AMPA glutamatergic receptor-immunoreactive subunits are expressed in lumbosacral neurons of the spinal cord and neurons of the dorsal root and pelvic ganglia controlling pelvic functions in the rat.

Sacral preganglionic neurons innervate the pelvic organs via a relay in the major pelvic ganglion. Pudendal motoneurons innervate striated muscles and sphincters of the lower urinary, genital and digestive tracts. The activity of these spinal neurons is regulated by sensory afferents of visceral and somatic origins. Glutamate is released by sensory afferents in the spinal cord, and interacts with a variety of receptor subtypes. The aim of the present study was to investigated the presence of AMPA glutamate receptor subunits (GluR1-GluR4) in the neural network controlling the lower urogenital and digestive tracts of male rats. We performed double-immunohistochemistry directed against a neuronal tracer, the cholera toxin beta subunit (Ctbeta) and each of the four receptor subunits. GluR1, GluR2 and GluR3 subunits were present in many sacral preganglionic neurons retrogradely labelled with Ctbeta applied to the pelvic nerve, and in some dorsolateral and dorsomedian motoneurons retrogradely labelled with Ctbeta injected in ischiocavernosus and bulbospongiosus muscles. The four subunits were detected in postganglionic neurons of the major pelvic ganglion retrogradely labelled with Ctbeta injected in the corpus cavernosum, and in some somata of sensory afferents of the L6 dorsal root ganglion labelled with Ctbeta applied to the dorsal penile nerve or injected in corpus cavernosum. The results provide a detailed knowledge of the neural targets expressing the various AMPA receptor subunits and suggest that part of the neural network that controls pelvic organs, including sensory afferents and postganglionic neurons, is sensitive to glutamate through the whole family of AMPA subunits.

Neurons that express the AMPA receptor GluR2/3 subunits in suprachiasmatic nuclei of Syrian hamsters colocalize either vasoactive intestinal peptide, peptide histidine isoleucine or gastrin-releasing peptide.

In mammals, many circadian rhythms are driven by a clock located inside the suprachiasmatic nucleus of the hypothalamus. They are synchronized to environmental light-dark cycles by information coming directly from the retina via glutamatergic afferents. In rodents, retinal fibres make direct synaptic contacts with neurons synthesizing vasoactive intestinal peptide and gastrin-releasing peptide. These two neuropeptides, administered alone or combined with the peptide histidine isoleucine, phase-shift the clock in the same way that light does. Using ICC and light and electron microscopy, our study demonstrates that subunits 2 and 3 of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamatergic receptors are colocalized in neurons expressing one or other of these three neuropeptides. Double-labelled neurons were located in the ventral and lateral ventral parts and near the symmetrical plane of the intermediate and caudal thirds of the nucleus. In light microscopy, brown and granular blue stainings of chromogens revealing both antigens were easily identifiable and spatially separated in perikarya. In electron microscopy, almost all the cells observed in these zones expressed the receptor subunits. A few labelled dendritic profiles, some of them post-synaptic, were observed; axon terminals were always unlabelled. Colocalization with vasoactive intestinal peptide and gastrin-releasing peptide was confirmed by the immunogold technique in perikarya and some dendrites. The present study suggests that peptidergic neurons expressing the AMPA receptors are involved in photic entrainment of the clock by the retina without excluding some glutamatergic information coming from other hypothalamic nuclei.

Circadian rhythm of AMPA receptor GluR2/3 subunit-immunoreactivity in the suprachiasmatic nuclei of Syrian hamster and effect of a light-dark cycle.

