Parvalbumin interneuron deficiency in the prefrontal and motor cortices of spontaneously hypertensive rats: an attention-deficit hyperactivity disorder animal model insight.

Background: Attention deficit hyperactivity disorder (ADHD) is characterized by impairments in developmental-behavioral inhibition, resulting in impulsivity and hyperactivity. Recent research has underscored cortical inhibition deficiencies in ADHD via the gamma-aminobutyric acid (GABA)ergic system, which is crucial for maintaining excitatory-inhibitory balance in the brain. This study explored postnatal changes in parvalbumin (PV) immunoreactivity, indicating GABAergic interneuron types, in the prefrontal (PFC) and motor (MC) cortices of spontaneously hypertensive rats (SHRs), an ADHD animal model. Methods: Examining PV- positive (PV+) cells associated with dopamine D2 receptors (D2) and the impact of dopamine on GABA synthesis, we also investigated changes in the immunoreactivity of D2 and tyrosine hydroxylase (TH). Brain sections from 4- to 10-week-old SHRs and Wistar Kyoto rats (WKYs) were immunohistochemically analyzed, comparing PV+, D2+ cells, and TH+ fiber densities across age-matched SHRs and WKYs in specific PFC/MC regions. Results: The results revealed significantly reduced PV+ cell density in SHRs: prelimbic (~20% less), anterior cingulate (~15% less), primary (~15% less), and secondary motor (~17% less) cortices. PV+ deficits coincided with the upregulation of D2 in prepubertal SHRs and the downregulation of TH predominantly in pubertal/postpubertal SHRs. Conclusion: Reduced PV+ cells in various PFC regions could contribute to inattention/behavioral alterations in ADHD, while MC deficits could manifest as motor hyperactivity. D2 upregulation and TH deficits may impact GABA synthesis, exacerbating behavioral deficits in ADHD. These findings not only shed new light on ADHD pathophysiology but also pave the way for future research endeavors.

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.

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.

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.

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.

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.

The amygdala of the common shrew, guinea pig, rabbit, fox and pig: five flavours of the mammalian amygdala as a consequence of clade-specific mosaic-like evolution.

The amygdala is a part of neural networks that contribute to the regulation of emotional behaviours and emotional learning, stress response, and olfactory, pheromonal and reproductive functions. All these various functions are processed by the three main functional systems, frontotemporal, autonomic and olfactory, which are derived from different telencephalic sources (claustrum, striatum and olfactory cortex) and are represented, respectively, by the basolateral complex (BLC), the central complex (CC) and corticomedial complex (CMC) of the amygdala. The question arises of how these three functional systems evolved during mammalian phylogeny to fit the amygdala to specific needs of various animals. In the present study, we provide quantitative information regarding the individual volumes and neuron numbers in the BLC, CC and CMC of the common shrew, guinea pig, rabbit, fox and pig, a series of animals arranged according to increasing size and complexity of the brain. The results show that, in this series of animals, the BLC underwent a gradual size increase in volume and number of neurons, whereas the CMC was gradually reduced with regard to both these measures. The CC was more or less conserved across studied species. For example, the volume of the amygdala in pigs is ~250 times larger than that in shrews and it also has almost 26 times as many neurons as the amygdala of shrews. However, the volumes of the BLC, CC and CMC were ~380, 208 and 148 times larger, respectively, in pigs than in shrews. The number of neurons in these three regions was ~38, 23 and 20 times greater, respectively, in pigs than in shrews. The results also show striking morphometric similarities of the amygdala in the guinea pig and rabbit as well as fox and pig. For example, the percentages of neurons in the fox and pig are 42.23% and 42.78%, respectively, for the BLC, 16.64% and 16.58%, respectively, for the CC, and 41.12% and 40.64%, respectively, for the CMC. In conclusion, our results indicate that the amygdala does not evolve as a single unit but, instead, the three main functional systems evolved independently, which suggests that brain structures with major functional links evolve together independently of evolutionary changes in other unrelated structures. The size progression of the BLC parallels the size progression of the neocortex with which it is strongly functionally linked, whereas the CMC is strongly connected to olfactory regions, and all these structures follow the same regression course. Remarkable morphometric similarity of the amygdala in the guinea pig and rabbit as well as in the fox and pig, however, suggest that there must also be another mechanism shaping the morphology of the amygdala and the brain during evolution. The gradual nature of size changes in the BLC and CMC support this hypothesis as well.

