Hearing and tinnitus in head and neck cancer patients after chemoradiotherapy.

Head and neck cancer patients treated with high-dose cisplatin and radiotherapy will suffer from hearing deficits. The current low-dose regimen seldom causes hearing threshold decrease. Tinnitus in this patient population has not been investigated earlier. We aimed to evaluate the possible ototoxicity of low-dose (40 mg/m(2)) weekly administered cisplatin with concomitant radiotherapy. Twenty-two patients with locally advanced head and neck cancer were prospectively recruited to participate the study after treatment recommendation for chemoradiotherapy with low-dose cisplatin and intensity-modulated radiotherapy. They filled in a Tinnitus Handicap Inventory and undertook audiologic evaluations before and after treatment. Ototoxicity was determined by >10 dB threshold shift at frequencies 4 and 8 kHz or in pure tone average. A historical cohort of nine patients treated with high-dose (100 mg/m(2)) cisplatin and radiotherapy was used for comparison. After treatment, study patients demonstrated no significant changes in their hearing over frequencies 0.5-4 kHz, and the threshold shifts were minor at 4 and 8 kHz. More than 50 % of patients reported no tinnitus after treatment and the remainder only had slight to moderate tinnitus causing no interference with their daily activities. In contrast, five of the nine patients having received high-dose cisplatin reported disturbing tinnitus. Further, changes in pure tone averages were exhibited in three of these patients and six had significant threshold shifts at 4 and 8 kHz. Head and neck cancer patients treated with concomitant intensity-modulated radiotherapy and low-dose cisplatin seem to experience only minor audiological sequelae and therefore, these patients appear to require no routine audiological monitoring. Such evaluation could be performed only when needed.

Comparison of MR imaging findings in paediatric and adult patients with acute mastoiditis and incidental intramastoid bright signal on T2-weighted images.

OBJECTIVES: To compare MR imaging features in patients with incidental mastoid T2-hyperintensity with those of clinical acute mastoiditis, to ascertain characteristic differences between them. METHODS: MR images of 35 adult and paediatric patients with clinical acute mastoiditis and 34 consecutive age-matched controls without relevant middle ear pathology and with incidental T2-hyperintensity that covered ≥ 50 % of the mastoid were retrospectively analysed with regard to signal, diffusion, and enhancement characteristics, and presence of complications. RESULTS: Incidental mastoid T2-hyperintensity that covered ≥ 50 % of the mastoid volume was found in 4.6 % of reviewed MR scans (n = 2341), and associated significantly (p < 0.05) less with the involvement of the tympanic cavity (38 % vs. 74 %) and mastoid antrum (56 % vs. 80 %), hypointense-to-CSF signal intensity on T2 FSE (6 % vs. 86 %), intramastoid diffusion restriction (0 % vs. 62 %), intense intramastoid enhancement (0 % vs. 51 %), periosteal enhancement (3 % vs. 69 %), perimastoid dural enhancement 3 % vs. 43 %), bone destruction (0 % vs 49 %), intratemporal abscess or cholesteatoma (0 % vs. 24 %), labyrinth involvement (0 % vs. 14 %), and extracranial abscesses (0 % vs. 20 %). CONCLUSION: Hypointense-to-CSF signal intensity on T2WI, restricted diffusion, intense intramastoid enhancement among other MR imaging characteristics favoured an acute mastoiditis diagnosis over clinically non-relevant incidental mastoid pathology. KEY POINTS: • Intramastoid T2-hyperintensity alone is not a reliable sign for acute mastoiditis. • In acute mastoiditis, intramastoid T2-weighted signal intensity is usually hypointense to CSF. • Diffusion restriction and intense intramastoid enhancement are absent in incidental mastoid effusion. • An ADC value ≥ 1.72 × 10 (-3) mm (2) /s contradicts the AM diagnosis.

Effect of S53P4 bone substitute on staphylococcal adhesion and biofilm formation on other implant materials in normal and hypoxic conditions.

