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Fulfilling the dreams of older people requires specific support from society and institutions, especially the economic and health sectors. Fulfilling those dreams also requires knowledge about older people's rights. If people are not aware of their rights, they cannot demand these rights through legally established processes and institutions.
In cooperation with the Older People's Group (OPG) in Banthi District, Lamphun Province, the EU supported project organised a team of volunteers called 'older people's rights promotion volunteer team of Banthi'. The team is working to increase knowledge about older people's rights and to encourage, monitor and protect those rights. The volunteer team also works with government agencies in 10 pilot areas to raise their awareness about the the topic.
Mae Janfong Maha-mai is the president of the volunteer team. 'Gathering a strong group of older people brought us the power of negotiation with key organisations such as municipal offices', she said. Workshop-style meetings were held in various villages to educate older people about 13 critical rights and entitlements for older people in accordance with Thailand's Older Persons Act 2003. The workshops also helped to strengthen Older People's Groups, placing them as invaluable sources of knowledge on the issues that affect their lives and their communities.
In the past, older people lacked useful knowledge and information that could assist their daily lives, especially knowledge of their rights. This meant a lack of knowledge around access to health services and access to the Elderly Fund provided by the government, among other important issues.The Older Persons Act was a start but there is still a gap between the act and its implementation.
Since the volunteer team was established a few years ago, Mae Janfong Maha-mai and her Banthi OPG team have been distributing information at monthly meetings. They also raise the awareness of the public at large.
Realising and being able to access their rights brings an important change for older people, such as access to comprehensive healthcare. For example, they can request shorter waiting times to visit a doctor.