The cytokine alterations/abnormalities and oxidative damage in the pancreas during hypertension development.

The aim of the present study was to compare the content of cytokines, chemokines, and oxidative stress markers in the pancreas of spontaneously hypertensive rats (SHRs) and Wistar Kyoto Rats (WKYs) serving as controls. Enzyme-like immunosorbent assay (ELISA) and biochemical methods were used to measure pancreatic levels of interleukin-1ß, interleukin-6, tumor necrosis factor α, transforming growth factor ß, RANES, monocyte chemoattractant protein 1, interferon gamma-induced protein 10, malondialdehyde, and sulfhydryl groups. The results showed that the pancreatic concentrations of all studied cytokines and chemokines did not differ between 5-week-old SHRs and WKYs, except RANTES which was significantly reduced in juvenile SHRs. In 10-week-old animals, except interleukin-1ß, the levels of all these proteins were significantly reduced in SHRs. The pancreatic levels of malondialdehyde were significantly reduced in 5-week-old SHRs and significantly elevated in 10-week-old SHRs while the contents of sulfhydryl groups were similar in both rat strains at any age studied. In conclusion, these data provide evidence that in maturating SHRs, the pancreatic levels of cytokines and chemokines are significantly reduced, while malondialdehyde significantly elevated. This suggests that in the pancreas of mature SHRs, the inflammation process is suppressed but there is ongoing oxidative damage.

ADHD pathogenesis in the immune, endocrine and nervous systems of juvenile and maturating SHR and WKY rats.

RATIONALE: Attention-deficit/hyperactivity disorder (ADHD) is one of the most common neurobehavioural disorders with morphological and functional brain abnormalities. However, there is a growing body of evidence that abnormalities in the immune and endocrine systems may also account for the ADHD pathogenesis. OBJECTIVES: To test ADHD pathogenesis in neurological, immune and endocrine systems, this study examined the concentrations of cytokines, chemokines, oxidative stress markers, metabolic parameters, steroid hormones and steroidogenic enzymes in the serum and/or tissues of spontaneously hypertensive rats (SHRs, animal model of ADHD) and Wistar Kyoto rats (WKYs, control animals). Moreover, the volume of the medial prefrontal cortex (mPFC) as well as the density of dopamine 2 (D2) receptor-expressing cells and tyrosine hydroxylase (TH)-positive nerve fibres in it was also elucidated. METHODS: Peripheral blood, spleen and adrenal gland samples, as well as brain sections collected on day 35 (juvenile) and day 70 (maturating) from SHRs and WKYs, were processed by ELISA and immunohistochemistry, respectively. RESULTS: The results show significant increases of serum and/or tissue concentrations of cytokines, chemokines and oxidative stress markers in juvenile SHRs when compared to the age-matched WKYs. These increases were accompanied by a lowered volume of the mPFC and up-regulation of D2 in this brain region. In maturating SHRs, the levels of inflammatory and oxidative stress markers were normalised and accompanied by elevated contents of steroid hormones. CONCLUSIONS: Significant elevations of serum and/or tissue contents of cytokines, chemokines and oxidative stress markers as well as volumetric and neurochemical alterations in the mPFC of juvenile SHRs may suggest the cooperation of neurological and immune systems in the ADHD pathogenesis. Elevated levels of steroid hormones in maturating SHRs may be a compensatory effect involved in reducing inflammation and ADHD symptoms.

