Regulation of Cholecystokinin Receptors in the Hypothalamus of the Rat: Reciprocal Changes in Magnocellular Nuclei Induced by Food Deprivation and Dehydration

Cholecystokinin (CCK) has been suggested to mediate satiety in a number of non-primate species via its peripheral actions as well as a possible central mechanism involving magnocellular and parvocellular oxytocin release. Quantitative in vitro autoradiography employing [¹²⁵l]-Bolton-Hunter labelled CCK-8S ([¹²⁵I]-CCK-8S) was used to examine the distribution and density of CCK receptors in sections of brain from normal rats and rats deprived of food, water or both food and water for 4 days. In food-deprived rats, specific [¹²⁵I]-CCK-8S binding was reduced by 64 ± 5% in the hypothalamic supraoptic nucleus (SON) and by 44±13% in the paraventricular nucleus of the hypothalamus (PVN). In contrast, water deprivation increased binding of [¹²⁵I]-CCK-8S by 128±15% in the SON and by 196 ± 24% in the PVN, while combined food and water deprivation produced smaller increases in both nuclei (30 ± 5% and 98 ± 26% in SON and PVN respectively). Changes in receptor density in the PVN appeared to be most prominent in the magnocellular (especially oxytocin-rich) subdivisions. None of the treatments employed produced changes in [¹²⁵I]-CCK-8S binding in the ventromedial hypothalamic nucleus or the reticular thalamic nucleus. Both CCK-A and CCK-B receptor subtypes were visualized in the nucleus of the solitary tract and the area postrema of normal rats, but levels of binding to both of these subtypes were unaffected by the experimental treatments. These selective alterations demonstrate the plasticity of CCK receptors in the SON and PVN, and are probably associated with changes in the level of neurochemical activity of magnocellular oxytocinergic neurones in these areas. These results, together with reports of changes in the level of CCK synthesis in cells of the SON and PVN after hyperosmotic stimuli, suggest that CCK may act in an autocrine fashion on these neurones and that both CCK receptors and peptide levels are altered in the same direction following cellular activation or inhibition.     

Adenylyl Cyclase mRNA Expression does not Reflect the Predominant Ca2+/Calmodulin-Stimulated Activity in the Hypothalamus

Only three (Types I, II, V) of the six currently-described subtypes of adenylyl cyclase are prominently expressed in the rat brain. These species are differently sensitive to Ca²⁺, βγ subunits of G-proteins and protein kinase C. A knowledge of the susceptibility of the cAMP-signalling system in particular brain regions to these diverse modes of regulation can shed light on the mechanism of action of the neurotransmitters that modify neuronal activity in such regions. Cyclic AMP is extensively involved in the physiological functions of the hypothalamus. We have used in situ hybridization histochemistry with synthetic oligonucleotides to examine the expression in the rat hypothalamus of the three major brain subtypes of adenylyl cyclase-Ca²⁺/calmodulin-stimulable (Type I), Ca²⁺-insensitive (Type II) and Ca²⁺ -inhibitable (Type V). The hypothalamus expresses high levels only of Type II mRNA, particularly in the supraoptic and paraventricular nuclei. Curiously, the strong expression of the Ca²⁺-insensitive Type II mRNA and the lack of expression of the major brain specific Type I mRNA does not correlate with the adenylyl cyclase activity, which is largely Ca²⁺/calmodulin stimulable in plasma membranes prepared from the hypothalamus.     

Regional expression of 5-HT1B receptor mRNA in the human brain

The regional mRNA expression pattern of 5-HT(1B) receptors has been extensively characterized in the rodent and guinea pig brain, but a detailed mapping of the 5-HT(1B) receptor mRNA expression in the human brain has not previously been performed. In the present study, the mRNA expression of 5-HT(1B) receptors was analyzed using in situ hybridization histochemistry and whole hemisphere sections of the human postmortem brain. The mRNA expression was compared with the autoradiographic distribution of 5-HT(1B) receptors. High levels of mRNA expression were found in the striatum, cortex, lateral geniculate nucleus, and raphe nuclei. The expression was higher in ventral than in dorsal striatal regions and was absent from the substantia nigra and pallidum, where high levels of 5-HT(1B) receptors were found. A layer-specific expression pattern was observed in cortical regions. The results extend previous knowledge about the localization of the 5-HT(1B) receptor in the human brain. This study provides evidence of a mismatch of the regional expression of 5-HT(1B) receptor mRNA and the 5-HT(1B) receptor distribution in human brain, similar to what has been demonstrated in other species. This is in line with the localization of this receptor subtype in nerve terminals. The results give support to species differences in the cortical mRNA expression pattern of this receptor subtype.     

