The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.

The evolutionary trajectories of the individual amygdala nuclei in the common shrew, guinea pig, rabbit, fox and pig: A consequence of embryological fate and mosaic-like evolution.

The amygdala primarily evolved as a danger detector that regulates emotional behaviours and learning. However, it is also engaged in stress responses as well as olfactory/pheromonal and reproductive functions. All of these functions are processed by a set of nuclei which are derived from different telencephalic sources (pallial and subpallial) and have a unique cellular structure and specific connections. It is unclear how these individual anatomical and functional units evolved to fit the amygdala to the specific needs of various mammals. Thus, this study provides quantitative data regarding volumes, neuron density and neuron numbers in the main amygdala nuclei of the common shrew, guinea pig, rabbit, fox and pig - species from across the mammalian phylogeny which differ in brain complexity and ecology. The results show that the volume of the amygdala and its individual nuclei scale with negative allometry relative to brain mass (an allometric coefficient below one). However, in relation to the whole amygdala volume, volumes and volumetric percentages of the lateral (LA) and basomedial (BM) nuclei scale with positive allometry, for the medial (ME) and lateral olfactory tract (NLOT) nuclei these parameters scale with negative allometry while the values of these parameters for the basolateral (BL), central (CE) and cortical (CO) nuclei scale with isometry. Moreover, density of neurons scales with strong negative allometry relative to both brain mass and amygdala volume with values of allometric coefficient below zero across studied species. This value for BL is significantly lower than that for the whole amygdala, for ME it is significantly higher while values for NLOT, CE, CO, LA and BM are quite similar to the value for whole amygdala. Finally, neuron numbers in the whole amygdala and its individual nuclei scale with negative allometry in relation to brain mass. However, in relation to the number of neurons in the whole amygdala, neuron numbers and percentages of neurons for LA and BM scale with positive allometry, for BL and NLOT they scale with negative allometry while the values of these parameters for CE, CO and ME scale with isometry. In conclusion, all of these results indicate that each of the nuclei studied displays a different and unique pattern of evolution in relation to brain mass or the whole amygdala volume. These patterns do not match with the various classical concepts of amygdala parcellation; however, in some way, they reflect diversity revealed by the expression of homeobox genes in various embryological studies.

GABAergic and Glutamatergic Phenotypes of Neurons Expressing Calcium-Binding Proteins in the Preoptic Area of the Guinea Pig.

The mammalian preoptic area (POA) has large populations of calbindin (CB), calretinin (CR) and parvalbumin (PV) neurons, but phenotypes of these cells are unknown. Therefore, the question is whether neurons expressing CB, CR, and/or PV are GABAergic or glutamatergic. Double-immunofluorescence staining followed by epifluorescence and confocal microscopy was used to determine the coexpression patterns of CB, CR and PV expressing neurons with vesicular GABA transporters (VGAT) as specific markers of GABAergic neurons and vesicular glutamate transporters (VGLUT 2) as specific markers of glutamatergic neurons. The guinea pig was adopted as, like humans, it has a reproductive cycle with a true luteal phase and a long gestation period. The results demonstrated that in the guinea pig POA of both sexes, ~80% of CB+ and ~90% of CR+ neurons coexpress VGAT; however, one-fifth of CB+ neurons and one-third of CR+ cells coexpress VGLUT. About two-thirds of PV+ neurons express VGAT, and similar proportion of them coexpress VGLUT. Thus, many CB+, CR+ and PV+ neurons may be exclusively GABAergic (VGAT-expressing cells) or glutamatergic (VGLUT-expressing cells); however, at least a small fraction of CR+ cells and at least one-third of PV+ cells are likely neurons with a dual GABA/glutamate phenotype that may coexpress both transporters.

Dopaminergic and cholinergic modulation of the amygdala is altered in female mice with oestrogen receptor ß deprivation.

The amygdala is modulated by dopaminergic and cholinergic neurotransmission, and this modulation is altered in mood disorders. Therefore, this study was designed to evaluate the presence/absence of quantitative alterations in the expression of main dopaminergic and cholinergic markers in the amygdala of mice with oestrogen receptor ß (ERß) knock-out which exhibit increased anxiety, using immunohistochemistry and quantitative methods. Such alterations could either contribute to increased anxiety or be a compensatory mechanism for reducing anxiety. The results show that among dopaminergic markers, the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and dopamine D2-like receptor (DA2) is significantly elevated in the amygdala of mice with ERß deprivation when compared to matched controls, whereas the content of dopamine D1-like receptor (DA1) is not altered by ERß knock-out. In the case of cholinergic markers, muscarinic acetylcholine type 1 receptor (AChRM1) and alpha-7 nicotinic acetylcholine receptor (AChRα7) display overexpression while the content of acetylcholinesterase (AChE) and vesicular acetylcholine transporter (VAChT) remains unchanged. In conclusion, in the amygdala of ERß knock-out female the dopaminergic and cholinergic signalling is altered, however, to determine the exact role of ERß in the anxiety-related behaviour further studies are required.

Expression of Calbindin, a Marker of Gamma-Aminobutyric Acid Neurons, Is Reduced in the Amygdala of Oestrogen Receptor ß-Deficient Female Mice.

Oestrogen receptor ß (ERß) knock-out female mice display increased anxiety and decreased threshold for synaptic plasticity induction in the basolateral amygdala. This may suggest that the γ-aminobutyric acid (GABA) inhibitory system is altered. Therefore, the immunoreactivity of main GABAergic markers-i.e., calbindin, parvalbumin, calretinin, somatostatin, α1 subunit-containing GABAA receptor and vesicular GABA transporter-were compared in the six subregions (LA, BL, BM, ME, CE and CO) of the amygdala of adult female wild-type and ERß knock-out mice using immunohistochemistry and quantitative methods. The influence of ERß knock-out on neuronal loss and glia was also elucidated using pan-neuronal and astrocyte markers. The results show severe neuronal deficits in all main amygdala regions in ERß knock-out mice accompanied by astroglia overexpression only in the medial, basomedial and cortical nuclei and a decrease in calbindin-expressing neurons (CB+) in the amygdala in ERß knock-out mice compared with controls, while other markers of the GABAergic system remain unchanged. Concluding, the lack of ERß led to failure in the structural integrity of the CB+ subpopulation, reducing interneuron firing and resulting in a disinhibitory effect over pyramidal function. This fear-promoting excitatory/inhibitory alteration may lead to the increased anxiety observed in these mice. The impact of neuronal deficits and astroglia overexpression on the amygdala functions remains unknown.