Distribution Patterns of Cocaine- and Amphetamine-Regulated Transcript- and/or Galanin-Containing Neurons and Nerve Fibers Located in the Human Stomach Wall Affected by Tumor.

The aim of the study was to investigate the distribution patterns of cocaine- and amphetamine-regulated transcript- (CART-) and galanin-immunoreactive (GAL-IR) neuronal structures in the human stomach wall, focusing on differences observed in regions directly affected by the cancer process, and those from the surgical margin. Samples from the stomach wall were collected from 10 patients (3 women and 7 men, the mean age 67.0 ± 11.9). Next, triple-immunofluorescence staining was used to visualize the changes in the frequency of neurons inside myenteric plexi and intramural fibers containing CART and/or GAL, as well as protein gene product 9.5 (as panneuronal marker). Tumor into the stomach wall caused a decrease in the number of CART-positive (+) nerve fibers in the longitudinal (LML) and circular muscle layers (CML). Notable changes in the dense network of CART+/GAL+ nerve fibers (an increase) were observed in the LML and lamina muscularis mucosae (LMM) within carcinoma-affected areas of the human stomach. Additionally, an elevated number of these nerve fibers from LMM were accompanied by an increase in the number of fibers containing GAL in the vicinity of the neoplastic proliferation. Obtained results suggest that a carcinoma invasion may affect the innervation pattern of the human stomach wall and their function(s).

Coexpression of Galanin and Nestin in the Chemoreceptor Cells of the Human Carotid Body.

The carotid body is a highly specialized chemoreceptive organ of neural crest origin whose role is to detect changes in arterial oxygen content. The sensory units are the chemoreceptor cells, which are neuronal-like cells, surrounded by sustentacular or glial-like cells. It is suggested that the carotid body contains self-renewing multipotent stem cells, which are putatively represented by glial-like sustentacular cells. The mechanisms of renewal of neuronal-like cells are unclear. Recently, we have demonstrated the expression of galanin, a peptide promoting neurogenesis, in chemoreceptor cells in the human CB. Thus, in the present study we seek to determine whether galanin expression in chemoreceptor cells could be matched with that of nestin, a peptide that is a marker of multipotent neural stem cells, or rather with the glial fibrillary acidic protein (GFAP), a marker for glial cells. The latter would underscore the pluasibly essential role of sustentacular cells in the self-renewal capability of chemorecetors. We found that galanin expression is matched with nestin in chemoreceptor cells of the human carotid body, but not with that of GFAP. Thus, galanin expression in chemoreceptor cells could provide a signal for neurogenesis and chemoreceptor cell differentiation in the carotid body.

Selective Expression of Galanin in Neuronal-Like Cells of the Human Carotid Body.

The carotid body is a neural-crest-derived organ devoted to respiratory homeostasis through sensing changes in blood oxygen levels. The sensory units are the glomeruli composed of clusters of neuronal-like (type I) cells surrounded by glial-like (type II) cells. During chronic hypoxia, the carotid body shows growth, with increasing neuronal-like cell numbers. We are interested in the signals involved in the mechanisms that underlie such response, because they are not well understood and described. Considering that, in literature, galanin is involved in neurotrophic or neuroprotective role in cell proliferation and is expressed in animal carotid body, we investigated its expression in human. Here, we have shown the expression and localisation of galanin in the human carotid body.

Encapsulated galanin-producing cells attenuate focal epileptic seizures in the hippocampus.

PURPOSE: Encapsulated cell biodelivery (ECB) is a relatively safe approach, since the devices can be removed in the event of adverse effects. The main objectives of the present study were to evaluate whether ECB could be a viable alternative of cell therapy for epilepsy. We therefore developed a human cell line producing galanin, a neuropeptide that has been shown to exert inhibitory effects on seizures, most likely acting via decreasing glutamate release from excitatory synapses. To explore whether ECB of genetically modified galanin-producing human cell line could provide seizure-suppressant effects, and test possible translational prospect for clinical application, we implanted ECB devices bilaterally into the hippocampus of rats subjected to rapid kindling, a model for recurrent temporal lobe seizures. METHODS: Two clones from a genetically modified human cell line secreting different levels of galanin were tested. Electroencephalography (EEG) recordings and stimulations were performed by electrodes implanted into the hippocampus at the same surgical session as ECB devices. One week after the surgery, rapid kindling stimulations were initiated. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) measurements prior to device implantation showed a release of galanin on average of 8.3 ng/mL/24 h per device for the low-releasing clone and 12.6 ng/mL/24 h per device for the high-releasing clone. High-releasing galanin-producing ECB devices moderately decreased stimulation-induced focal afterdischarge duration, whereas low-releasing ECB devices had no significant effect. SIGNIFICANCE: Our study shows that galanin-releasing ECB devices moderately suppress focal stimulation-induced recurrent seizures. Despite this moderate effect, the study provides conceptual proof that ECB could be a viable alternative approach to cell therapy in humans, with the advantage that the treatment could be terminated by removing these devices from the brain. Thereby, this strategy provides a higher level of safety for future therapeutic applications, in which genetically modified human cell lines that are optimized to produce and release antiepileptic compounds could be clinically evaluated for their seizure-suppressant effects.