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Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds—parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and -5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest that neural activity and signal transduction in vocal nuclei of vocal learners will be different relative to the surrounding brain areas.
Vocal learning is the ability to imitate sounds, usually species-specific vocalizations. It has been experimentally found in only six animal groups, all of which are distantly related. These are three groups of mammals (humans, bats, and cetaceans) and three groups of birds (parrots, hummingbirds, and songbirds; see Fig. 1A; Nottebohm, 1972; Jarvis et al., 2000). In other groups tested, including nonhuman primates, vocalizations are found to be only innate (Marler and Mitani, 1988). Learned vocalizations are usually composed of a set of complex syllables forming syntax that is modifiable, compared with innate vocalizations. The three vocal learning bird groups have seven similar but not identical cerebral vocal nuclei for learned vocalization (Fig. 1B; Jarvis et al., 2000). These nuclei have not been found in closely or distantly related vocal nonlearners. Most information about cerebral vocal nuclei is known in songbirds, the zebra finch in particular, where they form two major networks: 1) a posterior vocal pathway unique to vocal learners that is involved in production of learned vocalizations (see Fig. 1B, yellow-labeled nuclei) and 2) an anterior vocal pathway also unique to vocal learners that is involved in vocal learning and vocal syntax and is differentially regulated in different social contexts (see Fig. 1B, red-labeled nuclei). A third system, an auditory pathway, is involved in audition and auditory learning and has been found in both vocal learners and vocal nonlearners (see Fig. 1B, blue-labeled nuclei; Nottebohm et al., 1976; Scharff and Nottebohm, 1991; Yu and Margoliash, 1996; Jarvis et al., 1998, 2000; Hessler and Doupe, 1999b; Ribeiro and Mello, 2000; Kobayashi et al., 2001).
Each vocal nucleus has its own unique temporal and spatial firing pattern to control either the muscles of the syrinx and respiratory apparatus or other nuclei during singing or the practice of singing (Yu and Margoliash, 1996; Hessler and Doupe, 1999a,b; Hahnloser et al., 2002). Furthermore, singing induces expression of action-potential-regulated genes in these nuclei with different patterns for different genes, and this has been found for all three vocal learning groups (Jarvis and Nottebohm, 1997; Kimpo and Doupe, 1997; Jarvis and Mello, 2000; Jarvis et al., 2000; Li and Jarvis, 2001; Wada and Jarvis, unpublished results). Neurotransmission of activity and the regulation of these activity-dependent genes are thought to occur by neurotransmitter release onto neurotransmitter receptors (Worley et al., 1990; Lerea, 1997; Clayton, 2000; Jarvis, 2004a). For these reasons, we considered whether the neurotransmitter receptors are different for each vocal nucleus in vocal learners.
In vertebrates, one of the most important neurotransmitter receptors for excitatory transmission with a role in learning and generation of complex behavior is the glutamate receptor family (Abel and Lattal, 2001). In mammals, in total 26 receptor subunits/subtypes have been cloned to date, and they have been classified into four subfamilies based on their functions or pharmacological agents to which their receptor subunit combinations preferentially bind: three ionotropic receptor subfamilies, 1) α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA), 2) kainate (KA), and 3) N-methyl-D-aspartate (NMDA); and 4) the metabotropic subfamily (Pin and Duvoisin, 1995; Dengledine et al., 1999; Hollmann, 1999; Gustinci et al., 2003). Each ionotropic receptor is typically formed from hetero- or homotetrads of different receptor subunits encoded by different genes. Each metabotropic receptor is typically a monomer encoded by a single gene and, thus, is classified as a receptor subtype. Upon binding glutamate, the receptors directly gate ion channels (ionotropic receptors) and/or change conformation (metabotropic receptors) to transmit electrical signals between neurons and regulate expression of targeted genes via second messengers. Each receptor subunit/subtype has a unique brain expression pattern indicative of different brain subdivisions or cell types having different glutamate receptor functions (Bahn et al., 1994; Conti et al., 1994; Standaert et al., 1994; Testa et al., 1994). Furthermore, some of these subunits/subtypes have different mRNA splice variants contributing further to the diversity of receptor function.
In songbirds, pharmacological studies indicate the presence of glutamate receptors in vocal nuclei; specifically, NMDA receptors have been shown to be required for vocal learning (Basham et al., 1996) and have kinetics that change during vocal learning critical periods (Stark and Perkel, 1999; White et al., 1999; Dutar et al., 2000; Livingston et al., 2000). However, to date only three glutamate receptor subunits have been cloned from the songbird brain (NR1, NR2A, and NR2B, belonging to the NMDA subfamily; Singh et al., 2000; Heinrich et al., 2002). Here we report the identification and presence of nearly all glutamate receptor subunits/subtypes discovered in mammals within the songbird brain. Furthermore, we compared expression of four genes from each of the subfamilies that had most differential expression in zebra finches (GluR1, GluR5, NR2A, and mGluR2) in hummingbirds, parrots, and doves (the latter of which are vocal nonlearners). We found that several of these subunits/subtype show unique differential expression in cerebral vocal nuclei of all three distantly related vocal learning bird groups, suggesting that differential expression of neurotransmitter receptors in vocal systems contribute to learned vocal communication.
The following representative species were used for this study: zebra finches (Taeniopygia guttata), a songbird (n = 16; six for in situ hybridizations, six for immunohistochemistry, and four for Western blotting); sombre hummingbirds (Aphantochroa cirrochloris; n = 3); budgerigars (Melospititicus undulatus), a parrot (n = 3); and doves (vocal nonlearners)—ring doves (Stretophilia risoria; n = 3) and rock doves (pigeon; Columbia livia; n = 3). We chose these species because they are among the best studied in terms of the neuroethology of learned and innate vocal communication (Nottebohm, 1993; Striedter, 1994; Durand et al., 1997; Slabbekoorn et al., 1999; Jarvis and Mello, 2000; Jarvis et al., 2000). We examined only males, because this is the sex that most often demonstrates vocal learning. All birds were adults. The zebra finches, 150 days to ~1 year old, were obtained from our breeding colonies at the Duke University Medical Center and Tokyo Medical and Dental University. Because we wanted to examine expression of glutamate receptors in the brain in the absence of vocal behavior, birds were kept under relatively quiet conditions, not singing, for several hours to overnight before sacrifice. Brain sections of comparable quiet control sombre hummingbirds and budgerigars were obtained from our collection (Jarvis and Mello, 2000; Jarvis et al., 2000). Dove brains were collected from a parallel study conducted by Michael McElroy in the Jarvis laboratory. All animals were treated under the humane guidelines of Duke University and Tokyo Medical and Dental University, and experiments were approved by animal care committees.
Detailed cloning will be described separately (Wada et al., in preparation). Briefly, we attempted to clone cDNAs of 21 of 26 known mammalian glutamate receptor subunits/subtypes (Gustinci et al., 2003) from zebra finch brain mRNA [four AMPA (GluR1–4), five kainate (GluR5–7, KA1–2), six NMDA (NR1, NR2A–D, NR3A), and six metabotropic (mGluR1–5, mGluR8)]. Degenerate primers were made to conserved, unique regions of each subfamily among mammals (mouse, rat, and human) and when available with other vertebrates [Xenopus, chicken, and the previously described zebra finch NR2B sequence (Basham et al., 1999)]. We chose primers that would amplify regions with sequences specific to that subunit/subtype, to prevent cross-hybridizations to the related subunits/subtypes in in situ hybridization. Splice-variant-specific primers were also made to the alternative Flip and Flop domains of the AMPA subunits. RT-PCR was then performed on total RNA from adult male zebra finch brains with the appropriate degenerate primer pairs. PCR products were examined on 1.5% agarose gels and, if present, cut from the gel and cloned into the pGEMTeasy plasmid (Promega, Madison, WI). These plasmids were transformed into XL-1 blue supercompetent cells (Stratagene, La Jolla, CA), and Escherichia coli colonies were selected under ampicillin (100 μg/ml) agarose plates. Colonies were then picked and grown in LB broth with ampicillin (100 μg/ml), and DNA minipreps were prepared. Inserts were 5′- and 3′-end sequenced and sequences passed through BLASTN (DNA) and BLASTX (protein) GenBank searches. If a clone revealed homology to the expected glutamate receptor subunit/subtype of other species, then the entire clone was sequenced. All sequences have been deposited into GenBank (accession Nos. in Table 1).
Serial sagittal (right hemisphere) 10-μm sections were cut throughout the entire brains of adult quiet control zebra finch males. For other species, only sections known to contain vocal nuclei were cut and/or selected from our collection. 35S-labeled riboprobe in situ hybridizations were conducted. Riboprobes were made from T7 and SP6 promoter sites of pGEMTeasy with Promega RNA polymerases. For pGEMTeasy, high-concentration SP6 RNA polymerase (Progema) is necessary to generate high-quality riboprobes. Frozen sections were fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.0), acetylated, dehydrated in an ascending ethanol series, and air dried. We then used one of two different hybridization solutions: 1) 50% formamide, 2× SSPE, 2 μg/μl yeast tRNA, 1 μg/μl poly-A, 0.4 μg/μl bovine serum albumin (BSA), 100 mM dithiothreitol (DTT; Mello et al., 1997); 2) 50% formamide, 10% dextran, 1× Denhardt's, 12 mM EDTA (pH 8.0), 10 mM Tris-HCl (pH8.0), 30 mM NaCl, 0.5 μg/μl yeast tRNA, and 10 mM DTT (Yoshida et al., 1998). Both solutions gave similar results, except that the signals were stronger for the second. Either 32 μl (hybridization solution 1) or 100 μl (hybridization solution 2) with 35S-labeled probes (1 × 106 cpm/slide) were applied to each slide, coverslipped, and hybridized at 65°C for 4 hours in solution 1 or 16 hours in solution 2 under mineral oil. The mineral oil was removed by chloroform washes and excess probe removed by washing in 2× SSPE at room temperature for 1 hour; 2× SSPE, 50% formamide, 0.1% β-mercaptoethanol at 65°C for 1 hour; and 0.1× SSPE twice at 65°C for 30 minutes each. Slides were dehydrated in an ascending ethanol series and exposed to β-max hyperfilm (Kodak, Rochester, NY) for 2–3 days. They were then dipped in NTB-2 (Kodak) emulsion for ~2 weeks and developed, sections were counterstained with cresyl violet and then coverslipped. These exposure times allowed signal detection for imaging without signal saturation for quantification.
We measured gene expression levels by one of two methods: 1) from silver grains of emulsion-dipped slides (for Fig. 4A,B) and 2) from X-ray films (for Figs. 5C, 9). For emulsion-dipped slides, we calculated the average number of silver grains per cell, following a previously described semiautomated procedure (Jarvis and Nottebohm, 1997). First, images of brain regions from the dipped slides were captured with a Spot III CCD camera coupled to a Leica DMRXA2 microscope under a ×40 objective by using Spot software v3.2.4 (Diagnostic Instruments, Inc.). NIH Image software was then used to count silver grains from one or two cell fields (~0.2 mm2 per field, containing neurons, glia, and neuropil) in the central region of each nucleus (for lMAN 70–80% of the nucleus field was captured in one field). The average background number of grains per same-size field on the glass slide without brain tissue was then subtracted. The two counts from each field were averaged and then divided by the average number of cells (neurons and glia) per field for that nucleus (~60–100 cells depending on nucleus) to obtain grains/cell; the average number of cells was calculated separately from adjacent well Nissl-stained sections from the same animals as used for the in situ quantitations. Comparison using grains/cell controls for potential regional differences resulting from differences in cell density. For brain images on X-ray films, the exposed film was placed under a high-power dissecting scope (Wild M420) and captured to the computer with the Spot III camera. Images were transferred to Photoshop (Adobe) and converted to gray scale. Vocal nuclei and adjacent nonvocal areas were outlined with a highlighting tool, and the average pixel density was calculated. Compared with counting silver grains from emulsion-dipped slides, this method allowed us to perform high-throughput scanning and quantification of gene expression levels, and the results were comparable to silver grain counts (data not shown).
To graph ratios of differential expression in vocal and auditory regions relative to their surrounding brain subdivision (for Figs. 4A,B, 5C, 9), we divided the values of silver grain or pixel density of vocal and auditory regions with the respective adjacent nonvocal or nonauditory regions. To perform statistical analysis, we first performed an ANOVA to determine whether there was an overall significant difference in expression between vocal nuclei and the surrounding brain subdivisions. We followed the ANOVA with a Fisher's protected least significant difference (Fisher's PLSD) post hoc test for each gene. The Fisher's PLSD test performs pairwise comparisons (measurements from vocal nuclei and surrounding brain subdivisions come from the same animals), is less prone than other methods to making errors when considering the magnitude of differences between the means of two measures with a small n (three animals in our case), and accounts for repeated measures (more than one measurement from the same animal). After performing the Fisher's PLSD test, we then focused on those significant differences between the brain regions of interests, such as expression levels in a vocal nucleus compared with the respective adjacent nonvocal region surround. We chose a significance cutoff of P =.01, because we had more than 20 comparisons, but less than 100, for each gene; with a cutoff of .05, there is a probability that 1 of 20 comparisons could be significantly different by chance. For statistical analysis of expression ratio relationships between vocal nuclei (see Fig. 4C,D), we used a simple regression analysis with a 95% confidence interval.
Total brains of adult male zebra finches were homogenated in cold PBS, and the lysates were separated by SDS-PAGE (12.5%), transferred to a nitrocellulose membrane, and blocked with 5% skim milk in PBS for 1 hour. Membranes were then incubated with either rabbit anti-rat GluR1 or mGluR1 polyclonal antibodies made against the carboxyl terminal intracellular portions (1:500 dilution; Upstate Biotechnology, Lake Placid, NY; catalog Nos. 03-306 and 06-301, respectively). Membranes were then reacted with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:500; Zymed Laboratories, South San Francisco, CA), and binding was detected on X-ray films by using an ECL detection system (Amersham Bioscience, Piscataway, NJ). For immunohistochemistry, anesthetized birds were perfused with PBS and then 4% paraformaldehyde in PBS. Sagittal 20-μm sections were cut on a freezing microtome, freely floated in PBS, and reacted with 0.3% hydrogen peroxide for 20 minutes at room temperature. Sections were then washed three times with PBS and incubated for 2–3 hours in PBS containing 0.4% Triton X-100, 4% normal horse serum, and 1% BSA and then in fresh solution of the same composition overnight with the primary antibody. Colorimetric staining was performed with the avidin-biotin peroxidase complex (ABC) and nickel-intensified diaminobenzidine (DAB) detection kit from Vector Laboratories (Burlingame, CA).
By using RT-PCR with degenerate primers to conserved regions among mammals and/or other vertebrates, we attempted to and successfully cloned 21 of 26 known glutamate receptor subunits/subtypes described in mammals (Gustinci et al., 2003), covering all four glutamate receptor subfamilies, AMPA (GluR1–4), kainate (GluR5–7, KA1–2), NMDA (NR1, NR2A–D, NR3A), and metabotropic (mGluR1–5, mGluR8), from the zebra finch brain. We did not attempt to clone mGluR6 and mGluR7, because mammalian mGluR6 is known to be expressed in only the retina (Nakajima et al., 1993), and mGluR6 and -7 were too close in sequence to be distinguished reliably by initial PCR or by in situ hybridization. We did not attempt to clone the NMDA receptor subunit NR3B, because it was just being characterized at the time when we began our study (Nishi et al., 2001), or delta 1 and delta 2 subunits, because these are known to be involved mainly in cerebellar function (Morando et al., 2001). Without fail, we found all 21 glutamate receptor subunits/subtypes in the zebra finch brain. BLASTN and BLASTX searches revealed high cDNA (82–90%) and putative amino acid (83–100%) sequence identities between zebra finch and human (Table 1). The only exception was mGluR2 with 72% cDNA and 78% amino acid identity; this was due to an apparent splice difference in zebra finch, which has been not reported in mammals (data not shown). The spliced Flip and Flop regions of the AMPA subunits were nearly 100% identical between zebra finch and human (Table 1), showing conserved splicing recipient and donor sites. These results suggest 1) that the family and presence of glutamate receptors in the brain are highly conserved between birds and mammals and, thus, that the common stemamniote ancestor of birds and mammals presumably possessed these same glutamate receptor genes over ~300 million years ago, and 2) that the physiological function of most if not all glutamate receptors in songbirds is presumably similar to that in mammals, in support of electrophysiological and pharmacological findings (Basham et al., 1996; Stark and Perkel, 1999; White et al., 1999; Dutar et al., 2000; Livingston et al., 2000).
In situ hybridization revealed that each glutamate receptor subunit/subtype has a unique expression pattern in the songbird brain (Fig. 2). Within the cerebrum, the most apparent expression differences were coincident with Nissl-defined axonal–lamina boundaries [cell-sparse regions separating the hyerpallium (H), mesopallium (M), nidopallium (N), arcopallium (A), striatum (St), and pallidum (P)]. An exception to this rule was that expression in primary sensory areas [L2-auditory, entopallium (E)-visual, and basorostralis (B)-somatosensory] was similar among these areas and often different from the surrounding nidopallium (Fig. 2; entopallium not shown). There was no recognizable subfamily-specific brain expression pattern. Rather, several different receptor subunits/subtypes across subfamilies shared similar but not identical expression patterns. For example, GluR1 (AMPA type) and GluR7 (kainate type) were both higher in the mesopallium, caudal striatum (CSt), and arcopallium; GluR4 (AMPA type), mGluR2, and mGluR8 (metabotropic type) were higher throughout pallial regions vs. subpallial regions; mGluR4 and mGluR5 (metabotropic type) were lower in pallial relative to the subpallial striatum region (Fig. 2). Most glutamate receptor subunits/subtypes were low or absent in the subpallial pallidal region (P), an inhibitory area homologous to the mammalian globus pallidus (Reiner et al., 2004). The differential pallial vs. sub-pallial expression in the songbird brain matched that of the homologous subunits/subtypes expressed in mammalian cortical (pallial) vs. basal ganglia (subpallial) regions (Bahn et al., 1994; Conti et al., 1994; Standaert et al., 1994; Testa et al., 1994; Wada et al., in preparation).
A striking exception to the subdivision-specific expression patterns was that nearly all glutamate receptor subunits/subtypes showed highly differential expression, higher or lower, in one or more vocal nuclei relative to expression in the surrounding cells of each of their subdivisions (Figs. 2,,3).3). Each brain subdivision possesses at least one or more vocal nuclei (Av and MO in the mesopallium; MAN, HVC, and NIf in the nidopallium; RA in the arcopallium; Area X in the striatum; DLM in the thalamus; and DM in the midbrain). These expression differences in vocal nuclei are not the result of lamina differences, insofar as there are no known laminal boundaries separating vocal nuclei from the surrounding brain tissue. To evaluate the extent of this differential expression, we quantified in situ hybridization-labeled silver grains per cell within vocal nuclei relative to each subdivision immediately adjacent to the vocal nuclei. We found that 19 of 21 glutamate receptor subunits/subtypes had significant differential expression in one or more vocal nuclei (asterisk in Fig. 4A; the exceptions were KA1 and NR2D). In contrast, in nonvocal areas, such as auditory regions, fewer of these same subunits/subtypes had differential expression, and with lower magnitudes (Figs. 2, ,4B).4B). After further detailed examination of serial brain sections, we classified the vocal nuclei expression patterns into seven overlapping categories: 1) unique differential expression in large cerebral vocal nuclei, 2) differential expression in small cerebral vocal nuclei, 3) differential expression in noncerebral vocal nuclei, 4) correlations of expression among vocal nuclei, 5) lateral and medial differences in the anterior vocal pathway nuclei, 6) cell type uniqueness within vocal nuclei, and 7) individual animal differences.
Among the seven cerebral vocal nuclei, four of them (HVC, MAN, RA, and Area X; Fig. 4A) are larger than the others. All 19 of the 21 subunits/subtypes had differential expression in one or more these larger vocal nuclei (8 subunits/subtypes in HVC, 9 subunits/subtypes in RA, 11 subunits/subtypes in MAN, and 9 subunits/subtypes in Area X; Figs. 2, ,3A,3A, ,4A).4A). Of the two subunits that did not show differential expression in cerebral vocal nuclei, one (NR2C) was barely expressed in the cerebrum, and the other (NR2D) was selectively expressed throughout the pallidum (P), a brain subdivision with no known vocal nucleus (Fig. 2). These differential expression differences ranged from 7.7-fold lower (mGluR2 in RA) to 5.1-fold higher (GluR5 in RA) than the surrounding brain subdivisions. Indeed, among the 84 possible combinations (21 subunits × 4 cerebral vocal nuclei) close to half (37/84 = 44%) showed higher or lower differential expression in vocal nuclei (Fig. 4A).
To compare the differential expression of glutamate receptors in vocal areas relative to nonvocal areas, we quantified the extent of possible differential expression within cerebral auditory areas. We chose auditory regions, because a few subunits/subtypes appeared to have some differential expression relative to the surrounding subdivision (mGluR1 in caudal N for example; Fig. 2), and these regions are involved in auditory processing of learned vocalizations (Chew et al., 1995; Mello et al., 1995). We found 10 of 21 subunits/subtypes with statistically significant differential expression in auditory areas relative to the surrounding cells of each of their subdivisions (asterisk in Fig. 4B). However, the magnitude of these differences was much smaller (1.21-fold lower to 1.75-fold higher) than that found for vocal nuclei (7.7-fold lower to 5.1-fold higher). This suggests that, although both vocal and auditory pathways contribute to learned vocal communication in songbirds, high levels of differential expression of glutamate receptor subunits/subtypes within a brain subdivision are unique to vocal nuclei.
The three smaller vocal nuclei (NIf, Av, and MO; Fig. 2) appeared to have very few differences relative to the surrounding brain subdivisions. These were relatively low GluR3 and high mGluR4 in NIf, low mGluR3 in the medial part of the MO, and no recognizable differences in Av (Fig. 3B; mGluR4 in NIf not shown). The differences in MO were variable among animals. It is possible that there are more differences in the smaller nuclei that were not easily recognizable either because of: 1) the relatively smaller vocal nuclei and thus their potential for being missed in brain sections or 2) the location of NIf sandwiched between L2 and L1; the latter regions show expression differences between them, of which NIf expression could easily blend into the background with one or the other. Thus, the absence of differential expression in these small vocal nuclei should be viewed with caution.
A number of glutamate receptor subunits/subtypes also showed differential expression in the noncerebral vocal nuclei, DLM and DM. In DLM, nine subunits showed differential expression that ranged from 3.7-fold lower to 2.0-fold higher in the anterior part of this dorsal thalamic region (aDLM) compared with its cells more posteriorly (Figs. 3A, ,4A).4A). This included NR2D, the subunit not differentially expressed in the cerebral vocal nuclei, which was higher in aDLM. In birds, generally, the term DLM is used to refer to the entire lateral nucleus of the dorsal thalamus. In songbirds, DLM is also often used to refer to the entire region, but as a vocal nucleus. However, the exact boundaries of a putative vocal part of DLM in songbirds, we believe, might not have been consistently delineated well in prior connectivity studies (Okuhata and Saito, 1987; Johnson et al., 1995; Vates et al., 1997; Luo and Perkel, 1999a,b; Luo et al., 2001). Apparently, the differential expression patterns of glutamate receptor subunits/subtypes may have revealed well-defined boundaries of a vocal part of DLM (Fig. 3A). Only two glutamate receptor subunits, GluR1 and GluR5, appeared to show differential expression in the midbrain vocal nucleus DM compared with the adjacent midbrain (Figs. 3A, ,4A;4A; GluR1 not significant at P =.01).
We found on regression analysis that GluR5 and mGluR2 were consistent outliers (Fig. 4C; determined by using one-by-one simple regression analysis for an outliers search). When these outliers were removed from the analysis, significant correlations were found among the differential expressions of the remaining genes (Fig. 4D). The differential expression levels in the four cerebral vocal nuclei (HVC, RA, MAN, and Area X) all showed positive relationships with each other (Fig. 4D). These relationships were strongest among the pallial vocal nuclei (HVC, RA, and MAN; Fig. 4D) and between Area X and MAN (data not shown). The differential expression levels between Area X and aDLM showed a negative relationship with each other (Fig. 4D).
Differences were observed between the lateral and the medial parts of vocal nuclei in the anterior vocal pathway. Among the 11 subunits/subtypes with differential expression in lateral MAN (lMAN; Fig. 4A), only two subtypes had the same differential expression in medial MAN (mMAN), these being lower mGluR1 (Fig. 3C) and higher mGluR8 (not shown). Among the nine subunits/subtypes with differential expression in Area X (l-Area X; Fig. 4A), only six subunits had the same differential expression in medial area X (m-Area X), these being higher NR2A (Fig. 3C), lower mGluR3, and higher mGluR4 and mGluR5 (not shown). These findings are consistent with the previous observation that singing causes different patterns of motor-driven gene expression in the lateral and medial parts of MAN and Area X (Jarvis et al., 1998). There were no differential expression patterns in mMAN or m-Area X that were not present in their lateral counterparts.
The vocal nuclei are known to be composed of several types of neurons that perform different physiological functions and have different projections (Alvarez-Buylla and Kirn, 1997; Mooney, 2000; Perkel and Farries, 2000; Hahnloser et al., 2002). Some glutamate receptor subunits/subtypes appeared to have differential expression restricted to a particular cell type within a vocal nucleus. For example, GluR4 (AMPA type) and mGluR1 (metabotropic type) in Area X were expressed mainly in sparse, large cells (Fig. 3D, arrowheads), suggesting expression in Area X neurons that project to DLM. In the striatum surrounding Area X (Fig. 3D, area to the right of the dashed lines), both large and smaller cells expressed high levels of these same genes. This suggests that the unique differential expression in Area X is due to lower levels of these subunit/subtype in the smaller, spiny neurons. Both GluR1 and GluR4 (AMPA types) in HVC were expressed in a sparse cellular distribution (Fig. 3E, arrowheads), suggesting that the differential expression in HVC is due to lower expression in one of its cell types (X-projecting, RA-projecting, and/or interneurons) compared with the surrounding nidopallium. Double-labeling experiments are necessary to verify these suggestions.
The expression patterns that we observed were very stable among all animals examined, except for four subunits: GluR3 (in HVC, l-Area X, and aDLM), NR1 (in l-Area X and aDLM), NR2A (in lMAN) and mGluR3 (in RA; Fig. 4A, v for variable). For GluR3, expression was higher than, lower than, or the same as that in the surrounding subdivision, depending on the individual. For NR1, NR2A, and mGluR3, expression was always higher (four of six animals examined) or similar (two of six animals) to that in the surround. The source of these individual variations has not yet been identified, but potentially they could be due to recent behavioral experience, including social factors or the types of songs the birds produce. They do not appear to be due to recent singing activity, in that all birds did not sing in the 16 hours before sacrifice.
One gene can give rise to many different gene products through alternative mRNA splicing, thereby increasing diversity of the gene's functions. Among the best studied splice variants for glutamate receptors are the AMPA subunits. In mammals, all four AMPA receptor subunits (GluR1–4) are alternatively spliced into at least two forms, called Flip and Flop (Fig. 5A). The Flop spliced forms of the AMPA receptor subunits usually causes the receptors to desensitize faster after channel opening; the Flip forms have the opposite effect (Dengledine et al., 1999; Petralia et al., 1999). We found that, in most cases, either the Flip and/or the Flop splice-specific forms had the same direction of differential expression in vocal nuclei as revealed by the general probes, which hybridize to both Flip and Flop forms (Fig. 5B,C). For example, the low differential GluR1 expression in HVC appeared to be due to the Flip form and not the Flop form (Fig. 5C). However, in some cases, the Flip and Flop forms had differential expression in opposite directions (one higher and one lower than the surrounding subdivisions) that were not revealed by the general probe alone. For example, the general probe for GluR2 showed expression levels similar to those of the surrounding brain subdivisions in HVC and l-Area X, whereas the GluR2-Flip probe showed expression that was lower in HVC and l-Area X, and the GluR2-Flop probe showed expression that was higher in HVC and l-Area X relative to the surrounding brain subdivisions (Fig. 5B,C). This suggests that the general probe canceled the detection of the Flip and Flop differential expression in vocal nuclei. Similar opposite patterns of Flip and Flop expression were found for GluR3 in HVC and l-Area X (Fig. 5C). Thus, diversity of expression in splice variants leads to more diversity of differential gene expression in vocal nuclei. This is the first finding that we are aware of demonstrating differential mRNA splicing that is specific to vocal brain areas.
We obtained several antibodies that recognize specific subunits/subtypes of several glutamate receptors in mammals (GluR1 and mGluR1). We found that anti-rat GluR1 and anti-rat mGluR1 antibodies recognized proteins of similar molecular weight in Western blots of zebra finch and rat brains (Fig. 6A). For brain sections, we observed zebra finch protein expression patterns similar to the mRNA expression patterns, including differential expression in vocal nuclei (Fig. 6A). However, GluR1 protein levels appeared to be more differentially expressed in l-Area X (higher) and RA (lower), but less so in HVC and lMAN (Fig. 6A) relative to the mRNA (Fig. 2); mGluR1 protein levels appeared to be similar to the mRNA, with the exception that protein in RA was much lower than the surrounding arcopallium, whereas the mRNA was not. Both GluR1 and mGluR1 proteins were present in neuropil and cell bodies (Fig. 6B). However, GluR1 protein in Area X was much higher in its neuropil than in the neuropil of the surrounding striatum; the opposite pattern was seen for mGluR1 (Fig. 6B) with cell body expression restricted to sparse large neurons as seen with the mRNA (Fig. 3D). Expression in neuropil is expected of glutamate receptors, given that they are usually transferred to dendrites as parts of functional receptor proteins. In general, unique differential expression in vocal nuclei at the mRNA level appears to be reflected at the protein level.
We examined whether differential expression of glutamate receptor subunits/subtypes in vocal nuclei relative to the surrounding brain subdivisions is unique to songbirds or occurs in vocal nuclei of other vocal learners, hummingbirds and parrots. We selected one subunit/subtype that had the most differential expression in zebra finch vocal nuclei from each of the four families (GluR1 from the AMPA family, GluR5 from the kainate family, NR2A from the NMDA family, and mGluR2 from the metabotropic family) and hybridized them to brain sections of the sombre hummingbird and budgerigar. The zebra finch subunits/subtype cross-hybridized to the hummingbird and parrot homologs under the same high-stringency in situ hybridization conditions as used for zebra finch brain sections (Figs. 7, ,8).8). Although they are not identical, the general expression patterns of these four subunits/subtype in regions outside the vocal systems of the hummingbirds and parrots was similar to the pattern in the zebra finch, including in auditory areas (Figs. 7, ,8).8). As with the zebra finch, these other vocal learning species had differential expression of the glutamate receptor subunits/subtype in their vocal nuclei, with some similarities and some differences among them as follows.
