Neurogenic inflammation in the upper digestive tract of the mule duck: effect of a chemical algogen and force-feeding.

1.The objectives were to quantify the presence of neurogenic inflammation in 4 regions of the upper digestive tract of anaesthetised ducks (post-pharynx, pseudo-crop, transition between the pseudo-crop and the proventriculus, and proventriculus) after application of HCl stimulation of up to 4 M in the pseudo-crop. 2.The second objective was to quantify the presence of neurogenic inflammation in the same digestive tract regions as mentioned above during 4 feeding periods of foie gras production (rearing, preparation to force-feeding, and second and last meals of the force-feeding period). 3. Extravasation increased above a HCl stimulation threshold of 2 M. Furthermore, more extravasation was observed in the proventriculus compared to the other regions (P < 0·001). 4.Highest extravasation responses were observed in the proventriculus and the pseudo-crop at the end of the preparation period, and in the proventriculus after the second forced meal, compared with the rearing period (P < 0·01), with a return to rearing level at the end of force-feeding. 5.Such a kinetic could be indicative of a relative mildness of the irritant components associated with this feeding practice.

The effects of viscero-somatic interactions on thalamic mast cell recruitment in cystitic rats.

Mast cells accessing the brain parenchyma through the blood-brain barrier in healthy animals are limited to pre-cortical sensory relays - the olfactory bulb and the thalamus. We have demonstrated that unilateral repetitive stimulation of the abdominal wall generates asymmetry in midline thalamic mast cell (TMC) distribution in cyclophosphamide-injected rats, consisting of contralateral side-prevalence with respect to the abdominal wall stimulation. TMC asymmetry 1) was generated in strict relation with cystitis, and was absent in disease-free and mesna-treated animals, 2) was restricted to the anterior portion of the paraventricular pars anterior and reuniens nuclei subregion, i.e., the rostralmost part of the paraventricular thalamic nucleus, the only thalamic area associated with viscero-vagal and somatic inputs, via the nucleus of the solitary tract, and via the medial contingent of the spinothalamic tract, respectively, and 3) originated from somatic tissues, i.e., the abdominal wall where bladder inflammation generates secondary somatic hyperesthesia leading to referred pain in humans. Present data suggest that TMCs may be involved in thalamic sensory processes, including some aspects of visceral pain and abnormal visceral/somatic interactions.

Evidence for serotonin influencing the thalamic infiltration of mast cells in rat.

Serotonin (5-HT) is involved in neuroimmunomodulation. We analyzed the effects of sumatriptan, a 5-HT(1B/1D) receptor agonist, and ondansetron, a 5-HT(3) receptor antagonist, on thalamic mast cell (TMC) population, the only immunocytes known to infiltrate the brain in physiological conditions. Only sumatriptan was effective, significantly increasing TMC numbers versus controls, and especially those containing 5-HT. 5-HT(1B) receptors are concentrated in the median eminence on non-serotonergic axonal endings, probably hypothalamic terminal fibers, involved in hypothalamic-pituitary neuroendocrine modulating processes. TMC variations could reflect serotonergic actions on these fibers. TMCs would thus be cellular interfaces mediating immune action in the nervous system in relation with the hormonal status of the organism.

Neurotoxic effects of neonatal injections of monosodium L-glutamate (L-MSG) on the retinal ganglion cell layer of the golden hamster: anatomical and functional consequences on the circadian system.

In rodents, daily injection of neurotoxic monosodium L-glutamate (MSG) during the postnatal period induces retinal lesions, optic nerve degeneration with an alteration of visual pathway and an absence of the b-wave in the electroretinogram. Despite this damage, electrophysiological responses subsist in the lateral geniculate bodies and synchronization of circadian rhythms to the light/dark cycle can still occur. Using two formal properties of the circadian system (entrainment and phase-shift by light), we assessed the functionality of retinal projections to the circadian clock in MSG-treated hamsters. Displaced amacrine and ganglion cell populations were quantified and retinal terminals in the suprachiasmatic nuclei were estimated. Animals received daily doses of glutamate during the first ten days after birth according to two protocols. The two treatments similarly destroyed 56% of the overall population of the ganglion cell layer: 30% of displaced amacrine and 89% of ganglion cells. Surviving ganglion neurons (7,500 cells) were evenly distributed across the entire retina except in one area of high cell density located in the temporoventral quadrant. Retinal projections of the "image-forming" pathway were drastically reduced in the dorsal lateral geniculate bodies, less in their ventral part. The "nonimage-forming" pathway was also affected since the volume of labeled terminals in the suprachiasmatic nuclei was reduced by one-half to one-third. Nevertheless, treated hamsters exhibited a free-running locomotor activity rhythm after several months in constant darkness, could be entrained by the light/dark cycle and phase-shifted by light pulses. These results suggest that a damaged retinohypothalamic tract can still assume the photic entrainment of the circadian clock.