To study the effect of bioactive glass bone substitute granules (S53P4) on bacterial adhesion and biofilm formation on other simultaneously used implant materials and the role of the hypoxic conditions to the adhesion. Bacterial and biofilm formation were studied on materials used both in middle ear prostheses and in fracture fixtures (titanium, polytetrafluoroethylene, polydimethylsiloxane and bioactive glass plates) in the presence or absence of S53P4 granules. The experiments were done either in normal atmosphere or in hypoxia simulating atmospheric conditions of middle ear, mastoid cavity and sinuses. We used two collection strains of Staphylococcus aureus and Staphylococcus epidermidis. In the presence of bioglass and hypoxic conditions the adhesion of the planktonic bacterial cells was decreased for most of the materials. The biofilm formation was decreased for S. epidermidis on titanium and polydimethylsiloxane in both atmospheric conditions and on bioglass plates in normoxia. For S. aureus the biofilm formation was decreased on bioglass plates and polytetrafluoroethylene in normoxia. Hypoxia produces a decrease in the biofilm formation only for S. aureus on polytetrafluoroethylene and for S. epidermidis on bioglass plates. However, in none of the cases bioactive glass increased the bacterial or biofilm adhesion. The presence of bioglass in normoxic and hypoxic conditions prevents the bacterial and biofilm adhesion on surfaces of several typical prosthesis materials in vitro. This may lead to diminishing postoperative infections, however, further in vivo studies are needed.

Effects of S53P4 bioactive glass on osteoblastic cell and biomaterial surface interaction.

To study the effect of bioactive glass bone substitute granules (S53P4) and hypoxic atmospheric conditions on human osteoblastic cell adhesion on different biomaterials. Cellular adhesion and cytoskeletal organization were studied on titanium, polytetrafluoroethylene, polydimethylsiloxane and S53P4 plates in the presence or absence of S53P4 granules. Cells used were human osteoblast-like SaOS-2 cells. The experiments were done either in normal atmospheric conditions or in hypoxia which simulates conditions prevailing in chronically infected bone or bone cavities. Vinculin-containing focal adhesions, organization of actin cytoskeleton and nuclear staining of cells on biomaterial surfaces were studied at 4.5 h, 2 and 4 days. In normoxic conditions S53P4 granules alkalinized the cell culture medium but cellular adhesion and cytoskeletal organization were usually not affected by their presence. Hypoxic conditions associated with lower pH and impaired cellular adhesion, vinculin-containing focal adhesion formation and rearrangement of the actin filaments to actin cytoskeleton. On most materials studied in hypoxic conditions, however, S53P4 granules prevented this impairment of cellular adhesion and cytoskeletal reorganization. The S53P4 granules promote the adhesion of SaOS-2 cells to various biomaterial surfaces especially in hypoxic conditions, in which S53P4 granules increase pH. The presence of S53P4 granules may protect biomaterial surface from bacterial colonization and promote osteointegration of implants used together with S53P4 granules for fixation and weight bearing.

MR imaging features of acute mastoiditis and their clinical relevance.

BACKGROUND AND PURPOSE: MR imaging is often used for detecting intracranial complications of acute mastoiditis, whereas the intratemporal appearance of mastoiditis has been overlooked. The aim of this study was to assess the imaging features caused by acute mastoiditis in MR imaging and their clinical relevance. MATERIALS AND METHODS: Medical records and MR imaging findings of 31 patients with acute mastoiditis (21 adults, 10 children) were analyzed retrospectively. The degree of opacification in the temporal bone, signal and enhancement characteristics, bone destruction, and the presence of complications were correlated with clinical history and outcome data, with pediatric and adult patients compared. RESULTS: Most patients had ≥50% of the tympanic cavity and 100% of the mastoid antrum and air cells opacified. Compared with CSF, they also showed intramastoid signal changes in T1 spin-echo, T2 TSE, CISS, and DWI sequences; and intramastoid, outer periosteal, and perimastoid dural enhancement. The most common complications in MR imaging were intratemporal abscess (23%), subperiosteal abscess (19%), and labyrinth involvement (16%). Children had a significantly higher prevalence of total opacification of the tympanic cavity (80% versus 19%) and mastoid air cells (90% versus 21%), intense intramastoid enhancement (90% versus 33%), outer cortical bone destruction (70% versus 10%), subperiosteal abscess (50% versus 5%), and perimastoid meningeal enhancement (80% versus 33%). CONCLUSIONS: Acute mastoiditis causes several intra- and extratemporal changes on MR imaging. Total opacification of the tympanic cavity and the mastoid, intense intramastoid enhancement, perimastoid dural enhancement, bone erosion, and extracranial complications are more frequent in children.