Orexin-A regulates growth hormone-releasing hormone mRNA content in a nucleus-specific manner and somatostatin mRNA content in a growth hormone-dependent fashion in the rat hypothalamus

The orexins or hypocretins are two neuropeptides involved in the regulation of diverse biological processes such as feeding, sleep and neuroendocrine function. Recent findings suggest a possible functional interaction between orexins, somatostatin and growth hormone-releasing hormone (GHRH) in the rat hypothalamus. In order to understand the possible functional linkage between orexins and these neuropeptides, we determined the effects of intracerebroventricular orexin-A administration on hypothalamic somatostatin and GHRH mRNA levels. Furthermore, we examined whether growth hormone (GH) mediates these interactions by using two animal models that showed GH deficiency: hypophysectomized rats and dwarf Lewis rats. Using in situ hybridization, our data showed that GHRH mRNA levels in the paraventricular nucleus of the hypothalamus are decreased after orexin-A treatment, without changes in the arcuate nucleus of the hypothalamus. On the other hand, orexin-A treatment induces a GH-dependent stimulatory effect on somatostatin mRNA content in the periventricular nucleus of the hypothalamus. Finally, we demonstrated, for the first time, that hypophysectomized rats and dwarf Lewis rats, two classical models of GH deficiency with alterations in sleep patterns, showed a marked reduction in the GHRH mRNA levels in the paraventricular nucleus of the hypothalamus. These data improve our understanding of the interactions among the different systems involved in the control and pathophysiology of food intake, sleep and GH secretion.     

The C-Type Natriuretic Peptide Receptor is the Predominant Natriuretic Peptide Receptor mRNA Expressed in Rat Hypothalamus

The natriuretic peptide receptors (NPR) are membrane-bound guanylate cyclases with extracellular binding domains specific for particular members of the natriuretic peptide family. NPR-A binds atrial natriuretic peptide (ANP) with high affinity, whereas the NPR-B appears to be specific for C-type natriuretic peptide (CNP). Previous data indicating extensive overlap between localization of ANP and CNP in hypothalamic neuroendocrine circuits suggest the importance of determining whether specificity of natriuretic peptide action may be conferred via receptor type present on target cells. To address this issue, we used in situ hybridization histochemistry to localize NPR-A and NPR-B mRNA in the hypothalamus. NPR-A mRNA was not found in substantial abundance in any hypothalamic nucleus; however, detectable NPR-A signal was observed in other brain regions, including the subfornical organ and medial habenula. In contrast, NPR-B mRNA was expressed throughout the hypothalamus, including neurons of the magnocellular and parvocellular paraventricular, the arcuate, and the supraoptic nuclei. Expression was also seen in other nuclei essential to neuroendocrine control, including the median preoptic, anteroventral periventricular, tuberomammillary, ventromedial and suprachiasmatic nuclei. NPR-B mRNA was also observed in the neural lobe of the pituitary gland, suggesting expression by pituicytes. The results suggest that NPR-B is the primary natriuretic peptide receptor in hypothalamus, and by inference indicate that CNP is the primary active natriuretic peptide in neuroendocrine regulation.     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in situ Hybridization Study