Isofrequency labelling revealed by a combined [14C]-2-deoxyglucose, electrophysiological, and horseradish peroxidase study of the inferior colliculus of the cat.

Tonotopic sequences of single units were recorded in the central nucleus (ICC) of the inferior colliculus of cats. The sites of units with a particular characteristic frequency (CF) were marked histologically. After injection with [14C]-2-deoxyglucose (2-DG), each animal was stimulated with tone bursts, which corresponded to the CFs of the selected units. The position of a selected unit was found to correspond with the position of a band of discrete 2-DG labelling. In some animals, multiple penetrations revealed that more than one unit with the same CF was located on the same band, indicating that the 2-DG bands represent isofrequency contours. Single units were recorded in the pars lateralis (LV) of the medial geniculate body of cats and areas of consistent CF were found, which contained the same frequencies as some of those employed with 2-DG. Horseradish peroxidase (HRP) was ejected into these selected areas and labelled cells were identified in ICC after retrograde transport. The labelled cells were found to lie in a band which had a similar orientation to the 2-DG bands produced by the same frequencies. Both sets of data indicate that ICC is organized into three-dimensional isofrequency sheets, the orientation of which do not match the concentric model of frequency organization inferred from some Golgi studies. The isofrequency sheets slope from medial to lateral throughout ICC with high-frequency contours found more medially and caudally and low-frequency contours more laterally and rostrally. The organization of the isofrequency contours is largely in agreement with a recent Golgi model of the laminations in ICC put forward by Oliver and Morest ('84). The 2-DG contours do not agree with the orientation of laminae in the lateral part of ICC as described by this model. The 2-DG contours also extend further into the dorsal cortex than previous degeneration evidence would suggest. The functional isofrequency contours are often quite narrow and could represent two or three overlapping cellular laminae. The results show clearly that there is only one tonotopic organization extending throughout ICC and the dorsal cortex.

Circadian fluctuations in GFAP distribution in the Syrian hamster suprachiasmatic nucleus.

We examined the possibility that astrocytes reflect mammalian circadian rhythmicity by observing the astrocytes pattern using anti-glial fibrillary acidic protein (GFAP), within the suprachiasmatic nucleus of the Syrian hamster over the 24 h cycle. Our results reveal daily systematic changes in the distribution of GFAP-immunoreactivity (GFAP-ir) both in the light/dark cycle and in constant darkness. This rhythm is characterized by a change of the GFAP-ir distribution from a network pattern to isolated cells. Such a rhythm is in synchrony with the rhythm in SCN glucose consumption which we have previously reported. This is the first time a circadian rhythm in suprachiasmatic astrocytes has been observed, indicating that GFAP-ir can be used as a novel index of clock activity.

Photic induction and circadian expression of Fos-like protein. Immunohistochemical study in the retina and suprachiasmatic nuclei of hamster.

Fos-immunohistochemistry was performed in the retina and at four rostro-caudal levels of the suprachiasmatic nuclei (SCN) in hamsters. Animals were sacrificed at four circadian times (CT) relative to activity onset (CT12): CT07, 11, 14, 19 either in permanent darkness (DD) or 1 h after light stimulation. Quantification of immunoreactive nuclei showed (i) endogenous CT related changes exclusively within the rostral SCN with maximum immunoreactivity at CT07, (ii) CT related responses to light in retinal displaced amacrines, ganglion cells and caudal SCN (maximum at CT19), (iii) expression differences in four subsets of SCN cells according to CT. The rostral subset could be implicated in the endogenous clock mechanism since it exhibited a fluctuation of Fos immunoreactivity in DD and expression of Fos protein at CTs 06 and 18 when light provokes transients in the free-running period. In the caudal SCN, a ventro-laterally localized set responded to light at CTs 13 and 18, a dorsal crescent of cells responded only at CT18 and a group located laterally between these two responded at CT18. These cellular subsets may have different functions in the light-entrainment mechanism since light stimuli at CT13 induced phase-delays and, at CT18, phase-advances in the onset of the free-running locomotor activity rhythm.

