Immunohistochemical demonstration of cholinergic structures in central ganglia of the slug (Limax maximus, Limax valentianus).

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans.

Choline acetyltransferase of the common type immunoreactivity in the rat brain following different heroin treatments: a pilot study.

Previous studies suggest that behavioral consequences of heroin treatment depend on the drug history of the animals and that cholinergic neurotransmission is involved in both behavioral and motor sensitization induced by heroin and other drugs of abuse. Immunohistochemistry, using a recently developed antiserum, specific for choline acetyl-transferase of the common type (cChAT), was applied to four different groups of rats, differing in drug regimens. Two groups of rats were submitted to the same schedule of heroin sensitization and then challenged for vehicle or heroin before sacrifice, obtaining two distinct groups, namely heroin-vehicle (HV) and heroin-heroin (HH). The same challenge was applied to another group of rats, previously submitted to a treatment with vehicle, obtaining other two groups, vehicle-vehicle (VV) and vehicle-heroin (VH), respectively. The number of cChAT-positive neurons is significantly increased (p<0.05) in the diagonal band nuclei (with a consequent increase of cChAT positive fibers in the dentate gyrus) and notably, even not significantly (p>0.05), increased in the nucleus accumbens core of heroin-sensitized rats (HV, HH). Instead, acute heroin treatment significantly increase (p<0.05) the number of cChAT-positive cells in the nucleus accumbens shell of both heroin-naïve (VH) and heroin-sensitized (HH) rats. In heroin-sensitized rats (HV, HH), moreover, staining intensity of cChAT-positive fibers is significantly increased in the dorsal striatum, and basolateral amygdala (p<0.05). Unlikely, cChAT positive fibers in the central amygdala are significantly increased (p<0.05) by acute heroin treatments (VH, HH). The increase of cholinergic fibers in the dentate gyrus of the heroin sensitized rats (HV, HH) seems accompanied by a evident reduction in calretinin immunoreactive neurons in the same area. Our results, in a small group of animals, support the view that cholinergic mechanisms are intimately associated with the development of addictive phenotype. Furthermore, they suggest that cholinergic system is differentially engaged, following different heroin treatments.

First visualization of cholinergic cells and fibers by immunohistochemistry for choline acetyltransferase of the common type in the optic lobe and peduncle complex of Octopus vulgaris.

This study provides the first immunohistochemical evidence visualizing cholinergic octopus neurons containing choline acetyltransferase (ChAT), the synthetic enzyme of acetylcholine. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, and initially used for studies in mammals, to validate antibody specificity for the octopus counterpart enzyme we therefore used three methods. Immunoprecipitation using Pansorbin indicated that immunoreactive octopus brain molecules were capable of synthesizing acetylcholine. Western blot analysis after denatured gel electrophoresis of octopus brain extracts revealed a single band at approximately 81 kDa. A gel slice containing the 81-kDa protein after native (nondenatured) gel electrophoresis exhibited high ChAT activity. All findings obtained with these three methods clearly indicated that the antiserum effectively recognizes octopus ChAT. The immunohistochemical use of the antiserum in the retina, optic lobe, and its neighboring peduncle complex detected enzyme-containing neuronal cell bodies in only two regions, the cell islands of the optic lobe medulla and the cortical layer of the posterior olfactory lobule. Immunoreactive fibers and probable nerve terminals were also found in the plexiform layer of the deep retina, within the stroma of the optic gland, and the neuropils of the optic lobe, peduncle lobe, and olfactory lobe. These results provide information on the morphology and distribution patterns of cholinergic neurons in the octopus visual system, a useful invertebrate model for learning and memory where the cholinergic system, as in higher vertebrates including mammals, plays an important role.

Comparative immunohistochemical study of the dopaminergic systems in two inbred mouse strains (C57BL/6J and DBA/2J).

