Feeding inhibition by urocortin in the rat hypothalamic paraventricular nucleus.

Ventricular administration of urocortin (UCN) inhibits feeding, but specific site(s) of UCN action are unknown. In the current studies we examined the effect of UCN in the hypothalamic paraventricular nucleus (PVN) on feeding. We tested UCN administered into the PVN in several paradigms: deprivation-induced, nocturnal, and neuropeptide Y (NPY)-induced feeding. We compared the effect of equimolar doses of UCN and corticotrophin releasing hormone (CRH) on NPY-induced and nocturnal feeding, determined whether UCN in the PVN produced a conditioned taste aversion (CTA) and induced changes in c-Fos immunoreactivity (c-Fos-ir) after UCN and NPY administration in the PVN. UCN in the PVN significantly decreased NPY and nocturnal and deprivation-induced feeding at doses of 1, 10, and 100 pmol, respectively. UCN anorectic effects lasted longer than those attributed to CRH. Ten and thirty picomoles UCN did not induce a CTA, whereas 100 pmol UCN produced a CTA. UCN (100 pmol) in the PVN neither increased c-Fos-ir in any brain region assayed nor altered c-Fos-ir patterns resulting from PVN NPY administration. These data suggest the hypothalamic PVN as a site of UCN action.

Construction of 1 mm microdialysis probe for amino acids dialysis in rats.

We describe here a microdialysis probe with 1 mm opening for precise and confined dialysis area in the awake, freely moving rat. This probe is designed to allow the local diffusion of the perfusion medium to an area approximately 175 microm high, 266 microm wide (mediolateral direction), and 305 microm in rostrocaudal direction. In addition, the probe allows the local application of drugs to the same precise area of interest. The probe was constructed from a piece of 25 gauge tubing with 1 mm hallowed opening located 0.5 mm from the distal (inserting) end. The dialysis fiber which was inserted into the stainless steel 25 gauge tubing and cemented into place has 200 microm diameter and 5000 molecular weight cut off. We tested the probe diffusion extent by direct infusion of fluorogold through the dialysis cannula. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine a sodium channel activator. All amino acids tested showed a significant 80% times decrease in their recovery concentration when compared to their respective concentrations recovered through 2 mm probe constructed earlier in our laboratory (Renno et al., 1992). Tests in awake rats with probes in the ventrocaudal PAG showed stable amounts of 12 different amino acids during repeated (6-8 times) 12 min samples at 3-5 microl/min collecting rate. Depolarization with 75 microM veratridine resulted in significant elevation in extracellular gamma-aminobutyric acid (GABA), aspartate, glutamate, taurine, glycine and citrulline. This design enables us to apply drugs of interest and measure the concentrations of amino acid neurotransmitters to a more precise, delineated and premeasured areas in the CNS.

Electrical stimulation of the sciatic nerve alters neuropeptide content and lymphocyte migration in the subcutaneous tissue of the rat hind paw.

To study the possible mechanism by which peripheral nerves mediate immune responses in target tissues, electrical stimulation of the sciatic nerve was combined with subcutaneous microdialysis of the hind paw. Following unilateral stimulation of the sciatic nerve, an ipsilateral rise in substance P and a bilateral rise in VIP levels were observed in dialysate samples from experimental vs control animals. Electrical stimulation of the sciatic nerve induced a marked hyperemia and swelling of the ipsilateral paw. Quantitative immunocytochemical analysis of paraffin-embedded sections of the hind foot pads demonstrated T lymphocyte migration ipsilateral to the stimulated nerve. These findings suggest that peripheral nerves can directly modulate local immune and inflammatory responses.

Basal release of Met-enkephalin and neurotensin in the ventrolateral periaqueductal gray matter of the rat: a microdialysis study of antinociceptive circuits.

The periaqueductal gray (PAG) contains neural circuits that participate in descending antinociception. Anatomical and electrophysiological evidence suggests that these circuits might employ opioid peptides and GABA in series to remove a tonic inhibition of descending PAG output neurons. The present studies examined the release of the antinociceptive peptides Met-enkephalin and neurotensin in the ventrolateral PAG, and investigated the interaction between GABA and Met-enkephalin release. In awake and freely moving rats the ventrolateral PAG was dialysed using 25 ga. concentric probes. Basal release of peptide in 12 min or 40 min fractions was determined using radioimmunoassays. To establish how the ventrolateral PAG responds to nociception, dialysis was performed following unilateral hindpaw inflammation using Complete Freund's Adjuvant. Twenty-four hours after inflammation was induced, neurotensin release was increased 133% and Met-enkephalin release was increased 353% compared to control animals. Seven days after inflammation was induced, neurotensin release declined precipitously, while basal Met-enkephalin release remained elevated 313% above controls. Thus, unlike enkephalin, increased basal neurotensin release is not sustained with persistent tonic nociception. In addition, we confirmed in normal animals that the ventrolateral PAG is induced to release Met-enkephalin by systemic morphine. A 43% increase in basal Met-enkephalin release was observed immediately following a 12 mg/kg i.p. morphine injection. Morphine should have the opposite effect (inhibit peptide release) if it acts directly on the enkephalinergic neurons. Thus, we examined the hypothesis that GABAergic interneurons in the PAG mediated morphine-stimulated enkephalin release. When the GABAantagonist bicuculline (0.25 microM to 25 microM) was co-infused with the dialysis medium, Met-enkephalin release increased in a dose-dependent fashion and peaked 68% above pre-infusion levels. These data elucidate the reciprocal inhibitory relationship between GABA and enkephalin in the ventrolateral PAG. We hypothesize that, when nociception induces Met-enkephalin release within this region, the tonic GABAergic inhibition is overcome, resulting in greater sensitivity of PAG enkephalinergic neurons. Ultimately, this enhanced enkephalin release should result in greater excitability of the descending PAG output neurons that are responsible for antinociception.