We have used in situ hybridization to investigate estradiol regulation of estrogen receptor (ER) mRNA in regions of the mediobasal hypothalamus which contain ER and are related to specific neuroendocrine functions. Ovariectomized rats were treated with oil or 10 μg estradiol benzoate for 2, 4, 18 or 24 h. Brains were sectioned and hybridized with a [³ H]single-stranded DNA probe prepared from the pORF cDNA of the human ER gene and exposed to autoradiographic emulsion for 4 months. Specificity of labeling was determined by counting the number of grains over cells in hypothalamic regions known to bind estradiol, compared to cells in the thalamus and cortex, and by comparing with sections pretreated with ribonuclease or hybridized with a [³ H]single-stranded message-sense (control) probe. Labeling for ER mRNA was distributed differently than glucocorticoid and thyroid hormone receptor mRNAs, and was regulated by estrogen differently than progestin receptor mRNA. These differences indicated specific hybridization for ER mRNA. ER-expressing cells constituted 11.5% of the cells in the dorsomedial nucleus, 30% of the cells in the arcuate nucleus and 43% in the ventromedial nucleus, in close accordance with previous studies of ER autoradiography and binding. Quantitative analysis showed that the highest level of ER mRNA was present in the ovariectomized controls. ER mRNA levels fell 42% (ventromedial), 64% (arcuate), or remained unchanged (dorsomedial) 18 h following estradiol benzoate treatment. The pattern of decrease was similar for cells in the ventromedial nucleus and arcuate nucleus. These data show that estrogen regulation of ER mRNA in brain parallels that reported for MCF-7 cells and rat uterus. These results also demonstrate that in situ hybridization can be used to detect and measure the relative level of a low abundance mRNA in a heterogeneous tissue in which only 12% to 40% of the cells in limited regions express the message.     

Distribution of GLP-1 Binding Sites in the Rat Brain: Evidence that Exendin-4 is a Ligand of Brain GLP-1 Binding Sites

The distribution and biochemical properties of glucagon-like peptide (GLP)-1(7–36)amide (GLP-1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [¹²⁵I]GLP-1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [¹²⁵I][Tyr39]exendin-4, a GLP-1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [¹²⁵I]GLP-1 to the NTS was inhibited concentration-dependently by unlabelled GLP-1 and [Tyr39]exendin-4 with K ₁ values of 3.5 and 9.4 nM respectively. Cross-linking of hypothalamic membranes with [¹²⁵I]GLP-1 or [¹²⁵I][Tyr39]exendin-4 identified a single ligand-binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP-1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP-1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP-1 receptor resembles the peripheral GLP-1 receptor. Furthermore, the presence of GLP-1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood-borne GLP-1 and GLP-1 in the nervous system.     

Simultaneous Visualization of Vasopressin and Oxytocin mRNA- Containing Neurons in the Hypothalamus Using Non-Radioactive in situ Hybridization Histochemistry

In situ hybridization histochemistry (ISHH) using synthetic oligodeoxynucleotide probes has been used to demonstrate the sites of expression of mRNA for vasopressin (AVP) and oxytocin (OXT) in the rat brain. ISHH was performed with two types of non-radioactive probes, labelled with either alkaline phosphatase or 5'-biotin. Simultaneous detection of the mRNAs for both AVP and OXT was achieved using an alkaline phosphatase substrate for the AVP probe and an anti-biotin monoclonal (mouse) antibody for the OXT probe. These probes revealed two non-overlapping populations of AVP and OXT neurons on the same section.     

Galanin Antagonists Block Galanin-induced Feeding in the Hypothalamus and Amygdala of the Rat

Galanin significantly increased food intake when microinjected into the region of the central nucleus of the amygdala as well as into the paraventricular nucleus of the hypothalamus. In the amygdala this effect was specific to feeding; no change in grooming, resting, or other behaviour was observed after galanin treatment. These results provide evidence that the amygdala may be an important site in the mediation of galanin-induced feeding. The galanin receptor antagonists, C7 and M40, antagonized galanin-induced feeding, while having no effect alone on food consumption in free-feeding rats. These new galanin receptor antagonists provide useful tools for further investigating the role of endogenous galanin in the regulation of feeding.     

Food or Water Deprivation Modulate Nitric Oxide Synthase (NOS) Activity and Gene Expression in Rat Hypothalamic Neurones: Correlation with Neurosecretory Activity?