We observed that two (NR2A and mGluR2) of the four subunits had differential expression in the sombre hummingbird cerebral vocal nuclei with some similarities to zebra finches (Figs. 7, 9A,B). This included higher NR2A expression in VLN (HVC-like nucleus), VA (RA-like), and VAN (MAN-like); higher mGluR2 expression in VLN (HVC-like); and lower mGluR2 expression in VA (Figs. 7, ,9B).9B). In addition, for mGluR2, the posterior part of the hummingbird VAN nucleus (VAN-P) had lower expression, whereas the anterior part (VAN-A) had higher expression relative to the surrounding nidopallium, and this dual pattern was unique to hummingbirds (Figs. 7, ,9B).9B). The magnitude of the differential expression levels in hummingbird vocal nuclei was not as great as that seen for zebra finches (Fig. 9A,B); however, the differential expressions in vocal nuclei were still prominent (Fig. 7, NR2A and mGluR2). Unlike the case for zebra finches, there was no detectable differential expression of the four subunits/subtypes in the hummingbird VASt vocal nucleus (Area X-like structure in the striatum; Figs. 7, ,9B).9B). For noncerebral brain areas, similar to zebra finches, GluR5 expression was differentially and prominently expressed in DM (Fig. 7).
We observed that two (NR2A and GluR1) of the four subunits had differential expression in budgerigar cerebral vocal nuclei with some similarities to zebra finches (Figs. 8, 9A,C). This included higher NR2A expression in NLC (HVC-like) and lower GluR1 in NLC (HVC-like) and NAO (MAN-like) nuclei relative to the surrounding brain subdivisions (Figs. 8, 9A,C). There were, however, no other differences similar to the zebra finch. The MO vocal nucleus in parrots is much larger than in songbirds and hummingbirds, and this nucleus in parrots showed relatively prominent lower GluR1 and higher NR2A differential expression (Figs. 8, ,9C).9C). As with hummingbirds, there was no detectable differential expression of any of the four subunits/subtypes in the parrot MMSt vocal nucleus (Area X-like structure in the striatum; Figs. 8, ,9C9C).
In considering together the results from the three species, whenever NR2A was differentially expressed in vocal nuclei, it was always higher than in the surrounding brain subdivision; mGluR2 was higher in the nidopallium vocal nuclei (HVC-like and/or MAN-like) and lower in the arcopallium vocal nuclei (RA-like); and GluR1 tended to be lower, except in the striatal vocal nucleus of zebra finches (higher in Area X). Zebra finches showed more differential expression differences than the hummingbird and parrot species examined, with GluR5 being exceptionally high in RA of zebra finches.
We wondered whether differential expression of glutamate receptors in vocal nuclei is unique to vocal learners. We hybridized the same set of four glutamate receptor subunits/subtype to pigeon and ring dove brain sections containing the midbrain vocal nucleus DM, the thalamic region DLM, and cerebral regions in which forebrain vocal nuclei have been found in vocal learners. Doves produce only innate vocalizations (Nottebohm and Nottebohm, 1971; Baptista, 1996). The general brain expression pattern, including auditory areas and primary sensory regions (L2, entopallium, and basorotralis) of these vocal nonlearning species, was similar to that of songbirds, hummingbirds, and parrots (Fig. 10). However, there were no cerebral differential expression patterns in areas where vocal nuclei are found in vocal learners (Figs. 9D, ,10).10). Thus, highly differential expression within cerebral subdivisions appears to be unique to vocal nuclei of vocal learners. The doves had differential expression in thalamic and midbrain regions as found in the vocal learners. This included lower GluR1 and higher NR2A in the anterior part of DLM in a location similar to aDLM of songbirds and the conserved higher GluR5 in DM (Fig. 10, insets; DM data not shown). The lower GluR1 in the aDLM-like region was similar to that in the zebra finch, but the higher NR2A was in the opposite direction to that found in the zebra finch. Thus, differential expression in thalamic and midbrain vocal nuclei appears not to be unique to vocal learners.
We believe these findings to be a striking example of differential gene expression of nearly an entire gene family in systems that control a complex behavior, learned vocalization. Nearly all glutamate receptor subunits/subtypes showed differential expression in one or more cerebral vocal nuclei. Prior studies have shown differential expression of some genes in songbird and parrot vocal nuclei (Arnold et al., 1976; Bottjer, 1993; Ball, 1994; Casto and Ball, 1994; Aamodt et al., 1995; Holzenberger et al., 1997; Durand et al., 1998; Denisenko-Nehrbass et al., 2000), including the three previously cloned glutamate receptor subunits, NR1, NR2A, and NR2B from songbirds (Singh et al., 2000; Heinrich et al., 2002). However, none have shown systematic differential expression of nearly one entire gene family and across all vocal learning orders. We note that, in these other studies, the differential expression tends to be of genes involved in neural transmission (tyrosine hydroxylase, D1 dopamine receptor, and enkephalin). We suggest that the neural transmission machinery is generally specialized in the vocal nuclei of vocal learners. Below we present the significance of our findings for zebra finch vocal nuclei synaptic transmission and plasticity, for the possible mechanism of vocal nuclei differential gene expression, and for the evolution of vocal learning.
When glutamate binds to its receptors, two types of events occur: 1) chemical transmission of electrical activity for perception and production of behavior and 2) plasticity via second messengers that alters molecules and gene expression. For the ionotropic receptors (AMPA, kainate, and NMDA), this involves influx of cations (K+, Na+, and Ca2+), whereas, for the metabotropic receptors (mGluR1–8), this involves activation of intracellular signals via G-proteins coupled to their intracellular side (Pin and Duvoisin, 1995; Dengledine et al., 1999). Because glutamate receptors are highly conserved across species, as reinforced by this study, their functions across species are thought to be homologous (Dengledine et al., 1999). Thus, because we determined the brain expression patterns of nearly the entire known gene family in the zebra finch, although not in specific cell types, we believe that it is possible to make informative and testable predictions about relative synaptic transmission functions in zebra finch vocal nuclei. We discuss each receptor subfamily separately.
AMPA receptors are responsible for fast synaptic transmission (milliseconds). They are thought to form homo- and heterotetramers (Dengledine et al., 1999), where GluR2 combines with GluR1, GluR3, or GluR4. In the mammalian cerebrum, GluR2 is the most uniform and ubiquitous, whereas GluR1, -3, and -4 are more selectively expressed (Petralia et al., 1999). This is similar to the zebra finch pattern found here (Fig. 2, AMPA panel). GluR4-containing receptors generally have the fastest desensitization time and GluR1 the slowest (Dengledine et al., 1999), and GluR2 combined with GluR3 has a faster desensitization time than GluR2 combined with GluR1 or GluR4 (Dengledine et al., 1999). Thus, given the zebra finch expression patterns (Fig. 2, AMPA panel), the motor vocal nuclei HVC and RA potentially could be dominated by GluR2/GluR3 heteromers resulting in faster desensitization times than the surrounding brain areas, with higher GluR2/GluR1 and GluR2/GluR4 heteromers. The zebra finch vocal nucleus lMAN has less expression of all four AMPA subunits relative to the surround, which would suggest that fast transmission is not as prominent a feature of zebra finch lMAN. AMPA currents have been found in zebra finch lMAN, HVC, and RA (Stark and Perkel, 1999), but relative desensitization and synaptic transmission times have not been compared. Area X would be expected to have a lower number of GluR2/GluR4 receptors relative to the surrounding striatum that is compensated for by a higher number of GluR2/GluR1 receptors (Fig. 2, AMPA panel), potentially giving Area X cells (probably its small spiny neurons) more slowly desensitized AMPA receptors than those in the surrounding striatum. The large cells of Area X, presumably the projection neurons to DLM (Perkel et al., 2002), appear to have high levels of GluR4, as does the surround (Fig. 3D) and, thus, should have as fast a desensitization time as the surrounding striatum. The anterior part of DLM, with its selectively high expression of GluR4 (Fig. 3A), would be expected to have faster desensitization times. The Flop spliced form of the AMPA receptor subunits usually causes the receptors to desensitize faster after channel opening than the Flip form (Dengledine et al., 1999; Petralia et al., 1999). Zebra finch vocal nuclei tend to have higher levels of the Flop forms and lower levels of the Flip forms relative to the surrounding brain subdivisions in HVC and Area X (except GluR1 in Area X; Fig. 5C), and this may lead to even faster desensitization than the surrounding subdivisions. Both forms are consistently low in lMAN.
Kainate receptors also form homo- and heterotetramers, where GluR5–7 usually combine with either KA1 or KA2 (Hollmann, 1999). In the mammalian cerebrum, only KA2 is abundant and uniformly distributed (Wisden and Seeburg, 1993). This is the case for KA2 in the zebra finch cerebrum found here (Fig. 2, kainate panel). A difference from mammals is that zebra finch KA1 is also relatively abundant and uniform. As with AMPA receptors, kainate receptors are rapidly activated (milliseconds); GluR5 desensitizes the most quickly but takes the longest to recover (seconds; Dengledine et al., 1999). Thus, it is likely that cells in zebra finch RA, with a high level of GluR5, have rapid activation and slow recovery of kainate receptors relative to the surrounding arcopallium. In the mammalian hippocampus, kainate receptors down-regulate GABAergic interneuron inhibition (Rodriquez-Moreno and Lerma, 1998). During singing, zebra finch RA has phasic firing governed by brief bursts (Yu and Margoliash, 1996). RA's GABAergic interneurons were proposed to control this phasic firing, transforming afferent nonphasic HVC activity into phasic RA activity (Spiro et al., 1999). Perhaps GluR5 is regulating RA's GABAergic inhibition in RA as it does in the mammalian hippocampus.
NMDA receptors are in part responsible for slow synaptic transmission (milliseconds to seconds) and also form homo- and heterotetramers, where NR1 combines with NR2A, -B, -C, or -D (Cull-Candy et al., 2001). As in mammals (Standaert et al., 1994), zebra finch NR1 expression is the most uniform and ubiquitous, whereas NR2A and NR2B are more restricted and are present mainly in the cerebrum, and NR2C and NR2D are in mainly cerebral pallidal cells, as well as in the thalamus, brainstem, and cerebellum (Fig. 2, NMDA panel; cerebellum not shown). The different NR1 receptor combinations from fastest to slowest in deactivation times are NR1/NR2A > NR1/NR2B > NR1/NR2C > NR1/NR2D (Dengledine et al., 1999). NR3A has been proposed to modulate the activity of NR1/NR2A and NR1/NR2B receptors (Das et al., 1998). Thus, given that adult zebra finch pallial vocal nuclei (HVC, RA, and lMAN) have higher NR2A and lower NR2B relative to their respective brain subdivisions, they may have faster NMDA deactivation times relative to the rest of the cerebrum. This suggestion is supported by differences we noted from another study (White et al., 1999) in which adult NMDA excitatory postsynaptic potential (EPSP) decay times are on average three to four times faster in vocal nuclei than in the hyperpallium (or Wulst; 58 msec in RA, 48 msec in lMAN, and 185 msec in the hyperpallium). Pharmacological studies do indicate that zebra finch lMAN axons synapse onto NMDA receptors of RA neurons and HVC axons synapse onto AMPA and NMDA receptors of RA neurons (Mooney and Konishi, 1991; Stark and Perkel, 1999). Given this knowledge, our expression findings suggest that the lMAN input will be found mainly on NR1/NR2A NMDA receptors in RA, whereas the HVC input mainly on both NR1/NR2A NMDA and GluR2/GluR3 AMPA receptors in RA. The zebra finch striatal vocal nucleus (l-Area X) has NR2A and NR1 levels usually higher than those in the surround. This would theoretically result in l-Area X having slower NMDA deactivation times than the pallial vocal nuclei.
Our findings also provide insight into behaviorally induced immediate early gene synthesis. NMDA receptors in mammals regulate activity-dependent induction of ZENK (an acronym for Zif-268, Egr-1, NGFI-A, and Krox-24) and c-fos synthesis in neurons (Lerea, 1997). As singing induces expression of these genes in cerebral vocal nuclei (Jarvis and Nottebohm, 1997; Kimpo and Doupe, 1997), our results suggest that the induction is likely mediated through NR1/NR2A receptors for all four large cerebral vocal nuclei, and additionally through NR1/NR2B receptors for Area X (Fig. 2, NMDA panel). Interestingly, all brain areas that do not express high ZENK regardless of activity (i.e. DLM, Ov, L2, E, B, and P; Jarvis et al., 1998) have selective high NR2D expression (Fig. 2, NMDA panel, Ov and E not shown).
These receptors are classified into three groups. Group I (mGluR1 and 5) couples phosphatidylinositol hydrolysis to increased intracellular Ca2+ release, cAMP activation, and K+-channel inhibition (Pin and Duvoisin, 1995). Groups II (mGluR2 and 3) and III (mGluR4, -6, -7, and -8) tend to have the opposite effect. Because of these indirect actions, metabotropic glutamate receptors take longer to affect membrane currents than ionotropic receptors. Furthermore, they are usually farther away from sites of glutamate release and as such need high-frequency presynaptic stimulation to be activated postsynaptically. In this manner, metabotropic receptors have a strong modulatory role in synaptic transmission via signal transduction (Geiger et al., 1999). We found that group I receptors are generally lower in zebra finch cerebral vocal nuclei relative to the surround, whereas groups II and III are higher, mainly in HVC and/or RA. This suggests that the overall effect of metabotropic receptor activity in zebra finch vocal nuclei will be stronger inhibition of cAMP and intracellular Ca2+ release and higher activation of K+-channel outflux. Such modulation would result in stronger hyperpolarization relative to the surrounding brain areas. This prediction is supported by strong and lasting inhibition seen in HVC and RA after singing (McCasland and Konishi, 1981; McCasland, 1987; Yu and Margoliash, 1996) and by relatively unique pharmacological findings in HVC, where group II and III agonists generate long-lasting hyperpolarizing currents, whereas group I agonists have no effect (Dutar et al., 2000).
Many groups have reported that large numbers of genes are expressed with region specific patterns in the vertebrate brain (Eberwine et al., 1992; Watson and Margulies, 1993; Livesey and Hunt, 1996; Berke et al., 1998; Zirlinger et al., 2001). Some of this variability is thought to be due to differences in promoter regions among genes. Some studies have found unique species-specific differences in promotor regions in the same gene, such as the vasopressin neurotransmitter receptor, which control species-specific differential brain expression patterns (Young et al., 1999). In the case of glutamate neurotransmitter receptors, many receptor subunits/subtypes are thought to possess multipromotor sequences that are regulated by various combinations of transcription factors and CpG methyl islands, the latter of which are regulated by methylation and acetylation (Myers et al., 1998; Huang et al., 1999, 2002). Our findings suggest that cells located in the vocal nuclei may possess a unique set of transcription factors and/or genetic modifications to glutamate receptor promoters allowing for unique differential expression in those cells. In support of this idea, a vocal-nucleus-specific protein has been found in Estrilidine finches (Akutagawa and Konishi, 2001). Neurotransmitter receptors also regulate expression of different genes via cell signal transduction cascades (Sheng and Greenberg, 1990; Bading, 1999), so the differential expression of glutamate receptors in vocal nuclei is expected to lead to downstream consequences in the expression of the genes they regulate. In support of this idea, the BDNF gene, which requires activation through NMDA receptors for its expression (West et al., 2001), is synthesized at lower basal levels in songbird nuclei HVC, RA, and lMAN than in the surrounding brain areas (Li et al., 2000; Li and Jarvis, 2001).
Several hypotheses have been proposed for the evolution of cerebral brain nuclei for vocal learning. One hypothesis is that, because cerebral vocal nuclei have not been found in vocal nonlearners, then songbirds, hummingbirds, and parrots each evolved their similar cerebral vocal nuclei independently from a common ancestor (Fig. 1A, red dots; Brenowitz, 1997; Jarvis et al., 2000). If true, our results then further suggest that differential expression of glutamate receptors in vocal nuclei coevolved in manners similar but not identical to the vocal neural systems of each group independently. This hypothesis is supported by the absence of such cerebral differential expression in vocal nonlearners. The second hypothesis is that, because the vocal nuclei are similar among vocal learners, then either vocal nonlearners lost their cerebral vocal systems multiple independent times or vocal non-learners have cerebral forebrain nuclei that have not been detected in prior studies (Fig. 1A, green dots; Brenowitz, 1997; Jarvis et al., 2000). If true, then our results would further suggest that differential expression of glutamate receptor subunits/subtypes in cerebral vocal nuclei were lost in vocal nonlearners and that the differences found in the vocal learners were the result of divergence.
The differential expression levels found in vocal nuclei of hummingbirds and parrots are not as prominent as they are in zebra finches. These differences could be related to the type of learned vocal behavior they display, and we are currently testing this hypothesis. Some differences could also be related to differences in cell types in comparable vocal nuclei among vocal learning orders. Given these possibilities, though, the commonality among vocal learners is still that the vocal nuclei show prominent differential gene expression relative to the brain subdivision in which they are located, a property not common in other brain regions of each subdivision.
Of the above two hypotheses, independent evolution of cerebral vocal nuclei and vocal learning is the most commonly accepted. If this is correct, then the mechanisms of how such differential expression in vocal nuclei evolved require explanation. We propose an explanation that we call the vocal connectional evolution hypothesis: To date, in vocal nonlearners, no cerebral motor connections have been found to project to brainstem vocal nuclei (nXIIts and DM; Wild, 1997; Wild et al., 1997). However, vocal non-learners have differential glutamate receptor expression in noncerebral vocal nuclei. We suggest that differential expression in cerebral vocal nuclei of vocal learners could have been stimulated after a mutational event caused a connection from the arcopallium into the brainstem vocal nucleus DM. Because connectivity of brain pathways outside the vocal system is similar to that within the vocal system (Karten, 1967, 1968; Margoliash et al., 1994; Mello et al., 1998; Iyengar et al., 1999; Farries, 2001; Iyengar and Bottjer, 2002), once the arcopallium is connected with the brainstem vocal nuclei, this presumably can induce a feedback cascade of specializations in a preexisting cerebral network. This hypothesis requires only a few mutational changes that can be selected upon (selection for specific neural connections), followed by normal interactions of neural activity and gene expression in pre- and postsynaptic neurons. This hypothesis is consistent with our correlation findings. When one vocal nucleus has higher or lower expression of a particular subunit, one or more of the connecting vocal nuclei tend to have similar specializations.
Parallels have been noted between avian vocal learning brain pathways with human language brain regions (Doupe and Kuhl, 1999; Jarvis, 2004a,b), including the presence of a connection from the human face motor cortex to the brainstem vocal motor neurons (nucleus ambiguous); this connection is weak to absent in nonhuman primates (Kuypers, 1958a,b; Deacon, 1997). Given our findings of vocal nuclei specializations in vocal learning birds, it is not going too far to suggest that vocal learning mammals, including humans, also evolved unique differential expression of glutamate receptors in vocal areas of their cerebrums and that these will not be found in vocal non-learning mammals.
Grant sponsor: the Whitehall Foundation (E.D.J.); Grant sponsor: the National Science Foundation (E.D.J.); Grant sponsor: the Japan Ministry of Education, Science, and Culture (M.H.); Grant sponsor: Japan Scholarship Association Award (K.W.).
We thank Michael McElroy for sectioning ring doves brains and Jarvis laboratory members for critical reading of the manuscript. We also thank Drs. Richard Mooney, Michael Ehlers, and V. Anne Smith for critical reading and useful comments and Dr. Minmin Luo for consultation on aDLM location in songbirds.
1This list incorporates new terminology recommended by the Avian Brain Nomenclature Forum (Reiner et al., 2004). Online translation from the old terminology can be found at www.avianbrain.org
Abbreviations: A, arcopallium; AAC, central nucleus of the anterior arcopallium; Area X, Area X of the striatum; Av, avalanch; B, nucleus basorostralis; Cb, cerebellum; CM, caudal mesopallium; CMM, caudomedial mesopallium; CMS, caudal medial striatum; CN, cochlear nuclei; CSt, caudal striatum; DLM, medial nucleus of dorsolateral thalamus; DLMv, medial nucleus of dorsolateral thalamus, vocal part; DM, dorsal medial nucleus of the midbrain; DMm, magnocellular nucleus of the dorsomedial thalamus; E, nucleus entopallium; H, hyperpallium; HVC, (a letter based name); MOc, oval nucleus of the mesopallium complex; ICo, intercollicular nucleus; INP, intrapeduncular nucleus; L2, field L2; MMSt, magnocellular nucleus of the anterior striatum; M, mesopallium; MAN, magnocellular nucleus of anterior nidopallium; MLd, mesencephalic lateral dorsal nucleus; N, nidopallium; NAOc, oval nucleus of the anterior nidopallium complex; NCM, caudal medial nidopallium; NIf, interfacial nucleus of the nidopallium; NLC, central nucleus of the lateral nidopallium; nXIIts, tracheosyringeal subdivision of the hypoglossal nucleus; OT, optic tectum; Ov, nucleus oviodalis; P, pallidum; PAm, nucleus parambiguus; PSL, pallial subpallial lamina; RA, robust nucleus of the arcopallium; RAm, nucleus retroambiguus; St, Striatum; Uva, nucleus uvaeformis; VA, vocal nucleus of the arcopallium; VAN, vocal nucleus of the anterior nidopallium; VASt, vocal nucleus of the anterior striatum; VLN, vocal nucleus of the lateral nidopallium
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Format: Xbox 360
Developer: Ruffian Games
Publisher: Microsoft Games Studios
Release date: (scheduled) 9 July 2010
The word most associated with the open-world genre of video games is "freedom". Each new sandbox title that is released into circulation promises non-linear gameplay and unmatched freedom, and yet players soon find themselves rubbing up against the boundaries of a plot or a series of objectives which enforce a structure on them. This structure isn't necessarily a bad thing. In fact, in some open-world games, such as the superb Red Dead Redemption, it works very well. It's just worth noting that unbridled freedom is usually not on offer to players, no matter what the blurb on the back of a game's packaging says.
A notable exception in this regard is Crackdown. Developed by Realtime Worlds, Crackdown put the player in the shoes of a lab-grown law enforcement Agent – a cross between RoboCop and an NFL linebacker – tasked with cleaning up a crime-ridden futuristic metropolis called Pacific City. While this premise contained all the dramatic heft of thin air, it translated into hours and hours of fun. Crackdown had only one objective – 'kill all criminals' – and it bestowed complete freedom to the player concerning the time in which they accomplished this and how exactly they went about it. Due to its loose, free-roaming gameplay, and also its lack of plot and characterisation, Crackdown placed very few constraints on the player. The only aspect that offered any sort of structure was the fact that the more carnage the player unleashed, the stronger their character in the game would become.
On its release in 2007, Crackdown's main appeal was that it came packaged with the beta code for Halo 3's multiplayer. However, the game became a cult favourite as its particular brand of cartoon mayhem seduced millions of players. With those sort of sales figures, a sequel was perhaps inevitable. With Realtime Worlds moving on to develop what sounds like an MMO version of Crackdown for the PC in the form of APB, Microsoft has tapped up British developer, Ruffian Games for Crackdown 2. According to producer James Cope, the sequel has been in development for about 15 months, and while Ruffian has made its own additions to Crackdown's template, he says that the game's overall sense of freedom remains intact.
"We wanted to make a game that kept the Crackdown core experience," he says, "but then make it bigger, better, more. It was about what was important in Crackdown – the feel of the game – and the fact that you got stronger the more you played. That's something we really wanted to expand on. The Crackdown experience was so great for players, that our mission was basically to ensure, more players got to experience it."
The 'core Crackdown experience' Cope speaks of is largely unchanged from the first game. Players once again take on the role of an Agent let loose on the nefarious elements of Pacific City. Once again the city is in chaos and there are very few constraints on the player in how they tackle the game's central mission of clearing up the streets. Crackdown 2 is a free-roaming, third-person-shooter with driving and parkour elements tossed in for good measure and as players explore the city, shooting, punching and kicking their way through armies of enemies, they grow stronger through the power-ups dotted around the city and those that spill out of their victims. Cope says that it was important that the player's experience remains as uncluttered by plot as the in the last Crackdown – even though, in Crackdown 2, the story has a couple more layers to it than in the first game.
"I think we're unique in that we are a true sandbox game in the sense that the environment is wide open and the player is completely free," he says. "What we've found we've had to do in terms of story is that we have to provide a lot of background and context for our world."
Crackdown 2 is set ten years after the events of its predecessor, which saw The Agency – a conglomerated law enforcement organisation – use its superpowered Agents to brutally wipe out the three criminal gangs controlling Pacific City. It seems, however, that the Agency's designs on urban improvement have backfired horribly. Pacific is now in the grip of two new threats. The first of these is The Cell, a group of self-styled freedom fighters trying to break The Agency's control in the city. The second is the Freaks, nightmarish creatures who can be hurt be UV rays, and so roam the streets of the city exclusively at night.
In order to destroy both groups, The Agency has dispatched its superpowered Agents (ie the player) to activate a massive weapon called Project Sunburst. In order to do this, Agents need to secure strongholds and realign radar dishes that are currently in the hands of The Cell. Once they manage to do this, the dishes transmit information about Freak hives – vast subterranean dens filled with Freaks – which the player then infiltrates. The Agency drops a giant UV bomb into the centre of it and the player has to defend the bomb against hordes of Freaks as it arms and then explodes, obliterating the contents of the hive.
While all of this sounds a little more complex than mowing down gang members in the first Crackdown, Cope says that Crackdown 2's plot is brutally simple, and that Ruffian are avoiding the danger of the story intruding too much on the central gameplay.
"We're very clear about the game's goals and the player's objectives right from the beginning; everyone knows what they're working towards," he says. "The player's the driving force here; they'll learn a lot about the world they're running around in – if they choose to. We can't tell the story through our character; the player's avatar is a nobody, he's a clone. We explain what happening in the world through the way the world looks."
As Cope points out, Pacific City's dilapidated veneers speaks volumes. The Agency has been battling both The Cell and the Freaks for some time now, and this has taken its toll on the city. While the streets were arguably no safer when the three crime cartels were in control in the first Crackdown, the Pacific City enjoyed at least a small measure of growth. Now it looks like a war zone; the city is filled with ruined buildings, segments of its freeway have collapsed and tower block windows glow as fires burn away inside them.
"It's a bleak time in Pacific City," says Cope. "It's gone through a horrible time in which what little prosperity it had in the first game is now completely gone. Now you have this war-torn environment where The Cell fighting The Agency has destroyed a lot of the city. You've also got these huge caverns under the city filled with the Freaks, who just run riot through the streets at night."
Pacific City's burned out skyline is just one of the many changes Ruffian Games has made. Cope says that aesthetic changes to the game's environment aren't simply for show. "We wanted the world to tell the story of what has happened and so the way it looks is very important," he says. "Everything in the city is there for a purpose. The world will change and evolve as you play the game."
"We believe in complete freedom and that Crackdown is a completely open-world," continues Cope. "We want to make sure that the player can approach any objectives in any order they choose – it's all down to them and how they play. The problem you have with that is, the moment you introduce a story into the free-form environment, you sacrifice some of the player's freedom – we don't want to do that."
The player's sense of freedom has obviously been a key consideration for the developers in Crackdown 2's construction. While the game boasts slightly more structure than its predecessor, Cope says that the overall remit for Ruffian was to remove anything that got in the way of the player having fun. One notable change that has been made from the first game is that players no longer need to worry about civilian casualties. In the first game, players gained no power-ups by killing innocent bystanders, and running rampant through the streets and indiscriminately killing innocent bystanders. This isn't the case in Crackdown 2, where players gain strength for dispatching any living creature unfortunate enough to get in their way. Ruffian has also made levelling-up the player's driving ability a lot easier. Agents are no longer forced to seek out enemies to run down to gain driving power-ups as not only are civilians fair game, but crowds of Freaks swarm the streets at night providing a huge number of opportunities to convert roadkill into levelling-up currency.
"We really hate slapping players," Cope explains, "if they're enjoying themselves, why slap them? It breaks the fiction of being a supercop, a little bit, but the outcome is more fun for the player, which is our key aim."
This is perhaps the biggest change Ruffian has made to Crackdown's brutally straightforward template; they've removed anything from the first instalment that got in the way of the player enjoying themselves. On top of this they've enhanced and widened the in-game experience by adding a plethora of new weapons, new equipment and new vehicles. Players have access to an arsenal which starts with standard machine-guns, shotguns and pistols and then gradually expands to include satisfyingly nasty items such as turret guns, rocket launchers and UV shotguns which reduce Freaks to ashes with one blast. They will be able to take to the skies with the Agency glide suit, which allows them to extend free-falls and glide the city, or they can use Mag Grenades to slingshot vehicles at opponents. Crackdown 2 promises a ton of vehicles too, including SUVs, supercars, buggies and tanks.
All told, things are shaping up well for Crackdown 2 ahead of its planned July release. Pacific City may look more rundown than ever, but it's still the perfect game for players who relish an unconstrained and joyously chaotic experience. Cope says that the only players who may be put off by it are those who expect a very regimented structure to their games. "It has structure in the missions, but it also has that goofing off, free-roaming nonsense."
"I really hope that everyone tries it out," Cope continues, "because using your imagination and having fun puts you right back to the experience of when you were a kid, running into the playground at break-time. It's that 'aaaaah! This is brilliant!' type of fun at the beginning, and then making your own enjoyment on your own terms."


