Parabrachial lesions in rats disrupt sodium appetite induced by furosemide but not by calcium deprivation.

An appetite for CaCl2 and NaCl occurs in young rats after they are fed a diet lacking Ca or Na, respectively. Bilateral lesions of the parabrachial nuclei (PBN) disrupt normal taste aversion learning and essentially eliminate the expression of sodium appetite. Here we tested whether similar lesions of the PBN would disrupt the calcium-deprivation-induced appetite for CaCl2 or NaCl. Controls and rats with PBN lesions failed to exhibit a calcium-deprivation-induced appetite for CaCl2. Nevertheless, both groups did exhibit a significant calcium-deprivation-induced appetite for 0.5M NaCl. Thus, while damage to the second central gustatory relay in the PBN disrupts the appetite for 0.5M NaCl induced by furosemide, deoxycorticosterone acetate, and polyethylene glycol, the sodium appetite induced by dietary CaCl2 depletion remains intact.

Concentration and state dependent reductions in corn oil intakes after glossopharyngeal nerve transections in rats.

Previous studies indicate a role for the glossopharyngeal nerve (GL) in the detection of dietary fats. The present experiments examined the effects of bilateral glossopharyngeal nerve transections (GLx) on the intake of low (4.8%), moderate (16%), and full-fat (100%) corn oil in non-deprived, food-deprived, and water-deprived rats. The rats had access to oils, 0.3 M sucrose, and water in a gustometer that measured number of licks and latency to the first lick during brief access trials. The behavioral measures were used as indices of the amount consumed and the motivation to ingest, respectively. After baseline intakes had stabilized, the rats received GLx or sham transections (Sham) and were then re-tested. Pre and post-surgery responses were compared to determine the impact of GLx on intake and the motivation to ingest. In non-deprived rats, GLx reduced the intake of 4.8% and 16% oils and decreased the motivation to ingest these oils. In food-deprived rats, GLx prevented increases in the ingestion of 4.8% and 16% oils and in the motivation to ingest these oils. In water-deprived rats, GLx reduced the intake of 100% oil and produced a general decrease in the motivation to consume low, moderate, and full-fat emulsions. These results indicate that GL is partially involved in corn oil intake and suggest an interactive effect of oil concentration with homeostatic state.

Parabrachial and hypothalamic interaction in sodium appetite.

Rats with bilateral lesions of the lateral hypothalamus (LH) fail to exhibit sodium appetite. Lesions of the parabrachial nuclei (PBN) also block salt appetite. The PBN projection to the LH is largely ipsilateral. If these deficits are functionally dependent, damaging the PBN on one side and the LH on the other should also block Na appetite. First, bilateral ibotenic acid lesions of the LH were needed because the electrolytic damage used previously destroyed both cells and axons. The ibotenic LH lesions produced substantial weight loss and eliminated Na appetite. Controls with ipsilateral PBN and LH lesions gained weight and displayed robust sodium appetite. The rats with asymmetric PBN-LH lesions also gained weight, but after sodium depletion consistently failed to increase intake of 0.5 M NaCl. These results dissociate loss of sodium appetite from the classic weight loss after LH damage and prove that Na appetite requires communication between neurons in the LH and the PBN.

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling.

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.

Gustatory reward and the nucleus accumbens.

The concept of reward is central to psychology, but remains a cipher for neuroscience. Considerable evidence implicates dopamine in the process of reward and much of the data derives from the nucleus accumbens. Gustatory stimuli are widely used for animal studies of reward, but the connections between the taste and reward systems are unknown. In a series of experiments, our laboratory has addressed this issue using functional neurochemistry and neuroanatomy. First, using microdialysis probes, we demonstrated that sapid sucrose releases dopamine in the nucleus accumbens. The effect is dependent on oral stimulation and concentration. We subsequently determined that this response was independent of the thalamocortical gustatory system, but substantially blunted by damage to the parabrachial limbic taste projection. Further experiments using c-fos histochemistry confirmed that the limbic pathway was the prime carrier for the gustatory afferent activity that drives accumbens dopamine release.

Regional activation in the rat brain during visceral stimulation detected by c-fos expression and fMRI.