Cone-beam computed tomography: a new method for imaging of the temporal bone.

BACKGROUND: Clinical cone-beam computed tomography (CBCT), used in diagnostics of dental and maxillofacial radiology for almost 10 years, allows three-dimensional (3D) imaging of a focused area, with reasonable radiation dose. PURPOSE: To clarify the applicability of CBCT in imaging of the temporal bone. MATERIAL AND METHODS: We imaged cadaver temporal bones, one non-operated and five postmortem operated, with CBCT to evaluate the accuracy of this method in showing clinically important landmarks and the positions of middle-ear implants. In addition, to clarify the imaging protocols for the best possible result, we conducted a contrast-to-noise ratio (CNR) analysis by imaging a specially built phantom insert with different protocols. RESULTS: For all the temporal bones, image quality was good and of diagnostic value, and the surgical landmarks as well as positions and details of the implants could be accurately observed. Based on measurements conducted with the phantom, the best possible clarity of the images with the machine used (3D Accuitomo; Morita Co., Kyoto, Japan) was achieved with a tube voltage of 80 kVp and a current of 4 mA. CONCLUSION: Cone-beam CT is a promising new method for otologic imaging, based on its accuracy and relatively low radiation exposure per investigation.

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV-CBA-Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1mul of the AAV-CBA-Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV-CBA-GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging. Mice injected with AAV-CBA-GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV-CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV-CBA-Rz, AAV-CBA-GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV-CBA-Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV-CBA-GFP group. In this study, we demonstrate that AAV-CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in spiral ganglion cells, which should also be susceptible to clarin-1 mRNA cleavage. This result may be due to the injection technique, the promoter used, or tropism of the AAV serotype 2 viral vector. These results suggest the role of apoptosis in the progression of USH3A hearing loss warrants further evaluation.

Limited cone-beam computed tomography imaging of the middle ear: a comparison with multislice helical computed tomography.

PURPOSE: To determine the applicability of cone-beam computed tomography (CBCT) in otological imaging, and to compare its accuracy with the routinely used multislice helical CT (MSCT) for imaging of the middle- and inner-ear areas. MATERIAL AND METHODS: Thirteen unoperated human cadaver temporal bones were imaged with CBCT and MSCT. Sixteen landmarks of the middle and adjacent inner ear were evaluated and compared for their conspicuity according to a modified Likert scale. Total scores and scores for subgroups including landmarks of specific clinical interest were also compared. RESULTS: No significant differences were found between the imaging techniques or subgroups when scores of individual structures were compared. While the middle ear itself was visible in all cases with CBCT, parts of the inner ear were "cut off" in four cases due to the limited field of view. For the same reason, the evaluation of the whole mastoid was not possible with CBCT. The cochlear and vestibular aqueducts were not visualized in either CT techniques. The contrast-to-noise ratio was more than 50% lower in CBCT than in MSCT, but still adequate for diagnostic task. CONCLUSION: CBCT proved to be at least as accurate as routinely used MSCT in revealing the clinically and surgically important middle-ear structures. The results show that high-quality imaging of the middle ear is possible with the current CBCT device.

Efficacy of gene transfer through the round window membrane: an in vitro model.

The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM. The round window including the bony niche of a CD1 mouse was removed under an operating microscope and fixed with adhesive on the base of a petri dish through which a hole had been drilled. Toluidine blue was injected into the niche containing a hyaluronic acid ester sponge against the round window membrane. The niche was closed with a fascia. A plastic tube containing PBS was fixed on the opposite side, from where the samples were collected at different time points. The concentration of toluidine blue was evaluated spectrophotometrically. An adenoviral vector containing green fluorescent protein (GFP) marker gene was injected into the niche. Samples were collected from the opposite side at different time points. The presence of the vector was studied with GFP PCR. We also modulated the permeability of the RWM by treating it with clinically applicable detergents, histamine or silver nitrate. Silver nitrate and trichloracetic acid caused destruction of the surface epithelium of the RWM as shown by light microscopy. Both toluidine blue and adenoviral vectors passed through the RWM in a time-dependent fashion. RWM cells expressed GFP after Ad-GFP treatment. The permeability of the RWM was decreased after treatment with different detergents, histamine or silver nitrate. RWM offers an atraumatic route to the inner ear. Compared with more invasive gene delivery methods, this technique represents a safer and clinically more viable route of cochlear gene delivery.