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Browse through modern bathroom photos and discover vanities, bathroom sinks, bathtubs, toilets and showers for your bath remodel. Whatever the case may be, we have 121 bathroom designs and décor ideas to help foster the right style you're searching for.
not so dark but love raw stone wall 30 Luxury Shower Designs Demonstrating Latest Trends in Modern Bathrooms
Country House is a residential project completed by Aleksandra Fedorova Bureau. The home is located in Moscow, Russia. Photos courtesy of Aleksandra Fedorova Bureau
A Cleverly Decorated Family Home In Ukraine Designed by Yevhen Zahorodnii & Sivak Trigubchak, this 180 square meter home offers some exciting inspiration. Fascinating decor stands out against its.
Like: Large tiles, space-saving toilet Source: 22 Small Bathroom Remodeling Ideas Reflecting Elegantly Simple Latest Trends
It's possible for you to freshen up your bathroom decor with the addition of a favourite plant or flower. Nautical bathroom decor is the simplest and most affordable theme available for an entire bathroom makeover
Turn to the vanity to introduce wooden element into the modern bathroom [Design: Urban Angles]
minimalist monochrome bathroom modern bathroom colors dark gray wall paint tile flooring Eyebrow Makeup Tips
Light grey tile floor running behind bath; then right side exposed brick painted white + chunky wooden shelves above hidden cistern displaying plants & hand towels; left side feature wall of dark grey with zuster cabinet
96 Golden Beach Drive is a project completed by SDH Studio. Located on a square foot lot in Golden Beach, Florida this house was designed around a 27 foot high space that would be the heart of this home.


