 dopamine receptor (D1R) gene expression in the human striatum during ontogeny by in situ hybridization, immunohistochemistry, and D1R ligand autoradiography. D1R mRNA, protein, and binding sites ([3H]SCH 23390) were detected in the striatum from week 12 of fetal life. At this time, D1R mRNA was predominant in the striosomal neurons; D1R immunoreactivity (D1R-IR) and D1R binding sites displayed a pattern similar to D1R mRNA. D1R-IR was essentially present in striosomal cell bodies and neuropil, whereas only a few cell bodies were detected in the matrix. From week 20 of fetal life, D1R gene expression developed in the matrix neurons as well, thus leading to an even D1R mRNA expression throughout striosomes and matrix compartments at birth. Comparative analysis of the expression of D1R and dynorphin mRNA show the same developmental patchy pattern up to week 26. Indeed, neurons expressing the D1R gene contain dynorphin mRNA; in contrast, they do not express the preproenkephalin A gene. At birth, the pattern of D1R mRNA expression level was sharply different from that of dynorphin (DYN) gene expression. High DYN mRNA expression was restricted to the striosomes, whereas high D1R mRNA expression was present in the whole striatum. These results demonstrate that, during human ontogeny, functional D1 receptors are expressed as early as week 12 in the striatum, developing initially in the striosomal neurons containing high dynorphin mRNA content. Toward the end of fetal life, there is a dissociation between D1R and DYN expression levels, suggesting that neuroanatomical or neurochemical modifications occur at this period, which may contribute to the regulation of the tone of the striatal D1R and DYN gene with topological specificity.

Comparative ontogenic profile of cholinergic markers, including nicotinic and muscarinic receptors, in the rat brain.

The ontogenic profiles of several cholinergic markers were assessed in the rat brain by using quantitative in vitro receptor autoradiography. Brain sections from animals at different stages of development were processed with [3H]AH5183 (vesamicol; vesicular acetylcholine transport sites), [3H]N-methylcarbamylcholine (alpha(4)beta(2) nicotinic receptor sites), [3H]hemicholinium-3 (high-affinity choline uptake sites), [3H]3-quinuclidinyl benzilate (total population of muscarinic receptor sites), [3H]4-DAMP (muscarinic M1/M3 receptor sites), [3H]pirenzepine (muscarinic M1 receptor sites), and [3H]AF-DX 116 and [3H]AF-DX 384 (muscarinic M2 receptor sites) as radiolabeled probes. The results revealed that, by the end of the prenatal period (embryonic day 20), the densities of nicotinic receptor and vesicular acetylcholine transport sites already represented a considerable proportion of those observed in adulthood (postnatal day 60) in different laminae of the frontal, parietal, and occipital cortices, in the layers of Ammon's horn fields and the dentate gyrus of the hippocampal formation, as well as in the amygdaloid body, the olfactory tubercle, and the striatum. In contrast, at that stage, the densities of total muscarinic, M1/M3, M1, and possibly M2 receptor and high-affinity choline uptake sites represent only a small proportion of levels seen in the adult. Differences were also observed in the postnatal ontogenic profiles of nicotinic, muscarinic, vesamicol, and high-affinity choline uptake sites. For example, between postnatal weeks 3 and 5, the levels of M1/M3 and M1 sites were at least as high as in the adult, whereas M2 and high-affinity choline uptake site densities appeared to be delayed and to reach adult values only after postnatal week 5. With regard to cholinergic innervation in the developing rat brain, the present findings suggest a temporal establishment of several components of the cholinergic systems. The first components are the vesicular acetylcholine transporter and nicotinic sites; these are followed by M1/M3 and M1 sites and, finally, by M2 and high-affinity choline uptake sites.