Nitric oxide (NO) is produced by the enzyme NO synthase (NOS) and may be involved in the regulation of nutrient and endocrine homeostasis via actions on neurones of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. The effects of water deprivation or food deprivation for 4 days on the abundance of messenger RNA encoding NOS in these nuclei in rats were examined using in situ hybridization. Water deprivation markedly increased the abundance of NOS mRNA in both the SON and PVN (225±11% of control, P<0.05 and 261±34% of control, P<0.01 respectively). NOS mRNA abundance also appeared to be increased in magnocellular accessory nuclei. Food deprivation decreased NOS mRNA abundance in the SON and PVN (42±6% and 52±7% of control respectively, both P<0.05), while withdrawal of both food and water produced no significant net changes in the abundance of NOS mRNA. Treatment-induced alterations in NOS mRNA abundance were reflected by changes in NOS activity, as assessed by NADPH-diaphorase histochemistry, and NADPH-diaphorase staining was observed in neurones both positive and negative for oxytocin-like immunoreactivity. These findings suggest that NOS mRNA abundance, NOS enzymatic activity and presumably NO production are modulated in an activity-dependent manner in hypothalamic (magnocellular and parvocellular) neurones by alterations in fluid and nutrient homeostasis, and support data from other studies suggesting a role for NO in the central regulation of water and food intake in the rat.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Correlations between Water Maze Performance and Cortical Somatostatin mRNA and High-affinity Binding Sites During Ageing in Rats

Somatostatin levels and high-affinity (somatostatin-1) binding sites are decreased in post-mortem cortical samples of Alzheimer's disease patients but the relationships between such modifications and the cognitive deficits remain to be established. We investigated these relationships in the ageing rat. Three age groups (3–4, 14–15 and 26–27 months) were tested in a modified version of the Morris water maze. Somatostatin mRNA levels were quantified by in situ hybridization and somatostatin binding sites by radioautography using the selective agonist octreotide (SMS 201995) as a competing drug to evaluate high-affinity (somatostatin-1) and low-affinity (somatostatin-2) binding sites. The number of somatostatin mRNA-containing cells was not modified with age or memory performance in cortical, hippocampal and hypothalamic regions, but somatostatin mRNA densities were significantly decreased with age and with memory performance in the frontal and parietal cortex. In the frontal cortex somatostatin mRNA densities were already decreased in 14- to 15-month-old rats, whereas the decrease was observed only in 26- to 27-month-old rats in the parietal cortex. A decrease in somatostatin-1 binding was observed with memory performance, independently of age, in the basolateral amygdala only, while somatostatin-2 binding sites were not affected. In the frontal and parietal cortex, a significant correlation occurred between the latency to find the invisible platform in the water maze and somatostatin mRNA ( r = -0.54 and 0.59 respectively, P > 0.02). These results indicate that ageing rats with memory impairments display some of the features of the somatostatinergic deficits observed in Alzheimer's disease.     

Estrogen-Sensitive Oxytocin Binding Sites are Differently Regulated by Progesterone in the Telencephalon and the Hypothalamus of the Rat

The localization at the cellular level and the regulation by progesterone of the estrogen-sensitive oxytocin binding sites was studied in the rat telencephalon and the hypothalamus by using quantitative film-autoradiography and histoautoradiography. Male rats (castrated or not) and ovariectomized females (estradiol supplemented or not) were used to characterize these sites and to precise their localization. They were detected in the striatal cell bridges, the olfactory tubercle, the principal nucleus of the bed nucleus of the stria terminalis and the medial nucleus of the amygdala of the telencephalon and in the medial preoptic, the ventromedial and the ventral premammillary nuclei of the hypothalamus. Estrogen administration in addition induced expression of oxytocin binding sites in the major island of Calleja, the anterior hypothalamic area and the terete nucleus. The density of the estrogen-sensitive oxytocin binding sites varied during the estrous cycle, but differently in the telencephalon and the hypothalamus. in the telencephalon it peaked at proestrus 9 h and was already decreased at proestrus 21 h, whereas in the hypothalamus it was similarly high at proestrus 9h and proestrus 21 h, suggesting the intervention of progesterone in the regulation of the hypothalamic estrogen-sensitive oxytocin binding sites.