AIM: The aim of the study was to determine and compare the areas of brain activated in response to colorectal distention (CRD) using functional magnetic resonance imaging (fMRI) and c-fos protein expression. METHODS: For fMRI study (3.0 T magnet), anaesthetized rats underwent phasic CRD, synchronized with fMRI acquisition. Stimulation consisted of eight cycles of balloon deflation (90 s) and inflation (30 s), at 40, 60 or 80 mmHg of pressure. For c-fos study two sets of experiments were performed on anaesthetized rats: comparing (A) brain activation in rats with the inserted colorectal balloon (n = 5), to the rats without the balloon (n = 5); and (B) rats with inserted balloon (n = 10), to the rats with inserted and distended balloon (n = 10). The pressure of 80 mmHg was applied for 2 h of 30 s inflation and 90 s deflation, alternating cycles. RESULTS: Functional MRI revealed significant activation in the amygdala, hypothalamus, thalamus, cerebellum and hippocampus. Significant increase in c-fos expression was observed in amygdala and thalamus in the first set of experiments, and hypothalamus and parabrachial nuclei in the second. CONCLUSION: The two methods are not interchangeable but appeared to be complementary: fMRI was more sensitive, whereas c-fos had much greater resolution.

Intracranial renin alters gustatory neural responses in the nucleus of the solitary tract of rats.

Activation of the renin-angiotensin system in the brain is considered important in the arousal and expression of sodium appetite. To clarify the effects of directly activating this hormonal cascade, taste neurons in the nucleus of the solitary tract of rats were tested with a battery of sapid stimuli after intracerebroventricular injection of renin or its vehicle. The rats were chronically prepared but lightly anesthetized during the recording procedure. Eighty-five taste neurons were tested: 46 after renin injections and 39 after vehicle. Neural activity was counted for 5.0-s periods without stimulation (spontaneous) and during stimulation with water and sapid chemicals. The averaged responses to each of the standard stimuli (0.1 M NaCl, 0.3 M sucrose, 0.01 M citric acid, and 0.01 M quinine hydrochloride) did not differ significantly between the two conditions. When the rats were tested with a concentration range of NaCl, however, after renin the average responses to the hypertonic 0.3 and 1.0 M stimuli were reduced to 74 and 70%, respectively, compared with those after vehicle injections. A similar tendency was evident for the subsample of neurons that responded best to NaCl, but the effect was smaller. These data are consistent with, but not as dramatic as, those reported after dietary-induced sodium appetite.

Nuclear transfer technology in mammalian cloning.

The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

Cellular interactions in the development of the olfactory system: an ablation and homotypic transplantation analysis.

In the current study, we addressed two questions: First, is the olfactory placode necessary for the development of the olfactory bulb and the entire telencephalon? Second, does the olfactory placode contribute cells to the olfactory bulb? We addressed these questions by unilaterally ablating the olfactory placode in chick embryos before an olfactory nerve was produced and, in a second series of experiments, by replacing the ablated chick olfactory placode with a quail olfactory placode. Our results indicate that the olfactory placode is critical for olfactory bulb development, but is not necessary for the development of the rest of the telencephalon. Further, our results support the hypothesis that LHRH neurons and olfactory nerve glia originate in the olfactory placode, but do not support an olfactory placodal origin for other cell types within the olfactory bulb.

Parabrachial nucleus lesions block taste and attenuate flavor preference and aversion conditioning in rats.

Rats with ibotenic acid lesions of the parabrachial nucleus (PBN) failed to learn a taste aversion induced by lithium chloride (LiCl) toxicosis. The same rats also did not learn to prefer a taste that was paired with intragastric (IG) carbohydrate infusions during 22 hr/day trials. The PBN-lesioned rats did learn to prefer a flavor (odor + taste) paired with the IG carbohydrate infusions over a different flavor paired with IG water. The PBN-lesioned rats also learned to avoid a flavor paired with IG LiCl infusions during 22 hr/day trials. The flavor preference and aversion, however, were less pronounced than those displayed by control rats. These data indicate that the PBN is essential for forming orosensory-viscerosensory associations when taste is the primary cue but is less critical when more complex flavor cues are available.

Accumbens dopamine mechanisms in sucrose intake.