Mass spectrometry-based sequencing of protein C-terminal peptide using α-carboxyl group-specific derivatization and COOH capturing.

An approach to mass spectrometry (MS)-based sequence analysis of selectively enriched C-terminal peptide from protein is described. This approach employs a combination of the specific derivatization of α-carboxyl group (α-COOH), enzymatic proteolysis using endoproteinase GluC, and enrichment of C-terminal peptide through the use of COOH-capturing material. Highly selective derivatization of α-COOH was achieved by a combination of specific activation of α-COOH through oxazolone chemistry and amidation using 3-aminopropyltris-(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine). This amine component was used to simplify fragmentation in tandem mass spectrometry (MS/MS) measurement, which facilitated manual sequence interpretation. The peptides produced after GluC digestion were then treated with a COOH scavenger to enrich the C-terminal peptide that is only devoid of COOH groups, and the obtained C-terminal peptide was readily sequenced by matrix-assisted laser desorption/ionization (MALDI)-MS/MS due to the TMPP mass tag.

Neuropeptides of the human magnocellular hypothalamus.

Hypothalamic magnocellular nuclei with their large secretory neurons are unique and phylogenetically conserved brain structures involved in the continual regulation of important homeostatic and autonomous functions in vertebrate species. Both canonical and newly identified neuropeptides have a broad spectrum of physiological activity at the hypothalamic neuronal circuit level located within the supraoptic (SON) and paraventricular (PVN) nuclei. Magnocellular neurons express a variety of receptors for neuropeptides and neurotransmitters and therefore receive numerous excitatory and inhibitory inputs from important subcortical neural areas such as limbic and brainstem populations. These unique cells are also densely innervated by axons from other hypothalamic nuclei. The vast majority of neurochemical maps pertain to animal models, mainly the rodent hypothalamus, however accumulating preliminary anatomical structural studies have revealed the presence and distribution of several neuropeptides in the human magnocellular nuclei. This review presents a novel and comprehensive evidence based evaluation of neuropeptide expression in the human SON and PVN. Collectively this review aims to cast a new, medically oriented light on hypothalamic neuroanatomy and contribute to a better understanding of the mechanisms responsible for neuropeptide-related physiology and the nature of possible neuroendocrinal interactions between local regulatory pathways.

Age-dependent differences in galanin-dependent colonic fluid secretion after infection with Salmonella typhimurium.

Epithelial cells lining the colon do not normally express galanin type 1 receptors (Gal1Rs). However, subsequent to infection with enteric pathogens such as Salmonella typhimurium, the Gal1R is rapidly upregulated in colonocytes where it contributes to the excess fluid production associated with diarrhoea. Humans infected with non-typhoid Salmonella respond differently according to age: infants develop diarrhoea but not bacteraemia and survive, while the elderly become bacteraemic and die. Thus the aim of this study was to determine if age-related differences exist in response to S typhimurium infection in mice, and whether these differences are due to altered Gal1R expression. Wild-type C57BL/6J mice that were 2 and 15 months old, as well as 2-month-old Gal1R knockout mice, were infected by gavage. Young wild-type mice expressed Gal1R in response to infection, had increased colonic fluid secretion, low rates of bacteraemia and survived. In contrast, 15-month-old wild-type mice expressed fewer Gal1Rs in response to infection, had attenuated increases in colonic fluid secretion, high rates of bacteraemia and died. A similar profile was noted in 2-month-old Gal1R knockout mice. Addition of polyethylene glycol to the drinking water of 15-month-old wild-type mice increased colonic fluid secretion and reduced rates of bacteraemia to those observed in 2-month-old wild-type mice and eliminated fatalities. The difference in response to S typhimurium infection with age may be due, at least in part, to decreased Gal1R expression and decreased amounts of colonic fluid secretion.

Galanin immunoreactivity is sexually polymorphic in neuroendocrine and vocal-acoustic systems in a teleost fish.