Apoptosis in auditory brainstem neurons after a severe noise trauma of the organ of Corti: intracochlear GDNF treatment reduces the number of apoptotic cells.

We have studied the morphological and cellular changes in the cochlear nucleus (CN) after cochlear nerve degeneration and whether these changes can be prevented by rescuing the primary cochlear neurons from degeneration with local glial cell line derived neurotrophic factor (GDNF) treatment. Degeneration of spiral ganglion neurons was seen to lead to a reduction of the volume of the anteroventral cochlear nucleus (AVCN); the size of the cell nuclei in the AVCN also was reduced. No differences were observed in cell density. After intrascalar GDNF treatment the volume of the AVCN was significantly larger when compared to the untreated side, and the size of the cell nuclei in the AVCN was significantly larger on the treated side. After degeneration of spiral ganglion neurons, an increased number of apoptotic cell nuclei were seen in ipsilateral CN and superior olivary complex. This increase was significantly smaller after intrascalar GDNF treatment. Degeneration of primary cochlear neurons seems to lead to an increase in the number of CN neurons undergoing apoptotic cell death. This can be prevented partially by rescuing primary cochlear neurons from degeneration with local GDNF treatment.

Puromycin-induced lipid peroxidation in the cochlea of ApoE knockout mice.

Puromycin-treated apolipoprotein E (ApoE)-deficient mice were used to study lipid peroxidation (LPO) in the cochlea. Puromycin causes accelerated peroxidation of lipids and induces both inner ear and renal lesions in experimental animals presenting with abnormally high serum cholesterol. To prevent LPO, we used probucol, an effective inhibitor of LPO, and, simultaneously, also a lipid-lowering drug. The mice were given a single injection of the aminonucleoside of puromycin (25 mg/100 g). Polyclonal malondialdehyde and 4-hydroxynonenal antibodies were used to localize the LPO products. LPO products were mainly found in the stria vascularis of puromycin-treated mice. No LPO products were observed in the hair cells. LPO product immunoreactivity was clearly diminished in the animal group treated with both puromycin and probucol. In the cochlea of the ApoE-deficient mouse, puromycin affects mainly the stria vascularis due to the accelerated peroxidation of structural lipids. Probucol treatment prevented the formation of LPO products.

Efficiency of hyperbaric oxygen therapy in experimental acute acoustic trauma from firearms.

In acute acoustic trauma (AAT), excessive noise exposure causes rupture of cell membranes and decreased cochlear blood flow. This leads to decreased oxygen tension in inner ear fluids and reduction of a variety of different oxygen-dependent cellular activities. Hyperbaric oxygen treatment (HBO) may help the cells suffering from hypoxia to survive. We exposed male Wistar rats to 60 impulses of 162-dB SPL from a 7.62-mm assault rifle equipped with a blank adaptor. After the exposure, 15 animals were given HBO treatment for 90 min daily for 10 consecutive days at 0.25 MPa. After a survival time of 4 weeks, auditory brainstem responses were measured and the left cochleae processed for light microscopy. The impulse noise caused permanent damage to the cochlea of all animals, with the most severe lesions in the lower middle coil, where a significantly smaller number of hair cells was missing in the HBO-treated group. The morphological damage was also reflected in function, as measured by auditory brainstem responses, which showed the greatest threshold shifts at 6.0, 8.0 and 10.0 kHz.

Lack of calbindin-D28k does not affect hearing level or survival of hair cells in acoustic trauma.

