The distribution of dopamine D1 receptor and mu-opioid receptor 1 receptor immunoreactivities in the amygdala and interstitial nucleus of the posterior limb of the anterior commissure: relationships to tyrosine hydroxylase and opioid peptide terminal systems.

Mismatches between dopamine innervation and dopamine D1 receptor (D1) distribution have previously been demonstrated in the intercalated cell masses of the rat amygdala. Here the distribution of enkephalin and beta-endorphin immunoreactive (IR) nerve terminals with respect to their mu-opioid receptors is examined in the intercalated cell masses, along with a further immunohistochemical analysis of the dopamine/D1 mismatches. A similar analysis is also made within the extended amygdala. A spatial mismatch in distribution patterns was found between the mu-opioid receptor-1 immunoreactivity and enkephalin IR in the main intercalated island of the amygdala. Discrete cell patches of dopamine D1 receptor and mu-opioid receptor-1 IR were also identified in a distinct region of the extended amygdala, the interstitial nucleus of the posterior limb of the anterior commissure, medial division (IPACM), which displayed sparse tyrosine hydroxylase or enkephalin/beta-endorphin IR nerve terminals. Furthermore, distinct regions of the main intercalated island that showed dopamine/D1 receptor matches (the rostral and rostrolateral parts) were associated with strong dopamine and cyclic AMP regulated phosphoprotein, 32 kDa-IR in several D1 IR neuronal cell bodies and dendrites, whereas this was not the case for the dopamine/D1 mismatch areas (the rostromedial and caudal parts) of the main intercalated island. The lack of correlation between the terminal/receptor distribution patterns suggests a role for volume transmission for mu-opioid receptor- and dopamine D1 receptor-mediated transmission in distinct regions of the amygdala and extended amygdala. This may have implications for amygdaloid function, where slow long lasting responses may develop as a result of volume transmission operating in opioid peptide and dopaminergic communication.

The Sirt1 deacetylase modulates the insulin-like growth factor signaling pathway in mammals.

The lifespan of the nematode, Caenorhabditis elegans, can be extended by mutations affecting components of the insulin-like growth factor (IGF) signaling cascade or by overexpression of SIR2, an NAD+-dependent protein deacetylase. The mammalian homologue of SIR2, Sirt1, has been shown to modulate the activity of FoxO, a transcription factor that is downstream of the IGF signaling system. These results suggest that Sirt1 ought to affect the IGF pathway. We report here evidence that this is the case in mice. The loss of Sirt1 protein in mice results in increased expression of the IGF binding protein IGFBP1, a secreted modulator of IGF function. A number of the anatomical characteristics of Sirt1-null mice closely resemble those of transgenic mice overexpressing IGFBP1. Our data suggest that Sirt1 is part of a regulatory loop that limits the production of IGFBP1 thereby modulating IGF signaling.

Dynamics of volume transmission in the brain. Focus on catecholamine and opioid peptide communication and the role of uncoupling protein 2.

This review focuses on transmitter-receptor mismatches in the brain, which is one of the hallmarks of the Volume Transmission (VT) concept, and how this phenomenon may be related to local temperature gradients created by brain uncoupling protein 2 (UCP2), which uncouples oxidative phosphorylation from ATP synthesis, hereby generating heat. Recent studies on transmitter-receptor mismatches have revealed dopamine and opioid peptide receptor mismatches in the intercalated islands of the amygdala, which are GABAergic cell clusters regulating amygdaloid output. Such mismatches have also been found in regions belonging to the extended amygdala and the nucleus accumbens shell. Now substantial UCP2 immunoreactivity has been found within the above transmitter-receptor mismatch regions, suggesting that UCP2 may enhance diffusion and convection of DA and opioid peptides in such regions by generation of local temperature gradients, thereby contributing to a dynamic regulation of VT.

Vibration enhancement of slide-mounted immunofluorescence staining.

Immunofluorescence techniques allow the determination of protein and small molecule distribution within tissues and individual cells. There have been important, innovative modifications of these techniques since their introduction to the biosciences including the use of a mounting medium that prevents photo-bleaching, non-ionic detergents to permeabilize membranes, multiple immunofluorescence labeling and antigen recovery techniques for optimizing ligand-target interactions. While methods have been optimized for ligand-target accessibility in free-floating sections, little innovation has occurred to improve antibody access and epitope recognition in immunohistochemistry on slide-mounted sections or cell culture. During our studies of brain signaling pathways, we sought to improve the immunofluorescence signal to noise ratio in these specimens. We present here a minor modification of immunofluorescence procedures that significantly increases antibody access to epitopes within tissue and improves staining quality while significantly shortening incubation time. Antibody-epitope interactions are dependent on access and affinity. Our technique is based upon application of a vibration source during antibody incubation which increases epitope access, shortens incubation time and thereby minimizes background immunofluorescence. Data are presented on benefits evident with several antibodies raised against proteins and peptides localized in various subcellular compartments. Analysis of the quality of labeling was performed to show that signal intensity is enhanced and background intensity is often diminished when incubations are performed under gentle vibration. This, together with the significant saving of time, should make this procedure applicable to a wide range of neurobiological questions.

The dopamine D1 receptor-rich main and paracapsular intercalated nerve cell groups of the rat amygdala: relationship to the dopamine innervation.

The intercalated cell masses are GABAergic neurons interposed between the major input and output structures of the amygdala. Dopaminergic projections to the main and paracapsular intercalated islands were examined by determining the relationship of the dopamine nerve-terminal networks to the D1-receptor immunoreactive staining of cells within the intercalated islands, using double-fluorescence immunolabelling procedures in combination with confocal laser microscopy. The relationship of terminals positive for both tyrosine hydroxylase and dopamine beta-hydroxylase (noradrenaline and/or adrenaline) to terminals positive for tyrosine hydroxylase but negative for dopamine beta-hydroxylase (dopamine terminals) was studied in relation to the D1-receptor immunoreactivity in adjacent sections at various rostrocaudal levels. The microscopy and image analysis revealed that there was only a minor dopaminergic innervation of the D1 receptor-immunoreactive cells in the rostromedial and caudal component of the main intercalated island, suggesting volume transmission as the main communication mode for dopamine in these regions. In contrast, the D1 receptor-immunoreactive areas in the rostrolateral part of the main island and also the paracapsular intercalated islands showed a high degree of dopaminergic innervation, indicating that synaptic and perisynaptic dopamine transmission plays a dominant role in these regions. It is known that amygdala neurons are involved in the elicitation and learning of fear-related behaviors. We suggest that slow dopaminergic volume transmission in the rostromedial and caudal parts of the main intercalated island may have a role in tonic excitatory modulation in these parts of the main island, allowing GABAergic activity to develop in the central amygdaloid nucleus and thereby contributing to inhibition of fear-related behavioral and autonomic responses. In contrast, a faster synaptic and perisynaptic dopaminergic transmission in the rostrolateral part of the main intercalated island and in the paracapsular intercalated islands may have a role in allowing a more rapid elicitation of fear-related behaviors.