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Mission statements can be valuable if they articulate real targets. Otherwise, they sound too much like a corporate Hallmark card.
For example, consider Microsoft's big goal of "A computer on every desk and in every home, all running Microsoft software." Or Amazon's goal for the Kindle: "Every book ever printed, in any language, all available in less than 60 seconds."
Both statements do something crucial: They quantify the goal.
Nancy Lublin, CEO of Do Something, suggests that leaders write a mission statement with a goal that's an action, not a sentiment.
"If you're trying to sell a product, how and how many? If you're trying to change lives, how and whose?" she says. "Take your wonky mission statement and rip it to shreds. Then write and rewrite the thing until it reflects—in real, printable words and figures—the difference that you want to make."
--Adapted from "How to Write a Mission Statement That Isn't Dumb," Nancy Lublin, Fast Company.


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A most careless action is underestimating another person and using that to needlessly antagonize a potentially dangerous foe.
[quote text_size="small" author="JOHN V CAIN" author_title="6 Methods of Thinking Like an Operative" link="https://vinjabond.com//6-methods-of-thinking-like-an-operative/"]
It's better to overestimate a target, inversely, it's better to be underestimated by your target.
We live in a sad world that an article like this can exist and be useful. It shouldn't have to be said that it's not right to mess with a stranger for no good reason.
But this isn't about what's right, wrong or ethics, just why you shouldn't for self preservation.
Everyone knows someone or is that someone who offends / aggravates / provokes / abuses a complete stranger who just happens to be present and is undeserved of any malicious attack.
This usually occurs when one is aggressively drunk, in a furious mood or in a state of an unbalanced ego, typically late night on the streets, at a party or a bar.
So these 3 Australian muscle guys walk into a bar…
And realize the only women in the place is sitting with a small geek wearing thick glasses, an oversized sweater and shorts with flip flops at the end of the bar.
The Aussies sit at the other end and order drinks. When ordering another round, they also order 2 drinks for the women. When the waitress arrives and tells them the Aussies ordered the drinks for them, they decline while ignoring the Aussies' attempt at their attention from across the bar by raising their drinks. The women continues their conversation with the Geek.
Several drinks later, 1 of the Aussies walks to the Geek's table and right as his mouth opens up to say something, one of the women politely says "not interested, sorry". Trying to speak again, he's interrupted by the other woman as she simply says "no". He walks away.
Another drink later all 3 of them come up to the Geek's table and start talking to the women and gets shutdown again. They walk away. The entire bar laughs.
Now drunk, the biggest Aussie comes up to the Geek and puts his arm around him and asks him to introduce the women to them, the Geek says "sorry, they're clearly not interested". The Big Aussie tightens his hold on the Geek then throws him off his stool and on to the floor. The Big Aussie takes the Geek's seat and attempts to talk to the women.
The Geek gets up, picks up an empty stool and swings it to the legs of the stool that the Big Aussie was sitting on – not hitting his body as not to cause serious injury.
The stool collapses and the Big Aussie falls to the floor on his back. When raising his head to get up, the Geek smacks his forehead slamming the back of his head on the bar floor then gently rests his knee on his larynx just enough to bruise it. The Big Aussie is out.
The Small Aussie (but still much larger than the Geek) approaches the Geek as if it was an official boxing match with his fists in position. The Geek laughing, infuriates the Small Aussie causing him to set up an obvious haymaker, misses, and another, misses, and another and misses but then trips over the fallen Big Aussie.
The Geek about to be tackled by the Mid Aussie, ducks and right hooks the side of his right knee, tearing his ligaments. The Mid Aussie falls to the floor and cries. The Small Aussie gets up and runs out the bar.
[quote text_size="small" author="Iain Duncan Smith" author_title="" link=""]
Do not underestimate the determination of a quiet man.
That Geek was me. I was on assignment.
The professional move was to let it go, but I was young and still had my ego getting in the way.
You just don't know who you may be messing with.
It's not just the martially trained and physically capable strangers you may encounter, however.
It's the psychopaths and inherently violent people that's more of a concern. They don't wear name tags nor do they fit a specific physical description.
A random stranger, no matter how friendly, weak or innocuous they may seem, may have a blade and or a firearm and is not scared to use it. Or worse, they actively look for an excuse to do so.
And finally, there's the possibility that the person you're messing with is a plain clothed law enforcement officer or the relative of a prominent and powerful local figure.
It may not always be possible to avoid being messed with, but you can always avoid messing with others.
Stay up to date with newly published posts, newsletters, exclusives and giveaways by becoming a subscriber or by grabbing the RSS.