This study investigated possible neurochemical differences in the brain of two inbred mouse strains, C57BL/6J (C57) and DBA/2J (DBA) that in behavioral, memorization and learning tasks under normal and experimental conditions perform differently or often in an opposite manner. The immunohistochemical study, designed to investigate the dopaminergic system, identified many differences within the midbrain A10 area and less marked differences in areas A9 and A8. The number of dopamine transporter (DAT), vesicular monoamine transporter of type 2 (VMT) and tyrosine hydroxylase (TH) immunoreactive cell bodies was significantly higher in the midbrain of DBA mice than in C57 mice (on average +21.5%, P<0.001 in A10: +9.4% in A9, P<0.05: and +5.9% in A8, P<0.1). The distribution patterns of nerve fibres immunoreactive for same antisera also differed significantly in the two strains, especially at prelimbic, infralimbic and anterior cingulate cortical levels. In C57 mice these fibres were scanty whereas in DBA mice they were well represented. In the nucleus accumbens, also the territorial distribution of DAT immunoreactive nerve fibres differed in the two strains. In the midbrain, the galanin immunoreactive axons were more densely distributed in DBA than in C57 mice whereas neurotensin immunoreactive axons were more densely distributed in C57 than in DBA. These distinct immunohistochemical patterns could help to explain why performance differs in the two mouse strains.

Changes in neuropeptide FF and NPY immunohistochemical patterns in rat brain under heroin treatment.

Immunohistochemical distribution patterns of neuropeptide FF (NPFF) and neuropeptide tyrosine (NPY) were studied in the brain of rats submitted to two different protocols of heroin treatment. In drug-naive rats, acutely injected heroin significantly depleted NPFF-immunoreactive material within the neurons of the nucleus of solitary tract (NTS), significantly decreased the density of NPFF-immunoreactive nerve fibers within the median eminence, pituitary stalk, and neurohypophysis, and markedly increased NPY-immunoreactive neurons and nerve fibers in the thalamic paraventricular nucleus and bed nucleus of stria terminalis. In drug-sensitized rats, heroin significantly increased the number and immunostaining intensity of the NPFF-immunoreactive neurons within the NTS and induced minor changes in the NPFF-immunoreactive nerve fiber network of the median eminence, pituitary stalk, and neurohypophysis and a relatively minor increase in NPY neurons in the thalamic paraventricular nucleus and bed nucleus of stria terminalis. These heroin-induced changes suggest that NPFF is involved in regulating the effects of the heroin injection and in the mechanisms underlying behavioral sensitization. They also add further support to the key role of NPY in any conditions tending to change the animal homeostasis.

Guanylin-immunoreactive cells in the female and male rat adenohypophysis and their changes under various physiological and experimental conditions.

The peptide guanylin, first isolated from rat small intestine, is involved in the regulation of water-electrolyte transport between the intracellular and extracellular compartments of the epithelia. The main sites of guanylin expression are the intestinal, airway, or exocrine gland ductal epithelia where guanylin acts in a paracrine/luminocrine fashion. Because guanylin also circulates in the blood, sources of this peptide were sought in endocrine glands. Our group has already demonstrated the presence of guanylin-immunoreactive cells in the pars tuberalis of male rat adenohypophysis. In this study, we investigated whether guanylin-immunoreactive cells exist also in the adenohypophysial pars distalis and whether their appearance or distribution correlates with various physiological conditions in female rats or alters after gonadectomy in both sexes. These studies revealed that the rat pars distalis contains two guanylin-immunoreactive cell types, gonadotrophic cells, whose number varied notably during the estrous cycle, reached a peak in the proestrous phase, and increased consistently during pregnancy, in lactating animals, and after gonadectomy, and folliculo-stellate cells, a discrete number of which were found only in female rats at the estrous phase. These findings suggest that guanylin is involved in regulating gonadotrophic cell function. They also add important information on the controversially discussed functions of folliculo-stellate cells.

ARS Component B: structural characterization, tissue expression and regulation of the gene and protein (SLURP-1) associated with Mal de Meleda.

