Neuroanatomy of cerebellum and olfactory bulb in a substrain of C57BL/6J inbred mice carrying a spontaneous mutation.

Mice of the inbred C57BL/6JNmg substrain carry a mutation decreasing the size of the zinc-rich hippocampal intra- and infrapyramidal mossy fibre (IIPMF) terminal fields. In the present experiment, it was investigated whether this neurological mutation has also effects on other characteristics of the brain. No morphological differences were found in two other laminated neural structures, the olfactory bulb, where the accessory granular layer is also rich in zinc terminals, and the cerebellum. However, the mutants had a somewhat inferior performance on a motor function task known to test cerebellar involvement. The present findings confirm that previously found effects of this mutation on different types of behaviour are most probably due to the IIPMF. These substrains provide a powerful tool to localise the gene involved and subsequently investigate the plausible pathways leading from gene to behaviour.

Genetic dissection of gustatory sensitivity to bitterness (sucrose octaacetate) in mice.

Genes implicated in consumption of a bitter compound, sucrose octaacetate (SOA), were investigated using a full genomic scanning strategy. For a 0.1 mM concentration, two QTL reached 5.8 and 6.5 lod scores on chromosomes 2 (77 cM) and 11 (14 cM), respectively. For a 1 mM concentration, the Soa linkage on chromosome 6 (58 cM, lod score 9.4) was replicated, and another QTL was found on chromosome 19 (15 cM, lod score 3.2). Candidacy of previously identified genes in the close vicinity of the peak of the QTL was examined.

Localization of actin in Moloney murine leukemia virus by immunoelectron microscopy.

Immunoelectron microscopy was used to detect actin in wild-type (wt) Moloney murine leukemia virus (MoMuLV) and in virus-like particles (VLP) produced by recombinant Semliki Forest virus expressing only the MoMuLV gag polyprotein. Gold immunolabeling revealed the presence of actin on the surface of delipidized VLP and delipidized wt virus particles. Statistical evaluation of the number of colloidal gold particles per VLP revealed a large range of values and a prevalence of VLP with small numbers of gold particles. Labeling for actin was lost after prolonged treatment of VLP with 1% Nonidet-P40, high-pH buffer, or gelsolin. Gold immunolabeling with antibodies to gag proteins p15 (MA) and p12 and p30 (CA) was abundant and was not affected by treatment of VLP or wt virus with 1% Nonidet or gelsolin. VLP treated with a mixture of detergent and aldehyde fixatives showed more uniform and consistent labeling for actin than without fixatives. Negative staining or heavy metal shadowing revealed a globular surface of delipidized VLP. Stereomicrographs of gold-immunolabeled VLP showed that p15gag and p12gag were associated with the globular projections. Delipidized VLP were also well labeled with antibody to p30gag, which indicated that the gag shell permitted access of antibodies to p30gag and was therefore not a closely packed structure. Labeling for actin-binding proteins moesin and ezrin was negative in both the wt virus and the VLP. The absence of Gaussian distribution of actin in the sample of VLP suggests that actin is not a structural protein and its presence in MuLV virus particles may be fortuitous. This, however, does not rule out any possible role of actin in transport, assembly, budding, or release of virus particles, events which take place in the cytoplasm or at the plasma membrane. The site of actin in VLP is discussed in relation to the present knowledge of the molecular organization of the MuLV gag shell.

Incorporation of ethidium bromide in the Drosophila salivary gland approached by microspectrofluorometry: evidence for the presence of both free and bound dye in the nuclei of cells in viable conditions.