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A Police Services Act hearing has found two out of three Thunder Bay police drug unit officers guilty of unlawful and unnecessary exercise of authority.
Detective Sergeant William Wowchuck and Detective Constable Brad Bernst were both found guilty of the charge. Constable Ronald Popowich was found not guilty.
Hearing officer Morris Elbers dismissed a charge of discreditable conduct against all three officers.
The charges stem from the arrest of 50-year-old Richard Burns on September 7, 2011.
The drug unit officers had been staking out a house suspected of being a drug trafficking site. They saw several cars in the area, and decided to follow one of them. It was driven by Burns.
Wowchuck and Bernst arrested Burns as he was entering a MoneyMart on Memorial Avenue, and accused him of possessing a controlled substance. Popowich arrived on the scene after the arrest had already been made.
After a brief search, the officers found nothing and released Burns with no charges.
Hearing officer Morris Elbers said the police officers made a "rush decision" to arrest Burns and that they need to be more careful.
A disciplinary hearing for Wowchuck and Bernst will be held on October 10.
Prosecutor Bernd Richardt told CBC he wasn't surprised by Elbers' ruling.
"I think it simply reinforces that the officers need to be cautious, they need to have solid evidence before they arrest someone," he said.
The defence lawyer for the all three officers, Seth Weinstein, declined to comment, saying it wouldn't be appropriate until after the disciplinary hearing.