The ARS Component B gene (EMBL ID: HSARS81S, AC: X99977) encodes a 9 kD non-glycosylated polypeptide (also known as SLURP-1, SwissProt/TrEMBL: P55000), a soluble member of the human Ly6/uPAR superfamily. ARS Component B gene mutations have been implicated in Mal de Meleda. In this study we show by immunohistochemistry that SLURP-1 (secreted Ly-6/uPAR related protein, the protein product of the ARS Component B gene) is localized to human skin, exocervix, gums, stomach and esophagus. In the epidermis, keratinocytes underlying the stratum corneum are highly positive for SLURP1 immunostaining and cultured keratinocytes secrete the expected 9 kD protein. Circulating SLURP1 is detected in human plasma and urine. In the mouse, expression is evident in skin, eye, whole lung, trachea, esophagus and stomach. Human ARS Component B mRNA expression is regulated by retinoic acid, epidermal growth factor and interferon-gamma. The tissue localization and the association with Mal de Meleda suggest that ARS Component B and its protein product SLURP1 are implicated in maintaining the physiological and structural integrity of the keratinocyte layers of the skin.

Immunoreactive neurons in the brain of two mouse strains after incubation with an antiserum recognizing Asp-Val-Val-Gly.NH2 (DVVG), the C-terminal fragment of (D-Ala2)-deltorphin I.

D-Ala(2)-deltorphin I (DADTI) is a heptapeptide amide first extracted from frog skin that displays a high selectivity and affinity for delta opioid receptors. Previous studies using a polyclonal antiserum specific for its C-terminal tetrapeptide-amide (DVVG) have already described in rat and mouse brain the presence of immunoreactive neurons, most of them belonging to the mesencephalic dopaminergic neurons. C57BL/6J (C57) and DBA/2J (DBA) are two inbred strains of mice well known for showing marked genotype-dependent differences for phenotypes related to differential brain dopamine functioning. Brain specimens of both inbred mouse strains were frozen, cut and immunostained using the same antiserum. Some sections were also double immunostained with monoclonal anti-tyrosine hydroxylase (TH). DVVG-immunoreactive neurons were observed among both dopaminergic and non-dopaminergic neurons. DVVG- and TH-immunoreactive neurons were observed among the dopaminergic A8, A9 and A10 mesencephalic nuclei. They were on average 21.9% more numerous in DBA than in C57 mice. DVVG-immunoreactive nerve fibres could be seen in limbic, striatal, cortical and thalamic areas. The distribution patterns of DVVG-IR and TH-IR nerve fibres differed most conspicuously within the infralimbic, prelimbic and cingulate cortices, forming a dense network in DBA but rare in C57 mice. Non-dopaminergic DVVG-immunoreactive neurons did not differ significantly in the two strains. Our finding that the number and distribution pattern of this dopaminergic neuronal subpopulation differed in the two mouse strains could provide morphological support for the known behavioural differences between the DBA and C57 strains under normal and experimental conditions.

Dissociation in the effects of the D2/D3 dopaminergic agonist quinpirole on drinking and on vasopressin levels in the rat.

In the present study, we investigated the role of vasopressin in the development of quinpirole-induced hyperdipsia in the rat. We report that: (1), an acute intraperitoneal (i.p.) injection of 0.56 mg/kg of quinpirole increased plasma vasopressin (radioimmunoassay) at 15 min but not at 30 or 120 min; (2), nine daily injections of quinpirole (0.56 mg/kg, i.p.) progressively increased water intake and diuresis for a period of several hours after each treatment; (3), quinpirole hyperdipsia was associated with apparently normal levels of vasopressin (which might be considered inappropriately high in the presence of excessive drinking); (4), quinpirole reduced vasopressin and oxytocin, but not angiotensin, immunoreactivity in the supraoptic nucleus. These findings suggest that quinpirole hyperdipsia is a sound animal model of psychotic polydipsia.

Heroin sensitization as mapped by c-Fos immunoreactivity in the rat striatum.

Immunohistochemistry was used to map the induction of c-Fos protein in the forebrain of rats treated with heroin. Acute injection of heroin to drug-naive rats caused significant induction of c-Fos protein in the nucleus accumbens shell, whereas the same dose of heroin given to drug-sensitized rats significantly increased c-Fos immunoreactivity in the dorsomedial caudate-putamen. These results show that the heroin-induced pattern of c-Fos protein in the rat striatum differs according to the rat's drug history. These findings may represent a neural correlate of the motor components of heroin sensitization.