In mammals, the suprachiasmatic nuclei (SCN) of the hypothalamus are the site of the circadian clock that generates and coordinates many endogenous physiological and behavioral rhythms. SCN are normally entrained to light/dark (LD) cycle by direct retinal afferents using glutamate as neurotransmitter. N-Methyl-d-aspartate (NMDA) and non-NMDA receptors are involved in photic entrainment of SCN. In rodents, the presence of three of the four known 2-amino-3-(3-hydroxy-(-methylisoaxol-4-yl) propanoic acid (AMPA) receptor subunits has been demonstrated by in situ hybridization. This study analyzes the expression of GluR2/3 subunits in SCN of Syrian hamsters maintained under constant darkness (DD) or 12:12 LD cycle. In animals submitted to DD or LD, small immunoreactive neurons were located in the ventral and external latero-ventral parts of the rostral two-thirds of the SCN and along the symmetrical plane. The number of intensely labeled neurons with or without long process(es) were counted at six circadian times (CTs) in three groups of animals maintained in DD and six nycthemeral (zeitgeber time, ZT) times in one group of hamsters submitted to LD. In DD, we observed significantly more GluR2/3 subunit-immunoreactive (GluR2/3-ir) neurons during the subjective day than during the subjective night, with minima at CT 19-CT 23. The LD cycle significantly reduced the number of immunoreactive neurons, lessened the differences between LD phases and depressed immunoreactivity at light transition, i.e., at ZT 11 and ZT 23. This study demonstrates for the first time by immunohistochemistry the existence of a circadian dynamic of the expression of AMPA receptor subunits in SCN of rodents and the effect of the LD cycle on this dynamic.

Temporospatial characteristics of light-induced fos immunoreactivity in suprachiasmatic nuclei are not modified in Syrian hamsters treated neonatally with monosodium glutamate.

Neonatal treatment of rodents by intraperitoneal injections of monosodium glutamate (MSG) destroys many retinal ganglion cells whose neurons belong to the circadian system; howertheless, adults always synchronize their locomotor activity rhythm (LAR) to the light/dark cycle. Recent studies have shown that light-induced phase shifts of LAR are associated with the c-fos induction in suprachiasmatic nuclei (SCN) of nocturnal rodents. In this study, the circadian system was analyzed in treated and control hamsters maintained in constant darkness and exposed to light at circadian times (CTs) 13 and 18 during subjective night, 1 and 6 h after the onset of LAR. The period of the LAR and delay (CT13) and advance (CT18) phase shifts of LAR were not significantly different between MSG-treated and control hamsters. Temporospatial variations of Fos induction after light exposure were similar in both MSG-treated and control hamsters although the total number of Fos immunoreactive (Fos-ir) nuclei in the SCN was always lower in treated hamsters. However, the decrease in Fos-ir was significant only for the caudal third of the SCN of treated hamsters, the part where retinal afferents are most dense. The effect of light exposure on Fos expression in SCN of MSG-treated and control hamsters was the same at CT13 and CT18: (1) Fos-ir nuclei were significantly more numerous at CT18 than at CT13 in the rostral SCN; (2) dorsal Fos-ir cells were observed in the SCN only at CT18; (3) a ventral subgroup expressed Fos protein in intermediate SCN only at CT13. This study demonstrates that MSG-treatment does not significantly modify the phase-shifting effects of light on either the LAR or the associated cellular activation.

Retinal ganglion cells expressing the FOS protein after light stimulation in the Syrian hamster are relatively insensitive to neonatal treatment with monosodium glutamate.

In nocturnal rodents, the c-fos gene is directly involved in the light mechanism of resetting of the suprachiasmatic nucleus (circadian clock). Light also induces c-fos expression in the retinal ganglion cell layer (GCL), but no attempt has been made to study the retinal responses to the phase-shifting effects of light. The expression of the Fos protein in each of the two populations of the GCL (displaced amacrine cells [DACs] and ganglion cells [GCs]) was analyzed in hamsters after light stimulation delivered early (circadian time [CT13]) and in the middle (CT18) of the subjective night. To evaluate as accurately as possible the number of GCs able to phase shift the locomotor activity rhythm (LAR), neonatal hamsters treated with monosodium glutamate (MSG) were also used, an in vivo model which displays retinal degeneration and LAR normally entrained by light. In nontreated hamsters, the number of Fos-immunoreactive (Fos-ir+) nuclei in the GCL was significantly higher at CT18 than at CT13. In MSG-treated hamsters, the number of Fos-ir+ nuclei was the same at both CTs and nonsignificantly different as those of nontreated hamsters at CT13. MSG treatment destroyed as many Fos-ir+ DACs as Fos-ir- DACs or Fos-ir+ GCs. Fos-ir+ GCs were less sensitive to neurotoxic than other GCs, as only 37% of them were destroyed by treatment versus 92% for Fos-ir- GCs. At CT18, a maximum of 3,500 GCs expressed Fos protein in nontreated hamsters versus only 2,200 in MSG-treated hamsters. This minor subgroup was sufficiently potent to normally synchronize the circadian rhythms to the Light/dark cycle in treated hamsters.