Host resets phase of grafted suprachiasmatic nucleus: a 2-DG study of time course of entrainment.

The object of the present experiment was to examine whether in an intact animal implanted with a hypothalamic graft, the phase of the host and grafted suprachiasmatic nucleus (SCN) would become synchronized. To this end, we first established the time at which daily fluctuations in local cerebral glucose utilization were maximal in the SCN in our population of adult hamsters. Next, we verified that rhythms of (14C)2-deoxyglucose uptake could be measured on the day after birth in pups that were to provide donor tissue. Host and donor animals were housed in opposite light:dark cycles. We then transplanted fetal SCN tissue into the third ventricle of intact hamsters, placed the grafted animals in constant darkness with access to running wheels and examined the phase of metabolic activity in host and donor SCN. For several days after grafting, there was no circadian fluctuation in the metabolic activity of either the host SCN or of the grafted SCN. During this time, the circadian locomotor rhythms were not disrupted, suggesting that pacemaker activity was not interrupted. By day 14 after transplantation, metabolic activity in the host SCN was elevated during subjective day and host and donor SCN were in synchrony, invariably with the phase of the host animal. We conclude that a signal from the host SCN resets the grafted SCN and not vice versa and that pacemaker cells communicate with each other rather than exerting independent effects on target sites.

Decrease of [14C]2-deoxyglucose uptake at the intracerebellar nuclei during cerebellar cortex stimulation.

The effect of electrical stimulation of the cerebellar cortex at 10-30 c/s on metabolism in the intracerebellar nuclei has been studied using the [14C]2-deoxyglucose method. The experiments, performed on anesthetized or immobilized animals did not produce any detectable changes in the radioautographic labeling of the intracerebellar nuclei compared with controls. Experiments were also performed in animals pretreated with 3-acetylpyridine neurotoxin which selectively destroys the inferior olive and produces an intense labeling of the intracerebellar nuclei. Less marking was observed in restricted regions of the intracerebellar nuclei receiving the axon terminals of the stimulated Purkinje cells if the experiments were done within the first few hours. Following 3-acetylpyridine intoxication, in this early phase, destruction of the inferior olivary cell bodies occurs, but the climbing fibers remain intact. At 2 days or more following 3-acetylpyridine, changes in marking with stimulation could no longer be obtained. The finding is interpreted as being due to an actual reduction of the Purkinje cell activity upon stimulation of the cerebellar cortex, leading to a reduction of the metabolic activity at their presynaptic terminals.

Developmental study in the circadian clock of the golden hamster: a putative role of astrocytes.

The hypothalamic suprachiasmatic nuclei (SCN) house the circadian clock in the mammalian brain. A glial fibrillary acidic protein immunoreactivity (GFAP-ir) distribution rhythm has been observed in the SCN of adult Syrian hamster. The implication of astrocytes in photic entrainment was analyzed through developmental studies of the photic pathway and of SCN astrocytes appearance. Using anterograde tracing we studied the timing of penetration of the retinohypothalamic tract (RHT) fibers into the SCN. Using c-fos induction by light we followed the maturation of RHT synapses in the SCN. When RHT terminals reach the SCN, c-fos induction can be obtained. Using GFAP immunoreactivity we demonstrated that the number of astrocytes increased in parallel with RHT development from PN5 to PN15. At PN15, a time when pups can shift from maternal to photic entrainment, RHT terminals and GFAP-ir exhibit an adult-like pattern. One demonstrated role of astrocytes is to control extracellular glutamate concentration. Glutamate is the neurotransmitter released at RHT terminals; its content fluctuates according to a circadian rhythm within the SCN. Thus the present data tend to indicate that SCN astrocytes are participating in the circadian rhythm of SCN glutamate content.