Extracellular levels of dopamine (DA) and monoamine metabolites were measured in the nucleus accumbens (NAcc) during sucrose licking using microdialysis in freely moving rats. The converse relationship also was tested. Using bilateral reverse microdialysis, D1 and D2 receptor antagonists (SCH23390, sulpiride) and the DA uptake blocker nomifensine were introduced into NAcc while measuring both ingestive behavior and neurochemistry. Licking of 0.3 M sucrose caused a 305% (+/-69%) increase in NAcc DA compared with water intake. Reverse microdialysis of nomifensine at a dose that increased accumbens DA levels (1484+/-346%) led to an increase of sucrose intake (152.5+/-5.4%). Concurrent infusions of the D1 and D2 blockers with nomifensine brought sucrose ingestion back near to control levels (114.8+/-3.7%). The higher dose of the D2 antagonist sulpiride also increased DA levels and sucrose intake. In contrast, the lower dose of the D2, and both doses of the D1 antagonist had no chemical or behavioral effects. These results showed release of NAcc DA in response to sucrose licking and the converse, an augmentation of the behavior by uptake blockade. The same data, however, failed to prove that tonic, local accumbens D1 and D2 receptor activity influenced this ingestive behavior.

Pontine gustatory activity is altered by electrical stimulation in the central nucleus of the amygdala.

Visceral signals and experience modulate the responses of brain stem neurons to gustatory stimuli. Both behavioral and anatomical evidence suggests that this modulation may involve descending input from the forebrain. The present study investigates the centrifugal control of gustatory neural activity in the parabrachial nucleus (PBN). Extracellular responses were recorded from 51 single PBN neurons during application of sucrose, NaCl, NaCl mixed with amiloride, citric acid, and QHCl with or without concurrent electrical stimulation in the ipsilateral central nucleus of the amygdala (CeA). Based on the sapid stimulus that evoked the greatest discharge, 3 neurons were classified as sucrose-best, 32 as NaCl-best, and 16 as citric acid-best. In most of the neurons sampled, response rates to an effective stimulus were either inhibited or unchanged during electrical stimulation of the CeA. Stimulation in the CeA was without effect in two sucrose-best neurons, nine NaCl-best neurons, and one citric acid-best neuron. Suppression was evident in 1 sucrose-best neuron, 18 NaCl-best neurons, and 15 citric acid-best neurons. In NaCl-best neurons inhibited by CeA stimulation, the magnitude of the effect was similar for spontaneous activity and responses to the five taste stimuli. Nonetheless, the inhibitory modulation of gustatory sensitivity increased the relative effectiveness of NaCl resulting in narrower chemical selectivity. For citric acid-best neurons, the magnitude of inhibition produced by CeA activation increased with an increase in stimulus effectiveness. The responses to citric acid were inhibited significantly more than the responses to all other stimuli with the exception of NaCl mixed with amiloride. The overall effect was to change these CA-best neurons to CA/NaCl-best neurons. In a smaller subset of NaCl-best neurons (n = 5), CeA stimulation augmented the responsiveness to NaCl but was without effect on the other stimuli or on baseline activity. It appears that electrical stimulation in the CeA modulates response intensity, as well as the type of gustatory information that is transmitted in a subset of NaCl-best neurons. These findings provide an additional link between the amygdala and the PBN in the control of NaCl intake, modulating the response and the chemical selectivity of an amiloride-sensitive Na+ detecting input pathway.

TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neuroendocrine brain in a developmentally regulated and cell-specific fashion.