Neurons containing gastrin-releasing peptide and vasoactive intestinal polypeptide are involved in the reception of the photic signal in the suprachiasmatic nucleus of the Syrian hamster: an immunocytochemical ultrastructural study.

In mammals, the suprachiasmatic nuclei are involved in the generation of biological rhythms and are synchronized by light input coming from the retina. The targets of retinal afferents and the involvement of neurons containing gastrin-releasing and vasoactive intestinal peptides in photic reception were investigated in the suprachiasmatic nuclei of the Syrian hamster by using light- and electron-microscopic immunocytochemistry. Cholera toxin was used to trace retinal fibers and Fos immunoreactivity to visualize cellular response to light stimulation. Ultrastructural observations were made in the intermediate third of the nuclei, the area of highest overlap for the immunoreactivities investigated. Gastrin-releasing peptide and vasoactive intestinal peptide cell bodies were localized in the ventral part of the nuclei; their dense immunoreactive fiber network often displayed synaptic contacts. Both neuropeptides were colocalized in elongated cells observed near the optic chiasm. Following a light pulse in the middle of the subjective night, Fos protein was expressed in most gastrin-releasing peptide perikarya and in some vasoactive intestinal peptide cells. Retinal terminals mostly occurred in the midline zone between the suprachiasmatic nuclei. Symmetrical or asymmetrical retinal synapses were observed on gastrin-releasing peptide-immunoreactive dendrites and somata, but never on vasoactive intestinal peptide neurons. These results are discussed in relation to the photic entrainment of the circadian clock.

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.

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.

Deutocerebrum of the cockroach Blaberus craniifer Burm. Quantitative study and automated identification of the glomeruli.

The glomerular organization of the hemideutocerebrum is analyzed quantitatively, using only spatial position, in four individuals (eight hDTCs) after a visual identification of glomeruli on graphic reconstructions. In order to assess directly the invariance of the neuropil in an insect brain the following is done: (1) The position of each glomerulus is compared to the position A' it should occupy if the hDTCs were identical. It is shown that in 80 and 71% of the cases, respectively, intra- and interindividual comparisons in the studied glomerulus is the nearest to A'. (2) The actual position is equally compared to the theoretical location A" each glomerulus should occupy if its absolute position could be changed but not its relative position with respect to its neighbors. The calculation of A" is based on process (3). In 86 and 80% of the cases, respectively, it is found that the actual position is that which is nearest to A". (3) An automated identification process, based on absolute and relative locations, but completely independent of visual identification, is described. It allows the identification of 77 and 74% of glomeruli, respectively. These matchings are in 96 and 90% of the cases identical to the visual matchings. The location predictability of most glomeruli is discussed to show the existence, nature, and limits of the hemideutocerebral invariance.

The deutocerebrum of the cockroach Blaberus craniifer Burm. Spatial organization of the sensory glomeruli.

The objective of this work is to describe the glomerular organization of the deutocerebrum in Blaberus craniifer and to test the hypothesis that the glomeruli are identifiable. The problem is studied using the techniques of analytical geometry, i.e., by measuring the location of the glomeruli in cartesian coordinate systems. Computerized geometrical and statistical techniques are described for the three-dimensional reconstruction and quantitative study of these brain structures. The invariance of the glomerular organization, and consequently the identifiability of the glomeruli, is based on three criteria: (1) the number of glomeruli per hemideutocerebrum (hDTC) is a constant (109); (2) the position of the glomeruli is symmetrical in the two sides of one individual and identical in the same side of two individuals, except for small local variations,(3) the dimensions of two positionally homologous glomeruli are statistically equal in the same and in different individuals, except for a macroglomerulus which is found only in the male. The causes of variability, from experimental and biological origin, are discussed and quantitatively evaluated. The interindividual biological variability, not ascribable to differences in size between brains, expressed as a 95% interval, is estimated to be 27 micrometer for the location of glomeruli and 12 micrometer for the diamter of glomeruli.