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Ever ask yourself
These Questions
"My online marketing has never really worked. Is it really worth it?"
"Why are the leads from my website always poor quality?"
"How do I stand out in a crowded marketplace?"
"Is it possible for me to reach my niche audience?"
"How can I position my brand online and create awareness around it?"
"Which online channels should I be using?"
"Will a website redesign increase my traffic and leads?"
"Is my branding outdated? Will a rebrand really help?"
Guiding Businesses to Growth with Proven
Inbound and Digital Marketing Methodologies
It's frustrating to grow your business in the digital age and gain quality leads that convert. You feel like you've done everything to grow your revenue and expand your client base. You're feeling doubtful and overwhelmed by all the digital marketing options out there. You don't want to stall your business and lose out on opportunities, but you don't know what to do next. We're self-starters ourselves and know all too well the pain-points of trying to be successful online. We've helped countless companies all around the world turn their online marketing into gold.
At MO Agency, our team is small, but we are agile enough to adapt and are nimbler than our competition. We provide you with a custom-tailored experience. We are all about the personal touch and being hands-on. That's why we consider ourselves the biggest little agency. Our mission is to help companies just like yours grow further by implementing a solid HubSpot inbound methodology.
"The difference between an online marketing strategy that works and one that doesn't is a plan. We'll show you how to structure and scale your online marketing efforts cost effectively…" – Luke Marthinusen, Managing Director - MO agency
2006 - 2011
Back in 2006, our MD Luke Marthinusen developed a website, photocopiers.co.za while selling photocopiers and quickly became one of the most successful copier reps in South Africa. He used his website to generate inbound sales leads and soon realised his passion for digital marketing.
2011 - 2012
Speed up to mid-2011 and Luke starts Markets Online from his apartment in Illovo, Johannesburg. In 2012, Jaco van der Watt joined as a graphic designer and in 2015 became a partner in the business.
2013 -
2016 -
In May 2016, Markets Online moved into custom fitted offices in Hyde Park and that same year rebranded themselves as MO Agency. Fast forward to 2017, MO is now 15 people strong, with a focus on keeping small, and high quality output that is on a par and better than the larger and impersonal agencies.


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Miles Jones - Maybe Tomorrow
Update: The Graham Beasley, Scott Watson and Tom Nagy-directed visuals for Miles Jones's Maybe Tomorrow single have been added.
While many a rap record has described the grind's incompatibility with romance, Miles Jones isn't giving up on love without a fight. On Maybe Tomorrow, a freshly-released record off his next mixtape, the Canadian emcee struggles to patch up a relationship damaged by his careerism, praying that the desired reconciliation will come with the dawn of a new day. As usual, the artist paris his reflective, often abstract bars with some attention-grabbing production; this time around Joshua Sadlier-Brown does the honors, crafting an upbeat instrumental laced with 8-bit synth arpeggios. The Jones Act Part III is scheduled to drop October 23; in the meantime, fans can hear more on Miles' recent, Booth-hosted EP, Act So Strange.