The incorporation of 10(-6) M ethidium bromide (EB) was studied in viable Drosophila melanogaster salivary glands with a spatial resolution reaching a few microns3, using a confocal laser microspectrofluorometer designed for spectral analysis. Spectra were recorded with the 514 nm Argon laser line during excitation times of 1 second (20 microW on the preparation) at 5 min intervals for 30 or 60 min, either at points in determined cell sites or serially throughout the cells. The fluorescence intensity time-course indicated that the EB intake was not an all-or-none process, but rather a graded, sensitive indicator of the functional state of the cell. On the micrometer scale, the cytoplasm behaved as an homogeneous substrate with the fluorescence intensity depending on EB intake and intracellular diffusion. In the nucleus, however, localized enhancement of the emission intensity was observed. Spectral analysis allowed us to characterize the interactions. The mean values of lambda max in the cytoplasm (600 nm), in the nucleus (601 nm) and outside the glands (602 nm) were less than for free EB in aqueous solution (630 nm); values of full width at half maximum were between 92 and 96 nm, which is much lower than the 120 nm observed for free EB. The recorded spectra were analyzed using a linear combination of two spectral models, namely free and DNA intercalated EB. In the nucleus, the free EB model spectra was found to represent up to 10% of the recorded spectra whereas it was near zero in the cytoplasm. The present data suggest that the intranuclear concentration of free EB (allowing for its lower fluorescence quantum yield) might be at least equal to that of the bound EB.

Poly(pyrrolecarboxamides) linked to photoactivable chromophore isoalloxazine. Synthesis, selective binding, and DNA cleaving properties.

In an attempt to obtain DNA sequence-specific cleaving molecules, we have synthesized two types of hybrid groove binders composed of an isoalloxazine (flavin) chromophore linked through a polymethylenic chain to either a bis- or a tris(pyrrolecarboxamide) moiety related to netropsin and distamycin, respectively. In both types of molecules, the polymethylenic chain is linked to the alloxazine ring either in the N10 position or in the N3 position. As netropsin and distamycin, the hybrid derivatives preferentially bind to A + T-rich sequences and recognize sequences such as 5'-ATTT. Upon visible light irradiation the flavin moiety undergoes a redox cycling process generating superoxide anion and hydroxyl radical. Generation of oxy radicals appears to be more efficient with the hybrids in which the polymethylenic chain is linked at the N10 position. The generation of oxy radicals results in the occurrence of single strand break in supercoiled DNA. Breaks preferentially occur in the vicinity of A + T-rich sequences. The advantage of flavin relative to other oxy radicals generating compounds such as ferrous-EDTA is that it does not require chemical reduction but can be reduced either by visible light or by cellular enzymes, both conditions being compatible with pharmacological constraints.

Electron microscopy of the nucleocapsid from disrupted Moloney murine leukemia virus and of associated type VI collagen-like filaments.

To analyze the constituents of retroviruses, the Moloney murine leukemia virus was disrupted and observed by dark-field electron microscopy. Virus disruption was achieved by several methods: osmotic shock, freezing-thawing cycles, and exposure to urea up to 4 M, to NaCl up to 1 M, and to Triton X-100. Several components associated with broken Moloney murine leukemia virus were repeatedly found in preparations. These components have been described as rings, thick filaments, chain-like filaments, threads covered with proteins, threads with buckles, and naked threads. A quantitative analysis of the occurrence of these components has been carried out. Among them, the thick filaments composed of a compact helical arrangement of small beads 5 nm in diameter were considered to represent the nucleocapsid. The protease-sensitive buckles found on some threads could be a compact form of the viral RNA associated to the nucleocapsid protein NCp10. The RNase-sensitive naked threads are interpreted as the deproteinized viral RNA itself. The ubiquitous chain-like filaments possess a periodic structure identical to that of polymerized type VI collagen. It is proposed that this adhesive protein is associated with the viral envelope taken from the cell membrane during the budding process of retroviruses.

A liquid chromatographic preparation of retroviral RNA.