Galanin is a peptide that regulates pituitary hormone release, feeding, and reproductive and parental care behaviors. In teleost fish, increased galanin expression is associated with territorial, reproductively active males. Prior transcriptome studies of the plainfin midshipman (Porichthys notatus), a highly vocal teleost fish with two male morphs that follow alternative reproductive tactics, show that galanin is upregulated in the preoptic area-anterior hypothalamus (POA-AH) of nest-holding, courting type I males during spawning compared to cuckolding type II males. Here, we investigate possible differences in galanin immunoreactivity in the brain of both male morphs and females with a focus on vocal-acoustic and neuroendocrine networks. We find that females differ dramatically from both male morphs in the number of galanin-expressing somata and in the distribution of fibers, especially in brainstem vocal-acoustic nuclei and other sensory integration sites that also differ, though less extensively, between the male morphs. Double labeling shows that primarily separate populations of POA-AH neurons express galanin and the nonapeptides arginine-vasotocin or isotocin, homologues of mammalian arginine vasopressin and oxytocin that are broadly implicated in neural mechanisms of vertebrate social behavior including morph-specific actions on vocal neurophysiology in midshipman. Finally, we report a small population of POA-AH neurons that coexpress galanin and the neurotransmitter γ-aminobutyric acid. Together, the results indicate that galanin neurons in midshipman fish likely modulate brain activity at a broad scale, including targeted effects on vocal motor, sensory and neuroendocrine systems; are unique from nonapeptide-expressing populations; and play a role in male-specific behaviors.

Clinical value of a set of neuropeptides in term and preterm neonates with seizures: Brain derived neurotrophic factor, galanin and neuropeptide Y.

The aim of our study to investigate clinical value of a set of neuropeptides (brain derived neurotrophic factor-BDNF, galanin and neuropeptide Y-NPY) in critically ill neonates. A total of 53 neonates (preterm: 26, term: 27) evaluated with lumbar pucture for etiologic evaluation were consequtively included into the study. Serum and CSF levels of the neuropeptides were measured in the first 48 h of life. All infants were prospectively followed for prognostic outcome (survival and neurodevelopmental) at the first year of life. The study cohort was categorized into four groups with respect to seizure development; preterm neonates with or without seizure and term neonates with or without seizure. Mean CSF levels of NPY (pg/ml) were significantly higher in term neonates with than those without seizures (389.76 vs. 122.66) and galanin (3.31 vs. 1.55) respectively. Term neonates with seizures had significantly higher serum levels of NPY (ng/mL) as compared with neonates without seizures (54.00 vs. 9.10). No significant difference was noted in serum and CSF levels for the set of neuropeptides in neonates with respect to prognostic outcome. Serum NPY and CSF NPY and galanin levels have a potential role for detection of clinical seizures in term neonates.

Immunohistochemical distribution of regulatory peptides in the human fetal adenohypophysis.

We have studied here the cellular distribution of several regulatory peptides in hormone-producing cells of the human pituitary during the fetal period. Immunohistochemistry was used to show the expression of several regulatory peptides, namely Angiotensin-II, Neurotensin and Galanin, at successive gestational stages and their co-localization with hormones in the human fetal adenohypophysis. Somatotrophs, gonadotrophs and thyrotrophs were differentiated earliest. At gestational week 9, Angiotensin-II immunoreactivity was co-localized only with growth hormone immunoreactivity in somatotrophs, one of the first hormone-producing cells to differentiate. This co-localization remained until week 37. Neurotensin immunoreactivity was present in gonadotrophs and thyrotrophs in week 23, after FSH and TSH hormone differentiation. Galanin immunoreactivity was present in all hormone-producing cell types except corticotrophs. The different pro-opiomelanocortin-derived peptides were detected at different stages of gestation and adrenocorticotrophic hormone immunoreaction was the last to be detected. Our results show an interesting relationship between regulatory peptides and hormones during human fetal development, which could imply that these peptides play a regulatory role in the development of pituitary function.

Galanin immunoreactivity increased in chicken supraoptic neurons after activation of the vasotocin system at oviposition.

The avian arginine vasotocin (AVT) synthesized in the hypothalamic magnocellular neurons and released from the posterior pituitary is known to be involved in the regulation of uterine contractions for oviposition in chickens. However, regulation of AVT synthesis and release within the magnocellular hypothalamus has not been elucidated. Galanin, the oviposition inducing factor in the oviduct of the hen, has been demonstrated to have sexually dimorphic stimulatory action in oxytocin- and vasopressin neurons in the mammalian hypothalamus. In this study, galanin and AVT immunoreactivity was investigated around the time of oviposition in the supraoptic nucleus (SON) to determine if galanin modulates AVT synthesis and/or release. Within SON neurons increased AVT immunoreactivity before oviposition and the decreased AVT immunoreactivity after oviposition implied function-related peptide release. The significantly increased number of galanin neurons co-localizing with AVT immediately after oviposition suggests that galanin is involved in the negative feedback to limit AVT release in the SON. Thus, these data support the idea that AVT in the SON is involved in central regulation of oviposition and that AVT release could be modulated by the neuropeptide galanin.