Calbindin is a cytosolic calcium-binding protein abundant in the hair cells of the inner ear and in distinct neurons of the auditory pathway. It is suggested to speed the return of potentially toxic calcium levels to normal. In this study, we show the basic hearing functions and the result of noise trauma from the calbindin null mutant mice generated by gene targeting. Auditory brainstem evoked response and distortion product otoacoustic emissions appear similar as in the control group. A moderate noise-induced trauma produced a similar loss of hair cells in calbindin null mutant mice than in wild-type controls. The result suggests that although calbindin is abundant in hair cells, it is not essential for the main hearing function and it does not provide physiological protection against a moderate noise-induced inner ear trauma in mice.

Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli.

Neuropeptides FF (NPFF), AF (NPAF), and SF (NPSF) are homologous amidated peptides that were originally identified on the basis of similarity to the molluscan neuropeptide FMRF-amide. They have been hypothesized to have wide-ranging functions in the mammalian central nervous system, including pain modulation, opiate function, cardiovascular regulation, and neuroendocrine function. We have cloned the NPFF gene from human, bovine, rat, and mouse, and show that the precursor mRNA encodes for all three of the biochemically identified peptides (NPFF, NPAF, and NPSF). We demonstrate that NPFF precursor mRNA expression by Northern analysis and map sites of expression by in situ hybridization. We confirm the validity of the in situ hybridization by showing that its distribution in the brain and spinal cord matches the distribution of NPFF and NPSF immunoreactivity. We go on to show that the mRNA levels (as measured by in situ hybridization) in the spinal cord can be up-regulated by a model for inflammatory pain (carrageenan injection), but not by a model for neuropathic pain (lumbar nerve ligation). Our results confirm the evolutionary conservation of NPFF, NPAF, and NPSF neuropeptide expression in mammalian brain. They also provide a context for the interpretation of the pain-sensitizing effects of injections of these peptides that have been previously reported. Our results support a model for the role of these peptides in pain regulation at the level of the spinal cord.

Neuropeptide FF in the lateral spinal and lateral cervical nuclei: evidence of contacts on spinothalamic neurons.

Neuropeptide FF (NPFF, F8Famide) is best known for its modulating effect on opioid analgesia and morphine tolerance. However, the exact mode of action of NPFF in sensory transmission is not known. We compared the distribution of NPFF-immunoreactive (ir) fibers and terminal-like thickenings with the retrograde, tracer-filled spinothalamic (ST) neurons in the lateral spinal nucleus (LSN) and lateral cervical nucleus (LCN) of rat, areas where NPFF-containing nerve terminals are abundant. We injected fluorescent latex microspheres into the ventroposterolateral thalamic nucleus and more medial thalamic nuclei, which are innervated by ST neurons. We found NPFF-ir terminal-like thickenings and fibers apposing the tracer-filled neurons in the LSN and LCN. ST neurons filled with the retrograde tracer making contacts with NPFF-ir terminal-like thickenings, were found to terminate not only in the ventroposterolateral thalamic nucleus but also in more medial thalamic nuclei. The highest number of tracer-filled ST neurons having NPFF-ir terminal-like thickenings and fibers in apposition were found at the cervical level. Our results suggest that NPFF-containing systems in the spinal cord of rat are not limited to the substantia gelatinosa, and the sensory functions of NPFF may be mediated at least partly through the modulation of the ST system. NPFF-ir contacts in the LSN and LCN might play an important role in the somatic sensory transmission system. This study shows evidence for the first time that the spinal NPFF-containing system may be involved in mechanisms that control sensory input to the supraspinal levels.

Neuropeptide FF in the rat spinal cord during carrageenan inflammation.

The role of neuropeptide FF (NPFF) in the modulation of spinal nociception was studied in rats with carrageenan inflammation in the hind paw. Normally no NPFF-ir neuronal cell bodies are found in the spinal cord. During inflammation NPFF-neurons were seen in an area receiving innervation from the inflamed hind limb, but in rats pretreated with morphine no NPFF-ir neurons were found. NPFF or IgG from NPFF immunoserum administered intrathecally had no effect in thermal and mechanical nociceptive tests. Morphine produced significant antinociception in both tests in the inflamed paw, but the effect was not modified by NPFF. These findings differ from the effects of intrathecal administration of NPFF and opioids in acute thermal tests when no inflammation is present. The role of NPFF in the modulation of nociception in the spinal cord may be markedly changed during acute inflammation.