Liquid chromatography was used to prepare native dimer RNA from Moloney murine leukemia retrovirus by gel filtration on a TSK 6000PW column. Three RNA peaks were separated from viral lysate. RNA from the first eluted peak migrated in gel electrophoresis as a native dimer prepared by phenolic extraction and saccharose gradient separation. The last eluted RNA peak likely represents tRNA for proline. HPLC preparation was twice as fast and 20 times more productive than the other method, considering the quantity of pure RNA obtained for the same volume of viral lysate. Using several natural RNAs, it was verified that the dispersion coefficient was inversely proportional to RNA size, at least between 3.6 and 16.6 kb. Within the range of laboratory use, peak surface was in a direct ratio to the injected quantity of a given RNA species. Thus size exclusion chromatography could represent a valuable tool for preparation, analysis, and quantitation of large RNAs.

Bis(pyrrolecarboxamide) linked to intercalating chromophore oxazolopyridocarbazole (OPC): selective binding to DNA and polynucleotides.

We have investigated some properties related to interaction with DNA and recognition of AT-rich sequences of netropsin-oxazolopyridocarbazole (Net-OPC) (Mrani et al., 1990), which is a hybrid groove-binder-intercalator. The hybrid molecule Net-OPC binds to poly[d(A-T)] at two different sites with Kapp values close to 7 x 10(6) and 6 x 10(8) M-1 (100 mM NaCl, pH 7.0). Data obtained from melting experiments are in agreement with these values and indicate that Net-OPC displays a higher binding constant to poly[d(A-T)] than does netropsin. On the basis of viscometric and energy transfer data, the binding of Net-OPC to poly[d(A-T)] is suggested to involve both intercalation and external binding of the OPC chromophore. In contrast, on poly[d(G-C)], Net-OPC binds to a single type of site composed of two base pairs in which the OPC chromophore appears to be mainly intercalated. The binding constant of Net-OPC to poly[d(G-C)] was found to be about 350-fold lower than that of the high-affinity binding site in poly[d(A-T)]. As evidenced by footprinting data, Net-OPC selectively recognizes TTAA and CTT sequences and strongly protects the 10-bp AT-rich DNA region 3'-TTAAGAACTT-5' containing the EcoRI site. The binding of Net-OPC to this sequence results in a strong and selective inhibition of the activity of the restriction endonuclease EcoRI on the plasmid pBR322 as substrate. The extent of inhibition of the rate constant of the first strand break catalyzed by the enzyme is about 100-fold higher than the one observed in the presence of netropsin under similar experimental conditions.

Respiratory patterning of the rat olfactory bulb unit activity: nasal versus tracheal breathing.

To assess the role of nasal/tracheal (N/T) breathing in the respiratory patterning of the olfactory bulb (OB) neurons, the activity of 21 units was recorded in 6 anesthetized rats set with a cannula enabling reversible tracheotomy: the rats could inhalate either through nasal pathways or through trachea directly. Shift from tracheal to nasal breathing induced respiratory patterning in 7 units. The changes were steady, reversible and reproducible. The present data, matched with previous ones, indicate that tracheotomy and anesthesia decrease the occurrence of respiratory patterning in OB neurons. The experiments also suggest that peripheral as well as central structures may be a source of respiratory modulation in the olfactory bulb.

A centrifugal respiratory modulation of olfactory bulb unit activity: a study on acute rat preparation.

In behaving rats, unit activity in the mitral and granule cell layers of the olfactory bulb (OB) can be modulated by respiration. In order to determine whether central influences could take part in this phenomenon, respiratory rhythm and the activity of OB units were recorded in the present experiment and analyzed temporally in 18 anaesthetized tracheotomized rats. In spite of the interrupted nasal airflow, 30 of the 80 cells recorded in the mitral and granule cell layers, still displayed a significant respiratory patterning of their activity. Maximal neuronal discharges were time-locked with different phases of the respiratory cycle, most often synchronized with the end of expiration. This is in contrast with previous observations in intact animals. Possible underlying mechanisms are discussed.

Respiration and olfactory bulb unit activity in the unrestrained rat: statements and reappraisals.