Ontogeny of calcium-binding proteins in the cingulate cortex of the guinea pig: The same onset but different developmental patterns.

This paper compared the density of calbindin D28k (CB), calretinin (CR) and parvalbumin (PV) containing neurons in prenatal, newborn and postnatal periods in the cingulate cortex (CC) of the guinea pig as an animal model. The distribution and co-distribution among calcium-binding proteins (CaBPs) was also investigated during the entire ontogeny. The study found that CB-positive neurons exhibited the highest density in the developing CC. The CC development in the prenatal period took place with a high level of CB and CR immunoreactivity and both of these proteins reached peak density during fetal life. The density of PV-positive neurons, in contrast to CB and CR-positive neurons, reached high levels postnatally. The observed changes of the CaBPs-positive neuron density in the developing CC coincide with developmental events in the guinea pig. E.g. the eyes opening moment may be preceded by elevated levels of CB and CR at E50, whereas high immunoreactivity of PV from P10 to P40 with a peak at P20 may indicate the participation of PV in enhancement of the inhibitory cortical pathway maturation.

Parvalbumin, but not calretinin, neurons express high levels of α1-containing GABAA receptors, α7-containing nicotinic acetylcholine receptors and D2-dopamine receptors in the basolateral amygdala of the rat.

The generation of emotional responses by the basolateral amygdala is largely determined by the balance of excitatory and inhibitory inputs to its principal neurons - the pyramidal cells. The activity of these neurons is tightly controlled by g-aminobutyric acid (GABA)ergic interneurons, especially by those expressing parvalbumin (PV) and calretinin (CR). Although it is known that GABAergic, cholinergic and dopaminergic fibres make synapses on PV and CR cells, knowledge of the various receptors which are used by these cells is still incomplete. Thus, the present study investigates whether neurons expressing PV or CR co-express specific GABA, acetylcholine and/or dopamine receptors in the basolateral amygdala of the rat. The results show that almost two-thirds of PV neurons co-express high concentrations of α1 subunit of GABAA receptor, and more than half of them co-express high levels of α7 subunit of nicotinic acetylcholine receptor and/or D2-subtype of dopamine receptor. In contrast, a smaller percentage of CR neurons had detectable amounts of these receptors and at lower levels of abundance in most cases. In conclusion, the present results indicate that not only principal neurons but also GABAergic interneurons have specific receptors, which allow these cells to respond to the GABAergic, cholinergic and dopaminergic inputs coming to the basolateral amygdala of the rat. Since these cells receive intrinsic GABAergic inputs, they are strongly interconnected. Since they also receive extrinsic cholinergic and dopaminergic inputs, such stimulation may result in stimulus-driven feed-forward control of the principal neurons. The effects of such control may be either feed-forward inhibition of the principal neurons via α7 nicotinic acetylcholine receptors or disinhibition of these cells via D2-dopamine receptors.

The neurons expressing calcium-binding proteins in the amygdala of the guinea pig: precisely designed interface for sex hormones.

The generation of emotional responses by the amygdala is determined largely by the balance of excitatory and inhibitory inputs to its principal neurons. These responses are often sex-specific, and any imbalance in excitatory and/or inhibitory tones leads to serious psychiatric disorders which occur with different rates in men versus women. To investigate the neural basis of sex-specific processing in the amygdala, relationships between the neurons expressing calbindin (CB), parvalbumin (PV) and calretinin (CR), which form in the amygdala main subsets of γ-aminobutyric acid (GABA)-ergic inhibitory system, and neurons endowed with oestrogen alpha (ERα), oestrogen beta (ERß) or androgen (AR) receptors were analysed using double immunohistochemistry in male and female guinea pig subjects. The results show that in various nuclei of the amygdala in both sexes small subsets of CB neurons and substantial proportions of PV neurons co-express ERß, while many of the CR neurons co-express ERα. Both these oestrogen-sensitive populations are strictly separated as CB and PV neurons almost never co-express ERα, while CR cells are usually devoid of ERß. In addition, in the medial nucleus and some other neighbouring regions, there are non-overlapping subpopulations of CB and CR neurons which co-express AR. In conclusion, the localization of ERα, ERß or AR within subsets of GABAergic interneurons across diverse amygdaloid regions suggests that steroid hormones may exert a significant influence over local neuronal activity by directly modulating inhibitory tone. The control of inhibitory tone may be one of the mechanisms whereby oestrogen and androgen could modulate amygdala processing in a sex-specific manner. Another mechanism may be thorough steroid-sensitive projection neurons, which are most probably located in the medial and central nuclei.