Neurochemical characterization of intramural nerve fibres in the porcine oesophagus.

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4 ± 0.8 and 18.1 ± 3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3 ± 0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.

Short N-terminal galanin fragments are occurring naturally in vivo.

The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GAL1, GAL2, and GAL3, binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1-20), galanin(6-20), and tested on coronal rat brain sections competing with 125I-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced 125I-galanin(1-29) but galanin(6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species. BIOLOGICAL SIGNIFICANCE: This study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments.

Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus).

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P

Neurochemical properties of the middle cervical ganglion in the sheep.

The neurochemical properties of the ovine middle cervical ganglion (MCG) were studied using antibodies raised against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL). Double-labelling immunocytochemistry revealed that the vast majority (95.5 +/- 0.8%) of postganglionic sympathetic MCG neurons expressed simultaneously both catecholamine-synthesizing enzymes (neurons were TH/DbetaH-positive). A large population of noradrenergic neurons exhibited immunoreactivity (IR) to NPY (62.2 +/- 2.2%), but single NPY-positive perikarya-lacking noradrenergic markers were also observed (2.0 +/- 0.3%). None of the examined MCG neuronal somata contained SP, CGRP, GAL or VIP. A moderate number of noradrenergic nerve fibres located amongst neuronal cell bodies was also found. In small number of these terminals the presence of NPYor GAL (but not CGRP or VIP) was detected. The ovine MCG was numerously innervated with SP-immunoreactive nerve fibres which sometimes formed basket-like formations around postganglionic neurons. The MCG exhibited a sparse CGRP-immunoreactive innervation and lacked VIP-positive nerve terminals. In many aspects the chemical coding of MCG postganglionic neurons and nerve terminals resembles that found in other mammalian cervico-thoracic paravertebral ganglia, but some important species-dependent differences exist. The functional implications of these differences remain to be elucidated.

Activation of a subset of lumbar spinothalamic neurons after copulatory behavior in male but not female rats.

The precise pathways that convey copulation-related information to forebrain regions activated during male and female sexual behavior are poorly understood. Previous work from our laboratory and others has demonstrated the existence of a spinothalamic pathway that is a candidate to relay information to these areas. This pathway originates from a population of spinothalamic neurons in the lumbar spinal cord containing several neuropeptides including galanin, located in laminas 7 and 10 of the lumbar segments 3 and 4. To investigate the involvement of these lumbar spinothalamic neurons in conveying copulation-related information, we tested the hypothesis that these cells are activated after ejaculation in male rats and vaginocervical stimulation in female rats. This was assessed using galanin or cholecystokinin as a marker for this subset of spinothalamic neurons and Fos-immunoreactivity as a marker for neuronal activation. The results demonstrated that activation of these spinothalamic neurons is triggered by stimuli associated with ejaculation. Fos induction was specifically associated with ejaculation, because mounts or intromissions did not trigger expression. Moreover, these spinothalamic neurons were not activated by vaginocervical stimulation in female rats. Spinothalamic neurons have generally been associated with signaling pain and temperature information. The present findings demonstrate that a specific subpopulation of spinothalamic neurons signals information associated with ejaculation.

Morphological evidence of sensory neurons in the root of the rat tongue.

In our previous studies, a large number of substance P (SP)-immunoreactive (IR) nerve fibers were detected in the rat tongue and their number increased after inflammation, suggesting that these fibers might be involved in the axon reflex. Therefore, in this study, we have examined the different neuropeptide-containing nerve elements by light, electron, and confocal laser microscopy. SP, vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) IR varicose fibers were numerous compared with other ones. Small groups of ganglia with perikarya IR for SP, VIP, NPY, galanin, and somatostatin were observed. The SP-IR nerve cell bodies were mainly located in the tunica propria just below the epithelial lining. Double-labeling immunohistochemistry showed that the intrinsic SP-IR neurons did not colocalize VIP. The SP containing nerve terminals were observed in and below the epithelium as well as in very close contact to or making real synapses with other neurons in the intralingual ganglion. Our data confirmed the possibility of intrinsic sensory neurons, which might be the afferent branch of the intralingual reflex arch, while the VIP- and NPY-IR neurons located in the salivary glands, around the blood vessels, and in the muscle layer might constitute the efferent site of this reflex.