TTF-1 is a member of the Nkx family of homeodomain genes required for morphogenesis of the hypothalamus. Whether TTF-1, or other Nkx genes, contributes to regulating differentiated hypothalamic functions is not known. We now report that postnatal hypothalamic TTF-1 expression is developmentally regulated and associated with the neuroendocrine process of female sexual development. Lesions of the hypothalamus that cause sexual precocity transiently activate neuronal TTF-1 expression near the lesion site. In intact animals, hypothalamic TTF-1 mRNA content also increases transiently, preceding the initiation of puberty. Postnatal expression of the TTF-1 gene was limited to subsets of hypothalamic neurons, including LHRH neurons, which control sexual maturation, and preproenkephalinergic neurons of the lateroventromedial nucleus of the basal hypothalamus, which restrain sexual maturation and facilitate reproductive behavior. TTF-1 mRNA was also detected in astrocytes of the median eminence and ependymal/subependymal cells of the third ventricle, where it colocalized with erbB-2, a receptor involved in facilitating sexual development. TTF-1 binds to and transactivates the erbB-2 and LHRH promoters, but represses transcription of the preproenkephalin gene. The singular increase in hypothalamic TTF-1 gene expression that precedes the initiation of puberty, its highly specific pattern of cellular expression, and its transcriptional actions on genes directly involved in neuroendocrine reproductive regulation suggest that TTF-1 may represent one of the controlling factors that set in motion early events underlying the central activation of mammalian puberty.

Disruption of the olfactory placode and brain conditioned medium increase the number of LHRH immunostained neurons in explants.

The olfactory placode gives rise to both olfactory receptor neurons, which remain as a component of the peripheral nervous system, and to luteinizing hormone-releasing hormone (LHRH) neurons, which migrate to the central nervous system. In this study, we used chick olfactory placode explants to ask several questions regarding LHRH neuronal differentiation. We found that explants of ectoderm from the fronto-nasal region of embryos as early as Hamilton & Hamburger (HH) stage 12 gave rise to LHRH neurons, that explants from all regions of the olfactory placode were able to generate LHRH neurons, that both brain conditioned medium and disruption of the olfactory placode increase the number of LHRH neurons observed in explants, and that the combination of these two manipulations results in the production of more LHRH neurons than either treatment alone. We conclude that LHRH neurons originate in the olfactory epithelium and that some of the same factors which influence olfactory receptor neuron development also affect LHRH neuronal development.

Parabrachial lesions disrupt responses of rats to amino acid devoid diets, to protein-free diets, but not to high-protein diets.

Normal rats "reduce" intake of diets that lack an essential amino acid (THR-DEV), are protein free (PO%), or contain a high proportion of protein (P75%). We tested the importance of the parabrachial nuclei (PBN) in signaling such adjustments of food intake by placing electrophysiologically guided lesions in these nuclei at points that responded to gustatory stimuli. When fed the THR-DEV diet, rats with PBN lesions (PBNx) decreased their food intake significantly less than the controls (78.5 vs. 44.4%). When put on a P0% diet, PBNx animals decreased their intake only 8% compared with 23% for our CONT group. When put on a P75% diet, however, both groups decreased their intake in an equivalent amount. These experiments show that the PBN is involved in the learned aversion to an amino acid devoid diet.

Projections of the parabrachial nucleus in the old world monkey.

The efferent projections of the pontine parabrachial nucleus (PBN) were examined in the Old World monkey (Macaca fascicularis) using tritiated amino acid autoradiography and horseradish peroxidase histochemistry. Parabrachiofugal fibers ascended to the forebrain along three pathways: the central tegmental tract, the ventral ascending catecholaminergic pathway, and a pathway located on the midline between the medial longitudinal fasciculi. The PBN projected heavily to the central nucleus of the amygdala and the lateral division of the bed nucleus of the stria terminalis and moderately to the ventral tegmental area and the substantia nigra. Light terminal label also was present within the dorsomedial, ventromedial, lateral, supramammillary, and infundibular nuclei of the hypothalamus and the annular nucleus and the dorsal raphe nucleus within the brain stem. The overall pattern of terminal label was similar to that previously reported for nonprimate species, but several differences were notable. In monkey the projection to the ventrobasal thalamus did not coincide with the region that contains gustatory-responsive neurons. In rats, these parabrachiothalamic fibers convey gustatory activity but in the monkey these fibers may carry visceral afferent information. The projections from the PBN to the hypothalamus in the monkey were neither as widespread nor as intense as in the rat, and the monkey lacks a projection from the PBN to the frontal and insular cortices.

LHRH neuronal migration: heterotypic transplantation analysis of guidance cues.