The activity of 26 olfactory bulb units, including 19 mitral, 5 granular and 2 external plexiform cells, was recorded in unrestrained rats associating food odor stimuli/isoamyl acetate to a food reward/no reward. The respiratory activity was transduced from the intranasal air pressure and used as a time-base to analyze the unit discharge. The patterning of neuron activity was presented in histograms built from sequences of 30 successive cycles each resolved into 5 equal bins. 64 sequences were defined by the low or high respiratory frequency and by olfactory stimulation. In resting conditions, 15 (13 mitral) units displayed significant respiratory patterning, mainly characterized by the absolute and relative phases of the maximal and minimal activity in the cycle. Six typical groups of units could be defined accordingly. Increased respiratory frequency erased patterning, except in the 2 most typical units. The histograms from adjacent mitral cells showed that the various types were distributed as in a neuronal network with lateral recurrent inhibition, where noise was introduced at each inspiration. The data verified that the spatial and temporal distribution of the input activity elicited by the olfactory stimuli created local interferences, modifying the patterning of mitral activity. The odor-induced changes (R1 responses) were as consistent as the typology itself; they were selective and habituated rapidly. The transient R1 activity could also give rise to an R2 firing, atypical, regular and lasting, mainly when food odor elicited food intake. Possible functional interpretations of these phenomena are presented.

A removable head-mounted microdrive for unit recording in the free-behaving rat.

The microdrive described in this paper has been used for two years for chronic unit recording in the olfactory bulbs of unrestrained rats. It is a removable micromanipulator, suitable for tungsten microelectrodes, designed for stereotaxic exploration of cylindrical areas of nervous tissue 9 mm high and 1.6 mm in dia. The electrode is moved up and down without any rotation and with minimal vibration, with a degree of precision of 5 microns.

Unit responses changing with behavioral outcome in the olfactory bulb of unrestrained rats.

The unit activity of 16 neurons, including 12 likely mitral cells, was recorded in one olfactory bulb of 5 unrestrained rats, together with contralateral multiunit activity, and respiratory rhythm. The hungry animals were stimulated either by food odor (F) learned as a signal for one available food pellet, or by isoamyl acetate (IA), presented randomly. Eighty-four sequences, each with one stimulation, were analyzed to determine how the odors modified unit, multiunit and respiratory activities. The stimulation could change the variance and/or mean of the unit discharge and its correlation with the respiration phase or frequency or with multiunit activity. Besides the IA sequences, F+, F= and F-situations had to be distinguished, when the pellet was eaten spontaneously, accepted if presented at mouth or refused actively. The neuron responses were reproducible, in a given situation, but their occurrence within a series of identical stimuli was unpredictable from the controlled or observed events. The positive mitral responses were more probable in the F+ and F= than in the F-sequences; they were then associated with multiunit and respiratory activation. Response criteria and neuron typology are discussed. The functional involvements of neuron variability and modulation with internal state are considered.

Olfactory bulb excitability selectively modified in behaving rats after local 6-hydroxydopamine treatment.

Previous studies have shown that the olfactory bulb (OB) responsiveness was selectively enhanced towards food odor in free-moving hungry rats. this modulation of the olfactory input depended on the action of centrifugal fibers. The present study investigated the possible involvement of the noradrenergic (NA) projections from the locus coeruleus to the OB in this adaptative control of the OB excitability. After plugging of the olfactory lumen, 17 rats received discrete injections of 6-hydroxydopamine solution (10 micrograms of the salt; 40 nmol free base) into one OB (6-OHDA bulb) and the vehicle into the contralateral side (control bulb); 10 sham operated rats received intrabulbar isotonic saline solution injected bilaterally (sham bulbs). In the 12 6-OHDA bulbs with the noradrenaline content significantly reduced, the mean dopamine and noradrenaline levels were, respectively, 90% and 34% of the control values. One week after treatment, the average irritability score was significantly higher in the 6-OHDA treated animals than in the sham-operated ones. The main electrophysiological effect of the treatment was to amplify the selective enhancement of OB responsiveness to food odor normally occurring in hungry animals; this effect was restricted to the 6-OHDA bulbs. The results discussed considering the possible involvement of NA fibers in selective attention to odors at the OB level.