Increase in sensory neuropeptides surrounding the Achilles tendon in rats with adjuvant arthritis.

The Achilles tendon in rats with adjuvant arthritis was analyzed by radioimmunoassay (RIA) and semi-quantitative immunohistochemistry for the occurrence of two sensory neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and a sensory modulating peptide, galanin (GAL). The tissue concentration of SP and CGRP in the Achilles tendon and its envelope, i.e. the paratenon and bony insertion, as assessed by RIA was increased by 22% and 71%, respectively, compared to normal controls, whereas the level of GAL was unchanged. Semi-quantitative immunohistochemistry applied to different regions of the tendon in arthritic rats disclosed an increased occurrence of SP and CGRP positive nerve fibers in the paratenon and bone tendinous junction, whereas GAL fibers were only increased at the bone tendinous junction. Notably, neither neuropeptides nor inflammatory cells were seen in the tendon proper. The increased occurrence of SP and CGRP in the tendon envelope presumably reflects inflammatory actions, whereas that of GAL implies an endogenous anti-inflammatory response. The observed SP and CGRP upregulation in the paratenon and bony insertion suggests a pathophysiological role in paratenonitis and enthesitis often seen in patients with rheumatoid arthritis. Presumably Achillodynia originates in the tendon envelope rather than the tendon proper. The observations could be used to define new pharmacological targets for mitigating symptoms from tendons in rheumatoid arthritis and possibly also in other disorders. Whether a neuronal pathogenic mechanism underlies tendon overuse disorders in non-arthritic tendinopathies and the development of degeneration, i.e. tendinosis, remains to be studied.

Fasting-induced changes of neuropeptide immunoreactivity in the lateral septum of male rats.

The objective of the present study was to determine the rostrocaudal distribution and the effect of reduced food intake (60% of the average daily food intake for 1-4 weeks) on the amount of leucine-enkephalin (Leu-enk), neuropeptide Y (NPY) and galanin (Gal) in the lateral septum of male rat brain. Using pre-embedding immunocytochemistry combined with densitometry on 60 microm serial vibratome sections we found that in control animals Leu-enk-immunoreactive elements showed an increasing density from rostral towards the medial part of the septum, then a gradual decrease towards the caudal direction. The distribution of NPY proved to be rather even along the examined sequence of sections with two smaller peaks roughly at the 1/3 and 2/3 of the rostrocaudal axis. Gal showed similar distribution but the peaks were shifted to more caudal direction. We also found that Leu-enk forms the most dense plexus followed by a moderate amount of NPY-positive axonal meshwork. Gal was present in the lowest amount along the lateral septal nuclei. The effect of reduced food intake was marked and differential in the case of the three examined peptides. During the first 2 weeks of reduced food intake NPY-immunoreactivity was upregulated as compared to the control, then it was reduced close to the control value by the 4th week. The changes in Gal immunoreactivity showed similar pattern. The average relative density of Leu-enk-immunoreactive elements immediately decreased as a result of reduced food intake for 1 week and it gradually decreased by the end of the 4th week. These results indicate that reduced food intake affects the expression of NPY, Gal and Leu-enk not only in the relevant hypothalamic neuroendocrine centres, but also in the lateral septal area.

Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

On-column focusing is essential for satisfactory performance using capillary scale columns. On-column focusing results from generating transient conditions at the head of the column that lead to high solute retention. Solvent-based on-column focusing is a well-known approach to achieve this. Temperature-assisted on-column focusing (TASF) can also be effective. TASF improves focusing by cooling a short segment of the column inlet to a temperature that is lower than the column temperature during the injection and then rapidly heating the focusing segment to the match the column temperature. A troublesome feature of an earlier implementation of TASF was the need to leave the capillary column unpacked in that portion of the column inside the fitting connecting it to the injection valve. We have overcome that problem in this work by packing the head of the column with solid silica spheres. In addition, technical improvements to the TASF instrumentation include: selection of a more powerful thermo-electric cooler to create faster temperature changes and electronic control for easy incorporation into conventional capillary instruments. Used in conjunction with solvent-based focusing and with isocratic elution, volumes of paraben samples (esters of p-hydroxybenzoic acid) up to 4.5-times the column liquid volume can be injected without significant bandspreading due to volume overload. Interestingly, the shapes of the peaks from the lowest volume injections that we can make, 30nL, are improved when using TASF. TASF is very effective at reducing the detrimental effects of pre-column dispersion using isocratic elution. Finally, we show that TASF can be used to focus the neuropeptide galanin in a sample solvent with elution strength stronger than the mobile phase. Here, the stronger solvent is necessitated by the need to prevent peptide adsorption prior to and during analysis.