Zinc Transporter 3 (ZnT3) in the Enteric Nervous System of the Porcine Ileum in Physiological Conditions and during Experimental Inflammation.

Zinc transporter 3 (ZnT3) is a member of the solute-linked carrier 30 (SLC 30) zinc transporter family. It is closely linked to the nervous system, where it takes part in the transport of zinc ions from the cytoplasm to the synaptic vesicles. ZnT3 has also been observed in the enteric nervous system (ENS), but its reactions in response to pathological factors remain unknown. This study, based on the triple immunofluorescence technique, describes changes in ZnT3-like immunoreactive (ZnT3-LI) enteric neurons in the porcine ileum, caused by chemically-induced inflammation. The inflammatory process led to a clear increase in the percentage of neurons immunoreactive to ZnT3 in all "kinds" of intramural enteric plexuses, i.e., myenteric (MP), outer submucous (OSP) and inner submucous (ISP) plexuses. Moreover, a wide range of other active substances was noted in ZnT3-LI neurons under physiological and pathological conditions, and changes in neurochemical characterisation of ZnT3⁺ cells in response to inflammation depended on the "kind" of enteric plexus. The obtained results show that ZnT3 is present in the ENS in a relatively numerous and diversified neuronal population, not only in physiological conditions, but also during inflammation. The reasons for the observed changes are not clear; they may be connected with the functions of zinc ions and their homeostasis disturbances in pathological processes. On the other hand, they may be due to adaptive and/or neuroprotective processes within the pathologically altered gastrointestinal tract.

Tyrosine hydroxylase-immunoreactivity and its relations with gonadotropin-releasing hormone and neuropeptide Y in the preoptic area of the guinea pig.

The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA.

Cocaine- and amphetamine-regulated transcript and calcium binding proteins immunoreactivity in the subicular complex of the guinea pig.

In this study we present the distribution and colocalization pattern of cocaine- and amphetamine-regulated transcript (CART) and three calcium-binding proteins: calbindin (CB), calretinin (CR) and parvalbumin (PV) in the subicular complex (SC) of the guinea pig. The subiculum (S) and presubiculum (PrS) showed higher CART-immunoreactivity (-IR) than the parasubiculum (PaS) as far as the perikarya and neuropil were concerned. CART- IR cells were mainly observed in the pyramidal layer and occasionally in the molecular layer of the S. In the PrS and PaS, single CART-IR perikarya were dispersed, however with a tendency to be found only in superficial layers. CART-IR fibers were observed throughout the entire guinea pig subicular neuropil. Double-labeling immunofluorescence showed that CART-IR perikarya, as well as fibers, did not stain positively for any of the three CaBPs. CART-IR fibers were only located near the CB-, CR-, PV-IR perikarya, whereas CART-IR fibers occasionally intersected fibers containing one of the three CaBPs. The distribution pattern of CART was more similar to that of CB and CR than to that of PV. In the PrS, the CART, CB and CR immunoreactivity showed a laminar distribution pattern. In the case of the PV, this distribution pattern in the PrS was much less prominent than that of CART, CB and CR. We conclude that a heterogeneous distribution of the CART and CaBPs in the guinea pig SC is in keeping with findings from other mammals, however species specific differences have been observed.

The densities of calbindin and parvalbumin, but not calretinin neurons, are sexually dimorphic in the amygdala of the guinea pig.