During embryonic development, the olfactory placode (OP) differentiates into the olfactory epithelium (OE). Luteinizing hormone-releasing hormone (LHRH) neurons migrate out of the OE in close association with the olfactory nerve (ON) to the telencephalon. LHRH neuronal migration and ON extension to the telencephalon may be independent events which are correlated but do not represent a causal relationship. However, we hypothesize that LHRH neurons are dependent on ON axons to migrate to the brain. To test this hypothesis, we ablated the right trigeminal placode and replaced it with an OP from another chick embryo. After several days' additional incubation, the embryos were fixed, sectioned, and immunostained with antibodies against LHRH or N-CAM. The ectopic OPs were well integrated into the host and developed into relatively normal appearing OEs. The ONs extended from the OE to several different sites: the lateral rectus of the eye, the ciliary ganglion, and the trigeminal ganglion. In all cases, LHRH neurons were found in the OE and ON, regardless of where the ON terminated. When the ON extended to the trigeminal ganglion, LHRH neurons could clearly be seen entering the metencephalon. Our results support the idea that LHRH neurons are dependent on the ON for guidance as they appear to follow the nerve even when it extends away from the brain. The cues which direct the ON and LHRH neurons to the telencephalon do not appear to be unique to this brain region.

Effect of intraduodenal lipid on parabrachial gustatory coding in awake rats.

Intestinal fat differentially suppresses sham feeding of liquid diets and preferred gustatory stimuli. Although the behavioral effect is robust, no electrophysiological evidence exists to account for its neural basis. Therefore, we investigated the effect of intestinal fat on gustatory coding in the pontine parabrachial nuclei (PBN) by recording from single neurons in awake rats before, during, and after intraduodenal infusions of lipid (Intralipid; 10 ml, 5 kcal). Intraduodenal lipid did not alter the response profiles of PBN taste neurons. It did, however, produce an overall decrease in response magnitude (-16.25%; n = 43), with the largest reduction to sucrose (-30%; n = 43). The most pronounced suppression occurred in sucrose-best neurons in response to sucrose (-55%; n = 19), and this effect was largest for the sucrose-specific cells (-77%; n = 3). After lipid infusions, nonspecific neurons in both the sucrose-best and NaCl-best categories also responded less to their best stimulus (sucrose, -46%; n = 16; NaCl, -35%; n = 13). In contrast, no significant changes were found in NaCl-specific cells in response to NaCl. All effects appeared with short latency ( approximately 5 min) and were reversible within the time frame of a meal. In controls, duodenal infusions of saline did not cause any changes in taste responsiveness. These results suggest that intestinal fat has specific effects on taste coding in the PBN that may contribute to the intake suppression of palatable food observed in behavioral studies. The similar, short latency of both the behavioral and neural effects supports the hypothesis of a preabsorptive site of action.

The parabrachial nucleus is essential for acquisition of a conditioned odor aversion in rats.

Rats with bilateral ibotenic acid lesions of the gustatory zone of the parabrachial nuclei (PBN) failed to acquire a conditioned taste aversion (CTA) in Experiment 1. They also failed to acquire a conditioned odor aversion (COA) when the olfactory cue was presented on an odor disk in Experiment 2 or when it was presented in water in Experiment 3. The failure to acquire the COA was not due to an inability to detect or use olfactory stimuli because the lesioned rats displayed neophobia to a novel odor in Experiment 3 and used an olfactory cue to predict the availability of an aversive capsaicin solution in Experiment 4. Together, the results demonstrate that, as with CTA learning, PBN cell bodies are essential for the establishment of a specific association between an olfactory conditioned stimulus and a lithium chloride unconditioned stimulus.

Herpes simplex virus as a transneuronal tracer.

Determining the connections of neural systems is critical for determining how they function. In this review, we focus on the use of HSV-1 and HSV-2 as transneuronal tracers. Using HSV to examine neural circuits is technically simple. HSV is injected into the area of interest, and after several days, the animals are perfused and processed for immunohistochemistry with antibodies to HSV proteins. Variables which influence HSV infection include species of host, age of host, titre of virus, strain of virus and phenotype of infected cell. The choice of strain of HSV is critically important. Several strains of HSV-1 and HSV-2 have been utilized for purposes of transneuronal tract-tracing. HSV has been used successfully to study neuronal circuitry in a variety of different neuroanatomical systems including the somatosensory, olfactory, visual, motor, autonomic and limbic systems.