Functional changes in waking and sleeping rats after lesions in the olfactory pathways.

Food deprived control rats presented the following characteristics: (1) in a two-choice behavioral test, the animals explored significantly more the side of the cage odorized by a food odor (FO) than the non odorized one; (2) FO presentations in slow wave sleep (SWS) aroused them significantly more often than in a food satiated condition; (3) during wakefulness, the multiunit mitral cell responsiveness towards FO was selectively enhanced. The same 3 parameters have also been tested in animals where projections of one olfactory bulb were completely sectioned, or intact either in the medial or in the lateral part of one peduncle; the other olfactory peduncle was completely severed. The results showed that waking rats needed both medial and lateral projection pathways to perform normally in the food odor detection task, and to display the normal mitral cell excitability. However, in SWS, one medial pathway at least was needed to mediate normal rates of neocortical arousal in response to FO stimulations. The results are considered in terms of functional complementarity/redundance of the medial and lateral olfactory pathways.

Lesions of the olfactory pathways affecting neophobia and learned aversion differentially.

The contribution of ascending olfactory pathways in neophobia and learned aversion to the same food was investigated in male rats bearing lesions of both olfactory peduncles, or one olfactory peduncle and the opposite lateral olfactory tract or anterior limb of the anterior commissure. The animals were fed on usual stock diet (S) offered as a choice with novel vanilla food (V) on test days: during neophobia (N), then before and after aversive conditioning (Aa, At). Daily food intake was measured, and the preference was expressed as V/(V + S). Experiment 1 included a neophobia test, before aversive conditioning (3 mEq/kg LiCl, i.p.). In Experiment 2, aversion only was studied (0.9 mEq/kg). In the neophobia test, the preference ratio was 7% in unoperated controls, and 43-52% in the 3 lesioned groups. The same controls had preference ratios equal to 64% and 22%, before and after aversive learning. Similar drops were observed for any lesioned group in Expt. 1. The decrease was less obvious, although significant, in rats of Expt. 2 with asymmetric lesions; those with both olfactory peduncles cut through maintained the same preference ratio (48%) before and after LiCl treatment. The data are interpreted assuming that: (1) lateral olfactory tract and anterior commissure both contribute to information processing in neophobia and aversion; (2) olfactory cues subserve neophobia prepotently; and (3) one cannot account for the sensory determinism of neophobia and aversion calling for a single mechanism.

[Respiratory modulation of unit activity in the olfactory bulb of tracheotomized rats]. / Modulation respiratoire de l'activité unitaire dans le bulbe olfactif de rats trachéotomisés.

Unit activity, most probably from the mitral cells, has been recorded in the olfactory bulbs of anesthetized and tracheotomized rats. Two of 11 cells still increased their firing rate significantly during inspiration, although the airflow through the nasal cavity had been totally suppressed; the rest of the sample was not modulated according to respiratory activity.

Olfactory bulb responsiveness to an aversive or novel food odor in ;the unrestrained rat.

The mechanisms subserving neophobia and learned aversion have been investigated by recording multiunit olfactory bulb discharges either in hungry rats following food deprivation or in satiated rats. Under the two conditions, rats were stimulated with the smell of their familiar maintenance diet or that of a novel food or of control food-unrelated odor. Responses to the odor of the novel food were tested, following a pairing of the first or the second intake of that food with a LiCl injection, or following its first intake paired with a NaCl control injection. All rats exhibited enhanced level of discharges when they were stimulated in the hungry state with the smell of the familiar food and not when stimulated with the non-alimentary control odor. The hunger to satiety modulation of olfactory bulb discharges, also exhibited in rats tested with the smell of the novel food, previously paired with NaCl, was absent after a LiCl-induced taste aversion to this odor. The small, although significant, modulation observed when the conditioning of aversion occurred with the less novel food is consistent with the view that learned safety prevails upon learned harmfulness. Results are discussed in terms of relations of olfactory bulb electrical responses to odors with food palatability, neophobia and learned aversion.