In the amygdala, the calcium-binding proteins (calbindin, parvalbumin or calretinin) are useful markers of specific subpopulations of γ-aminobutyric acid (GABA) containing neurons. In the rat and monkey they together mark the vast majority of GABA-containing neurons in this brain region. As GABA involvement in the control of various behaviors in a sex-specific manner and sexual dimorphism of the GABAergic system itself were recently proven, the question is how much dimorphic may be various subpopulations of this system. Thus, the present study investigates for the first time the presence/absence of sexual dimorphism among neurons expressing calbindin (CB), parvalbumin (PV) and calretinin (CR) which form in the amygdala main subsets of GABAergic system. The results show that in the amygdala of the guinea pig the densities of CB and/or PV expressing neurons are sexually dimorphic with the female>male pattern of sex differences in the basolateral amygdala. In the medial and cortical amygdala respectively CB and PV values are also sexually dimorphic, favoring males. The densities of CR expressing neurons are in the amygdala of the guinea pig sexually isomorphic. In conclusion, the results of the present study provide an evidence that in the amygdala of the guinea pig the densities of neurons expressing CB and/or PV are sexually dimorphic what supports the idea that GABA participates in the mediation of sexually dimorphic functions, controlled by this brain area.

The amygdala in the guinea pig is sexually dimorphic--a morphometric study.

Previous studies have shown that sexual dimorphism in the brain can present two morphological patterns: one in which males present greater morphological measures than females (male>female) and another in which the opposite is true (female>male). These studies have also shown that at least the part of amygdala namely the cortical and medial amygdala, an olfactory region involved in the control of reproductive physiology and behavior, is sexually dimorphic in the rat and other rodents. However, data comparing the basolateral and central amygdala between the sexes is lacking. To my knowledge, the present study is the first morphological work that systematically describes sexual dimorphism throughout the entire amygdala in the guinea pig. The results show that sex differences were found in: (a) the medial amygdala (ME) and its dorsal (MEd) and ventral (MEv) subdivisions, males showing greater values than females in volume and number of neurons, (b) the cortical amygdala (CO) and especially its posterior (COp) subdivision. In the CO, males exhibited a greater number of neurons and in the COp, males showed a greater volume and number of neurons. No differences between the sexes were observed in the basolateral and central amygdala. The results of the present study indicate that in the guinea pig sex differences are present in the large part of the amygdala and they present the male>female pattern, as it was observed in other rodents (rat and hamster), but not in the rabbit. As some previous neurochemical and functional studies have indicated that all parts of the amygdala may be sexually dimorphic, further studies are required to elucidate how much this brain region differs in both sexes.

Axotomy-induced changes in the chemical coding pattern of colon projecting calbindin-positive neurons in the inferior mesenteric ganglia of the pig.

The present study examines the response of colon-projecting neurons localized in the inferior mesenteric ganglia (IMG) to axotomy in the pig animal model. In all animals (n = 8), a median laparotomy was performed under anesthesia and the retrograde tracer Fast Blue was injected into the descending colon wall. In experimental animals (n = 4), the descending colon was exposed and the bilateral caudal colonic nerves were identified and severed. All animals were euthanized and the inferior mesenteric ganglia were harvested and processed for double-labeling immunofluorescence for calbindin-D28k (CB) in combination with either tyrosine hydroxylase (TH), neuropeptide Y (NPY), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), Leu-enkephalin (LENK), substance P, vesicular acetylcholine transporter, or galanin. Immunohistochemistry revealed significant changes in the chemical coding pattern of injured inferior mesenteric ganglion neurons. In control animals, Fast Blue-positive neurons were immunoreactive to TH, NPY, SOM, VIP, NOS, LENK, and CB. In the experimental group, the numbers of TH-, NPY-, and SOM-expressing neurons were reduced, whereas the number of neurons immunoreactive to LENK was increased. Our data indicate that the colon-projecting neurons of the porcine IMG react to the axotomy in a similar, but not an identical manner in a comparison to other species, especially rodents. Further studies are needed to elucidate the detailed factors/mechanisms involved in the response to nerve injury.