Peptide GEGLSS-like immunoreactivity in the rat central nervous system.

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.

Evidence for cholecystokininA receptors in bovine adrenal chromaffin cells.

We have studied a possible role of cholecystokinin (CCK) in regulating adrenal medullary function. Caerulein (10(-10)-10(-7) M), a CCK receptor agonist, increased formation of inositol phosphates in primary cultured bovine adrenal medullary (BAM) chromaffin cells in a concentration-dependent manner. The effect of caerulein was antagonized by devazepide, a selective CCKA-receptor antagonist, but not by L-365.260, a selective CCKB-receptor antagonist. These results suggest that BAM cells possess functional CCK receptors of the CCKA-subtype. Stimulation of these receptors with caerulein activates a signal transduction pathway via phospholipase C. CCK may regulate catecholamine release in BAM cells.

Neuropeptide FF, a mammalian neuropeptide with multiple functions.

Neuropeptide FF (NPFF) and neuropeptide AF (NPAF) are two mammalian amidated neuropeptides which are highly concentrated in the posterior pituitary, spinal cord, hypothalamus and medulla. One precursor protein has been identified in mouse, rat, bovine and human brain. The precursor contains a single copy of both peptides, followed by a glycine residues necessary for amidation and flanked by basic residues necessary for processing by enzymes. In the brain, NPFF-like immunoreactive neurons are found in the hypothalamus and medulla. These systems may be associated with observed effects of NPFF on memory and autonomic regulation, respectively. A hypothalamo-pituitary pathway may be involved in neuroendocrine regulation. This is supported by lack of NPFF in the pituitary gland of vasopressin-deficient Brattleboro rats. It is also possible that NPFF acts as a hormone, as it has been detected in human plasma. The spinal cord contains an intrinsic NPFF-ir neuron system, with cell bodies in the dorsal horn and around the central canal. Nerve terminals are highly concentrated in the superficial laminae of the dorsal horn, where NPFF-immunoreactivity can be released by, e.g., potassium and substance P. One specific high-affinity binding site, distinct from binding sites for other peptides, has been characterized in the rat and human brain and spinal cord. The NPFF receptor appears to be coupled to a G-protein, but details of the second messenger systems have not been clarified yet. Intracerebroventricular injection of NPFF induces a vigorous abstinence syndrome in morphine-tolerant rats. Although clear antiopioid-like effects of NPFF on pain have been observed, some studies have also demonstrated long-lasting analgesic effects. These findings and the observed increase in NPFF-immunoreactivity in the cerebrospinal fluid during development of opiate tolerance render NPFF an interesting and challenging target of investigation.

Neuropeptide FF-containing efferent projections from the medial hypothalamus of rat: a Phaseolus vulgaris leucoagglutinin study.

Neuropeptide FF (FMRFamide-like peptide, morphine-modulating peptide) is an octapeptide isolated from the bovine brain. There is evidence that neuropeptide FF participates in the modulation of nociceptive mechanisms. Neuropeptide FF acts through its own receptors which are distinct from the opiate receptors. In the rat brain neuropeptide FF is found in two major cell populations. We have studied the efferent connections of the hypothalamic neuropeptide FF-containing cell group, which is located in the medial hypothalamus between the dorsomedial, ventromedial and periventricular hypothalamic nuclei. By using an anterograde tracing method (Phaseolus vulgaris leucoagglutinin) combined with double-staining immunohistochemistry we characterized the connections of this cell group with the limbic system, certain hypothalamic nuclei, periaqueductal gray and with the solitary tract nucleus. In the limbic system, the major targets were the lateral septal nucleus, bed nucleus of stria terminalis and certain subnuclei in the amygdala. These connections suggest that neuropeptide FF may act, in addition to its well-characterized action in the sensory system, in limbic functions. Efferent connections to the periaqueductal gray suggest that neuropeptide FF may modulate the opiate mediated analgesia at this site. Good correlation between our results and receptor autoradiography support the idea that the terminal areas which our results show are target areas of the neuropeptide FF-containing system.