A combined electrophysiological and [14C] 2-deoxyglucose study of the frequency organization of the inferior colliculus of the cat.

Single units were recorded in the inferior colliculus of 3 young cats. A tonotopic sequence of characteristic frequencies (CF) was obtained in response to contralateral stimulation. In each cat Pontamine sky blue was deposited from the recording pipette at the site of a unit with a particular CF. [14C] 2-Deoxyglucose was then injected into the cats, each of whom was presented with tone bursts at a frequency identical with the CF recorded at this site. Selective labelling was found in the contralateral inferior colliculus of each cat and the position of the band of marking coincided with the recording position of the chosen unit. These data demonstrated for the first time that cells in the region of [14C] 2-deoxyglucose marking have the differential physiological properties capable of producing the selective functional labelling. The bands of selective labelling found in response to different frequencies are produced by cells tuned to the particular stimulating frequency.

Demonstration of a direct projection from the retina to the hypothalamic supraoptic nucleus of the hamster.

Hamsters were injected in the left eye with unconjugated cholera toxin subunit B (CHB) and the tracer was localized using immunohistochemistry. A large number of immunoreactive fibers was found in the suprachiasmatic nuclei and the contralateral lateral hypothalamic area. Labeled fibers coursed around the supraoptic nucleus and nerve terminals accumulated in a zone dorsally and laterally to the nucleus. Single fibers from this plexus penetrated into the supraoptic nucleus, where few fibers arborized into delicate immunoreactive profiles possessing varicosities. Labeled fibers were identified only in the dorsal and lateral parts of the nucleus, and mostly at the caudal level of the optic chiasm. These results show a direct retinal innervation of the magnocellular neurons of the supraoptic nucleus, and indicate a direct photic influence on the hypothalamo-neurohypophysial system.

Host resets phase of grafted SCN: influence of implant site, tissue specificity and pineal secretion.

The suprachiasmatic nuclei (SCN) have daily fluctuations in energy consumption with glucose utilization high in the day, and low at night. In a previous study, we used [14C]2-deoxyglucose (2-DG) uptake to index the phase of the SCN, and found that in intact hamsters bearing SCN grafts in the third ventricle (3V), the native and grafted SCN, which were initially 12 h out of phase, became synchronized to the phase of the host clock [32]. In the present study, adult males (host animals) and pregnant females (source of donor tissue) were housed in opposite light-dark cycles. Host animals were sacrificed 14 days after implantation with either (1) SCN grafts into the lateral ventricle (LV), or (2) cortical grafts into 3V, or (3) SCN grafts in 3V of pinealectomized hamster. The results indicate that rhythms of 2-DG uptake are not induced in cortical tissue grafts, that the host SCN does not entrain SCN grafts located in the LV, and that entrainment of the grafted SCN (located in 3V) by the host circadian system occurs in the absence of pineal secretions.

Daily changes of cytochrome oxidase activity within the suprachiasmatic nucleus of the Syrian hamster.

Cytochrome oxidase (CO) activity was studied over a 24-h period in the Syrian hamster suprachiasmatic nucleus (SCN) (site of the biological clock), anterior hypothalamic area (AHA), and motor cortex. The SCN CO activity was highest at the middle of the day (Zeitgeber time (ZT) 05), decreased at the end of the light period (ZT 10) and continued at a low level during the night (ZT 13, 16, 21.5 and 24). AHA and motor cortex showed a similar profile of CO activity and no changes of CO activity were found in animals maintained under darkness (DD). We propose that photic input plays a role in the SCN neuronal activity that modulates metabolic activity on this area.

Autroradiographic demonstration with 2-[(14)C]deoxyglucose of frequency selectivity in the auditory system of cats under conditions of functional activity.

2-[(14)C]deoxyglucose was injected into 3 cats, each of whom was presented with a tone of a different frequency under controlled auditory conditions. Clear selective labelling was observed in autoradiographs of the cochlear nucleus, parts of the superior olivary complex, the nuclei of the lateral lemniscus and the inferior colliculus. No selective labelling was observed in autoradiographs of the medial geniculate body and the auditory cortex. The labelling observed in the cochlear nucleus and the inferior colliculus was in agreement with previous studies of both the physiology and anatomy of these regions and for the first time provides an anatomical demonstration of the organization of frequency selectivity under conditions of functional activity.