Chemical coding of zinc-enriched neurons in the intramural ganglia of the porcine jejunum.

Zinc ions in the synaptic vesicles of zinc-enriched neurons (ZEN) seem to have an important role in normal physiological and pathophysiological processes in target organ innervation. The factor directly responsible for the transport of zinc ions into synaptic vesicles is zinc transporter 3 (ZnT3), a member of the divalent cation zinc transporters and an excellent marker of ZEN neurons. As data concerning the existence of ZEN neurons in the small intestine is lacking, this study was designed to disclose the presence and neurochemical coding of such neurons in the porcine jejunum. Cryostat sections (10 mµ thick) of porcine jejunum were processed for routine double- and triple-immunofluorescence labeling for ZnT3 in various combinations with immunolabeling for other neurochemicals including pan-neuronal marker (PGP9.5), substance P (SP), somatostatin (SOM), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin (LENK), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), galanin (GAL), and calcitonin-gene related peptide (CGRP). Immunohistochemistry revealed that approximately 39%, 49%, and 45% of all PGP9.5- positive neurons in the jejunal myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, were simultaneously ZnT3(+). The majority of ZnT3(+) neurons in all plexuses were also VAChT-positive. Both VAChT-positive and VAChT-negative ZnT3(+) neurons co-expressed a variety of active substances with diverse patterns of co-localization depending on the plexus studied. In the MP, the largest populations among both VAChT-positive and VAChT-negative ZnT3(+) neurons were NOS-positive cells. In the OSP and ISP, substantial subpopulations of ZnT3(+) neurons were VAChT-positive cells co-expressing SOM and GAL, respectively. The broad-spectrum of active substances that co-localize with the ZnT3(+) neurons in the porcine jejunum suggests that ZnT3 takes part in the regulation of various processes in the gut, both in normal physiological and during pathophysiological processes.

Neurochemical characterization of zinc transporter 3-like immunoreactive (ZnT3(+)) neurons in the intramural ganglia of the porcine duodenum.

The SLC30 family of divalent cation transporters is thought to be involved in the transport of zinc in a variety of cellular pathways. Zinc transporter 3 (ZnT3) is involved in the transport of zinc into synaptic vesicles or intracellular organelles. As the presence of ZnT3 immunoreactive neurons has recently been reported in both the central and peripheral nervous systems of the rat, the present study was aimed at disclosing the presence of a zinc-enriched neuron enteric population in the porcine duodenum to establish a preliminary insight into their neurochemical coding. Double- and triple-immunofluorescence labeling of the porcine duodenum for ZnT3 with the pan-neuronal marker (PGP 9.5), substance P, somatostatin, vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin, vesicular acetylcholine transporter (VAChT), neuropeptide Y, galanin (GAL), and calcitonin gene-related peptide were performed. Immunohistochemistry revealed that approximately 35, 43, and 48 % of all PGP9.5-postive neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, of the porcine duodenum were simultaneously ZnT3(+). In the present study, ZnT3(+) neurons coexpressed a broad spectrum of active substances, but co-localization patterns unique to the plexus were studied. In the ISP, all ZnT3(+) neurons were VAChT positive, and the largest populations among these cells formed ZnT3(+)/VAChT(+)/GAL(+) and ZnT3(+)/VAChT(+)/VIP(+) cells. In the OSP and MP, the numbers of ZnT3(+)/VAChT(+) neurons were two times smaller, and substantial subpopulations of ZnT3(+) neurons in both these plexuses formed ZnT3(+)/NOS(+) cells. The large population of ZnT3(+) neurons in the porcine duodenum and a broad spectrum of active substances which co-localize with this peptide suggest that ZnT3 takes part in the regulation of various processes in the gut both in normal physiology and during pathological processes.