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Your satisfaction is our primary concern. All customers
are backed by our 100% money back guarantee for 30 days from receipt of order.
You may return the unused shoes in their original
condition along with the shoe box and accessories within 30 days for exchange or
Return Procedures:
Step 1 - Fill out an
RETURN AUTHORIZATION FORM for return or exchange.
Step 2 - We will email you the RA# (return authorization) within 1-2 business
Step 3 - Send the item back to us with the RA# indicated outside of the
Return Authorization Form URL:
Return Address:
Asia Focus Inc. 9900 Hayward WaySouth El Monte, CA 91733
Free UPS Return Label for US customers with items over $40
Now you can have a UPS Return
Label sent to you via e-mail.
Print the label and tape it
outside the return package. You can drop off at any UPS location.
Find the UPS
Center Nearest you.
UPS Return Label is FREE for
EXCHANGE and cost $10 toward REFUNDS.


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MOVIE SYNOPSIS [ +16 years-old ] TV Miniseries. 6 chapters. A group of teenagers painters and poets, Pre-Raphaelite Brotherhood, aims to revolutionize the art world forever. You can watch Desperate Romantics online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2009.
Rating: 5 by 220 users./
Runtime: 60 minutes
Aidan Turner, Rafe Spall, Samuel Barnett, Sam Crane, Amy Manson, Jennie Jacques, Zoë Tapper and Tom Hollander.
60-minute film (full movie) directed by Paul Gay and Diarmuid Lawrence.
The original audio of Desperate Romantics has dialogues in Dolby Digital, it was produced in UK.
It premiered in cinemas or television in 2009.
Screenplay written by Peter Bowker.
Alan Almond and Kieran McGuigan.
This film was produced and / or financed by British Broadcasting Corporation.
Composed by Daniel Pemberton.
Without commercial breaks, the full movie Desperate Romantics has a duration of 60 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Desperate Romantics in FULLTV Movies.


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Vince Camuto's $48M-plus Southampton home in contract
Fashion designer Vince Camuto's Southampton estate, on the market for more than $48 million, is in contract.
Wooldon Manor, Vince Camuto's Southampton estate that is in contract, is expected to be sold for more than the asking price of $48 million because the deal also includes an additional nine acres. (2013)
The Southampton estate Wooldon Manor sold last year for $75 million, and now it's back on the market for $98 million.
Besides shoe designer Vince Camuto, the owners of Wooldon Manor, an estate in Southampton, have included the Woolworth family and Edmund C. Lynch, founding partner of Merrill Lynch. (2013)
Wooldon Manor, Nine West co-founder Vince Camuto's estate in Southampton, has a pool as well as ocean frontage. (2013)
The property of Wooldon Manor, an estate in Southampton, includes elegant outdoor patios. (2013)
Wooldon Manor, shoe designer Vince Camuto's 10,000-square-foot oceanfront estate in Southampton that has been on the market for $48 million, is in contract. (2013)
The Wooldon estate, on 5.14 acres in Southampton, was put up for sale in June. (2013)


text: Re: New Cars coming with BOSCH music system? @vijay I asked FIAT around 3 days back regarding the 7 month old model(i put up a query on their) site..but as expected, now that I have already bought the car.. there was no response from them. My main concern was if they might have done some "secret" improvements in the newer model which might be missing in the older model. But I am really surprised if all the FoxT azure models would be June manufactured since its not a fast moving colour. May be anyone else who has a FoxT azure Punto 90bhp can comment. Regarding the rust part I will wait as you say to see if it improves. @livelyyoungman The dealer said he would give me the Form22 on Jan5th.. so anyway its too late . My major concern was regarding some secret improvements which I might miss. I am taking the car tothe service station right now to see what they say about the rust. I put the query to FIAT but till now there has been no response from them. Thanks for the inputs!!


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2003 BMW 3 Series Coupe - Consumer Review
Build Quality
Reliability Value
Build Quality
Reliability Value
Sheer Driving Pleasure!
By bmw4sho on
Vehicle2003 BMW 3 Series 330Ci Rwd 2dr Coupe (3.0L 6cyl 5M)
ReviewI recently purchased a 2003 BMW 330ci
Coupe, and let me tell you what, This
is the most amazing car I have driven
short of the M3. Previous to this I had
a 1998 BMW 328i, and I thought that was
a blast, but this car rekindles my
passion for driving every time I take
it out. I find myself going on midnight
runs just to avoid traffic and the
cops, to see what this thing can do.
Any review here that says BMW treats
you bad, or builds a bad car, doesn't
know waht they are talking about. BMW
as a company, and network of
dealerships never ceases to amaze me
with there passion to make my driving
experience better! I LOVE THIS CAR!!!
Best FeaturesEngine note, silky smooth nanual gearbox, fit and finish, EVERYTHING!
Worst FeaturesBesides the M3, I would like to get a BMW as a loaner car on the rare occasions my car is out of commision, but no biggy
Recommend (0) (0)
Are you the author of this review?Update Your Review
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Dreamworld Trailer (2012)
"a love story." 09 March 2012 Drama, Comedy, Romance 92 mins
Oliver Hayes, an aspiring animator whose confidence is at a low point, meets the captivating and impulsive Lily Blush, who encourages him to drop everything and go with her to Northern California in the hopes of fulfilling his fantasy of working for Pixar Animation Studios. As they travel up the coast, Oliver falls deeply in love but upon making certain startling discoveries he must decide if he wants to face reality or stay in dreamworld.
Watch the official Dreamworld 2012 trailer in HD below or find more Dreamworld videos on Vidimovie.


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Watch In the Realm of the Senses online free spacemov, Based on a true story set in pre-war Japan, a man and one of his servants begin a torrid affair. Their desire becomes a sexual obsession so strong that to intensify their ardor, they forsake all, even life itself.
Genre › Biography, Drama, History
Release › 1 April / 1976
IMDB Ratings › 6.6
Duration › 2h 49 min
Director by › Nagisa Oshima
Written by › Nagisa Oshima
Country › USA
Language › English
Production › Argos Films
Starcast › Tatsuya Fuji, Eiko Matsuda, Aoi Nakajima, Kanae Kobayashi


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Turbocharged! Gasoline! This is the vehicle for you if you're looking to get great gas mileage on your way to work! You can't beat a Ford for dependability, and you'll be glad to know that this 2018 Ford Escape is no exception.
Price may exclude tax, title, tags, governmental fees, any emissions testing charges and any finance charges (if applicable). Unless otherwise stated separately in the vehicle details, price does not include documentation, processing, administrative, closing or similar fees. Current mileage may vary due to test drives and vehicle relocation.


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Frances Bean Cobain
August 16, 2011 by Post Team
Frances Bean Cobain, Frances Bean Cobain, who turns 19 next August 18, starred in a photo shoot for Hedi Slimane, Dior photographer.
The daughter of Kurt Cobain and Courtney Love physically resembles his mother, with whom he remains just an idyllic relationship. However, those eyes and her pose godless nihilists and serene remember (much) to the parent that spawned it.
In the gallery, an exhibition of the collection of black and white portraits, you can see full on the website of the photographer Slimane.
Please feel free to send if you have any questions regarding this post , you can contact on
Disclaimer: The views expressed on this site are that of the authors and not necessarily that of U.S.S.POST.


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TOP STORY: KILLER USED A BASEBALL BATA baseball bat was used to bludgeon a 20-year-old man to death in a Derby street, police believe. Melvyn Hadley junior was battered and then left to die on a patch of waste ground close to his Normanton home in the early hours of Sunday.
I don't mean to make light of a murder, but since I started this blog, Google News searches for "baseball" have brought up stories about people killing or assaulting other people with baseball bats in the U.K. almost every single day. I'll admit that my first impulse upon reading these hasn't been "Oh, how awful." Rather, it's been "where are they getting all of these baseball bats in the U.K.?"


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Welcome to WordPress. This is your first post. Edit or delete it, then start blogging!
Category Archives: Uncategorized
Seafood paella looking lush tonight. Can't wait to dig in :-) #noms #slimmingworld #mattvfood
Marvel Cinematic Universe
Wow, its been a long time since my last proper blog post. So let's make a fresh start!!
I've been struggling to get into Agents of Shield. Tried really hard and watched the first few episodes a number of times. I've also found it difficult to watch CAptain America. Not sure what it is, just not that partial to that film.
Everything else Marvel Studios has released is epic. I really could watch the three Iron Man movies time and time again, as well as Thor, and certainly Guardians of the Galaxy will get a hammering when it comes out on Blu Ray at the end of the month. In fact, Guardians is my favourite to date!!!
So, in a quest to finally watch Captain America and get into Agents of Shield I have decided to watch the whole Marvel Cinematic Universe in Chronological order (rather than order of release). This includes a number of "Marvel One Shots" and post credit scenes.
I have to say, it is helping. Following the list that I sourced from this website, I am about to watch Iron Man 3 before embarking on Agents of Shield. Hopefully, having freshly watched all of the lead up releases, this will help. I hope!!!
Click here for the Marvel Cinematic Time Line


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Tolliver's Flooring in Barling, Arkansas Adds U-Haul Rentals
BARLING, Ark. (Sept. 26, 2012) — Michael Tolliver, owner of Tolliver's Flooring, located at 601 Church St., recently added U-Haul truck and trailer rentals to the floor covering business he has been operating since 1979.
Families needing the finest in moving services now will have increased convenience and a shorter distance to travel when moving, which not only will make their move easier but also will have the positive effect of reducing the amount of carbon emissions released into the atmosphere. U-Haul partnering with business owners across North America to increase convenience for customers while helping our environment is just one of the programs that support U-Haul Company's Corporate Sustainability initiatives.
Tolliver's Flooring can now offer its customers a variety of moving equipment and supplies designed specifically for moving household furnishings, including moving vans, open trailers, closed trailers, furniture pads, appliance dollies, furniture dollies, tow dollies and auto transports. Tolliver's Flooring also will offer sales items to protect their customers' belongings and make moving easier, such as heavy-duty boxes, which are made of up to 90 percent recycled content and are available in a variety of sizes.
"Moving and home renovations go together conveniently. Adding U-Haul rentals was upgrade for our customers," Tolliver said.
"U-Haul is proud to be partnering with a quality independent business such as Tolliver's Flooring," exclaimed Billy Proffitt, president, U-Haul Company of Arkansas. "Michael is a great example of the type of successful business relationship U-Haul has established in order to build and maintain a strong network of more than 15,000 independent dealers across North America."
For more information, or to rent your moving equipment today, call 479-221-9115. Visit www.tolliversflooring.com. Business hours of operation are: Mon. – Fri. 8 a.m. – 5 p.m. and Sat. 9 a.m. – 2 p.m. Please ask for Carrie or Mary and they will gladly assist you.
About U-Haul
Joanne Fried
Kelie Budd-Hale
E-mail: firstname.lastname@example.com
Phone: 602-263-6194