Both [1-14C]glucose and 2-[1-14C]deoxyglucose produce selective iso-frequency labelling in the inferior colliculus of the cat with short stimulation periods.

Both [1-14C]glucose and 2-[1-14C]deoxyglucose (2-DG) revealed selective autoradiographic labelling to tones in the inferior colliculus of the cat with short stimulation periods (5-15 min). With longer periods of stimulation (45 min), the selectivity disappeared with glucose but remained with 2-DG. At all stimulation intervals, 2-DG labelling was always more selective than that obtained with glucose. However, the selectivity seen with glucose was good enough to indicate that isotopes of glucose with short half-lives could still be employed to study human functional activity with the positron emission tomography technique, provided that short stimulation periods were used.

Tonotopic bands produced by tones commenced long after 2-deoxyglucose injection.

Presentation of two different tones at two different time periods each under two different types (14C or 3H) of 2-deoxyglucose (2-DG) produced two bands of labelling in the inferior colliculus (IC) of a guinea pig. Filtering tests revealed that both bands were produced by only one of the labels (14C). Four guinea pigs were given one injection of 2-DG and then stimulated with two tones, each presented at different time periods. Two bands of labelling were produced in IC, with one band being produced by tones given 55-90 min after injection. The implications of this long lasting effect of 2-DG are discussed for the general 2-DG model and for sequential double labelling experiments.

Increase of rat medial habenular mast cell numbers by systemic administration of cyclophosphamide.

Cyclophosphamide administration generates systemic toxicity having immune and nervous consequences. After focusing on nervous consequences by studying neuronal activity, we now consider cyclophosphamide impact on diencephalic mast cells as part of the brain immune system. Diencephalon, the ultimate sensory relay before neocortical processing, is the only brain structure containing mast cells. Single cyclophosphamide administration (100 mg/(kg 1 ml ip)) was performed in naturally behaving rats and diencephalic mast cell numbers were analyzed once all drug effects had developed (4 h postinjection). Significant increases were observed only in the medial habenular nucleus--bilaterally and especially in its caudal portion. Mast cell increase is temporally related to behavioral impairment and evoked neuronal activity in a restricted number of visceral/limbic extrathalamic structures. The medial habenular nucleus belongs to the limbic system involved in processing emotional reactions and regulation of the autonomic nervous system. Its involvement during toxic challenge is highly compatible with its presumed function in the maintenance of vital functions.

Deoxyglucose demonstration of in-utero hearing in the guinea pig foetus.

2-Deoxyglucose (2-[14C]DG) autoradiography was used to demonstrate central auditory function in the foetal guinea pig in-utero. The major advantage of this approach is that the experiment is carried out with the foetuses within the intact amniotic sac. Using pure tone stimuli at around 100 dB SPL, isofrequency bands of elevated metabolic activity were observed in the inferior colliculus of the majority of foetuses within the last 10 days of gestation.

The frequency organization of the inferior colliculus of the guinea pig: A [14C]-2-deoxyglucose study.

The [14C]-2-deoxyglucose (2-DG) technique was used to study the frequency organization of the inferior colliculus (IC) of the guinea pig. Discrete regions of heightened 2-DG labelling were observed in the ICs of animals exposed to a variety of pure-tone stimuli. Regions associated with 1, 4, 10 and 19 kHz pure tones were described and displayed in three-dimensional representations. The IC of the guinea pig was found to be arranged as a series of sheet-like, iso-frequency planes that extend throughout the nucleus from its caudal to its rostral pole. Iso-frequency planes associated with low frequencies are located dorsolaterally in the nucleus and those associated with higher frequencies are located progressively more ventromedially. The predominant orientation, in the frontal plane, of all iso-frequency planes is oblique from dorsomedial to ventrolateral. Most planes, however, twist about their caudal-to-rostral axis in a caudal-to-rostral, horizontal-to-vertical direction. The extent to which each plane twists is frequency-dependent; planes associated with low frequencies twist most and those associated with high frequencies do not twist at all.