Cultured olfactory interneurons from Limax maximus: optical and electrophysiological studies of transmitter-evoked responses.

1. The olfactory processing network in the procerebral (PC) lobe of the terrestrial mollusk Limax maximus exhibits a coherent oscillation of local field potential that is modulated by odor input. To understand the cellular basis of this oscillation, we developed a cell culture preparation of isolated PC neurons and studied the responses of isolated cells to stimulation with neurotransmitters known to be present in the PC lobe. 2. The distribution of PC soma diameters suggests at least two different populations of neurons. Approximately 95% of isolated cells had soma diameters of 7-8 microns, with the remaining cells having larger diameters (10-15 microns). 3. Extracellular measurements of action potentials and optical measurements of intracellular calcium concentrations in fura-2-loaded cells were made. Serotonin and dopamine excited PC neurons and promoted transitions from steady to bursty activity. Both amines elicited increases in intracellular calcium, presumably concomitant with the increase in action-potential frequency. 4. Glutamate suppressed action-potential firing and reduced intracellular calcium. This effect was seen most clearly when glutamate was applied to cells excited by high potassium medium. Quisqualate is an effective glutamate agonist in this system, whereas kainate is not. 5. Combined with anatomic and biochemical data and with studies of the effects of these neurotransmitters on the oscillating local field potential of the intact PC network, the data from isolated PC neurons are consistent with the hypothesis that dopamine and serotonin modulate network dynamics, whereas glutamate is involved in generating the basic oscillation of local field potential in the PC. 6. The optical studies of fura-2-loaded cells showed that several treatments that increase the rate of action-potential production lead to elevations in intracellular calcium. Optical studies of intracellular calcium may be useful for multisite measurements of activity in the intact, oscillating PC lobe network.

Chronic clorgyline treatment of Syrian hamsters: an analysis of effects on the circadian pacemaker.

Clorgyline, a type A monoamine oxidase inhibitor with antidepressant properties when administered to depressed patients, is often associated with disturbances of the human sleep-wake cycle. In order to assess its effects on the mammalian circadian system, this drug was administered chronically to Syrian hamsters. It was found to affect the hamster circadian system in four specific ways. Clorgyline increased the intrinsic period of wheel-running activity, altered the phase response curve to brief light pulses, altered the reduced waveform of running activity in animals maintained in light-dark cycles or constant darkness, and increased the activity-rest ratio in animals maintained in constant darkness. Our data support the interpretation that clorgyline exhibits direct or indirect input to the circadian pacemaker and alters the processing of photic information to the pacemaker.

Galactogen synthesis-stimulating factor in the slug, Limax maximus: cellular localization and partial purification.

Galactogen synthesis in the albumen gland of the slug Limax maximus is under direct endocrine control [J. van Minnen, J. Wijdenes, and P. G. Sokolove (1983) Gen. Comp. Endocrinol. 49, 307-314]. The present results show that dorsal body cells (DBC) present throughout the connective tissue around the circumesophageal brain are the major cellular source of a galactogen synthesis-stimulating factor (GAL-SF). The amount of GAL-SF varies during sexual maturation of L. maximus (a protandrous hermaphrodite): it is low when animals are immature and reaches a peak in the late female phase when the animals prepare to lay eggs. The morphology of DBC also changes during maturation. DBC are small and release little secretory product in immature and early male-phase animals; release is higher and DBC are larger in late female-phase animals. Gel filtration of homogenates of cerebral ganglia on Sephadex G75 yields one peak of GAL-SF activity with an estimated Mr of 4000-7000 Da. Subsequent anion exchange chromatography showed that GAL-SF could be eluted with a linear NaCl gradient; GAL-SF was found in fractions with a NaCl concentration between 0.04 and 0.07 M.

Immunocytological localization of a somatostatin-like substance in the brain of the giant slug, Limax maximus L.

Immunocytological tests reveal the presence of a somatostatin-like substance in perikarya and axons in the brain of the giant slug Limax maximus L. Controls carried out on adjacent sections with absorbed antiserum or different antibodies raised against several biologically active peptides of vertebrates (ACTH-17-39, alpha- and beta endorphin, alpha- and beta MSH, methionin-enkephalin, TRH) demonstrate the specificity of the "staining". However, some cells are both somatostatin- and FMRF-amide-positive. In the cerebral ganglia, the right Z-area cells, responsible for the synthesis of the maturation hormone (MH) are strongly somatostatin-positive. These results suggest a similarity between the MH and the somatostatin-like material contained in the Z-area cells. The simultaneous presence of two peptides in one and the same cell, the nature (elementary granules or soluble product) of the material, and its site of release are discussed.

Male gonadotrophic factor in brain and blood of photoperiodically stimulated slugs.

A pulmonate male gonadotrophic factor (MGF) has been described that is released from cerebral ganglia of male-phase slugs (Limax maximus). This factor produces, directly or indirectly, an increase in spermatogonial proliferation as determined by in vivo incorporation of [3H]thymidine into gonadal DNA. In the present investigation MGF activity was demonstrated in saline homogenates of male-phase cerebral ganglia by injecting homogenates into immature slugs for 5 consecutive days and assaying gonadal [3H]thymidine incorporation on Day 7. Dose-response data indicate that daily administration of as little as 0.1 brain equivalent can produce a significant stimulation in incorporation. Comparison of brain homogenates from immature (short-day) and male-phase (long-day) animals has shown that male-phase cerebral ganglia contain substantially more MGF activity than immature ganglia. Similar injection experiments using slug blood plasma showed that activity is present in male-phase blood but not in the blood of short-day immatures. MGF activity in long-day brain homogenates and blood plasma was found to be associated with a molecular weight fraction of 50 to 100 kDa obtained by ultrafiltration. Activity could be reduced or destroyed by treatment with trypsin or by heating. The present findings suggest that MGF is a proteinaceous factor of substantial size. It appears that both the synthesis and the secretion of MFG are stimulated in slugs that are in their male developmental phase as a result of prior exposure to long-day photoperiods.

Stimulation of spermatogonial DNA synthesis in slug gonad by a factor released from cerebral ganglia under the influence of long days.

Incorporation of [3H]thymidine into gonadal DNA was shown to increase 1 week after implantation into an immature slug (Limax maximus) of a "brain" (circumesophageal ring of ganglia) from a male-phase donor. Light microscope autoradiography revealed that in stimulated gonads labeling was localized primarily in the nuclei of spermatagonia. Implant-stimulated spermatogonial DNA synthesis was found to depend upon implantation of supraesophageal (cerebral) ganglia. Neither subesophageal ganglia implants nor immature supraesophageal implants had an effect. Thymidine incorporation could also be stimulated by exposure of slugs to long-day lightcycles (LD 16:8) for 3 to 4 weeks. Similar duration of long-day treatment was also adequate to trigger male-phase development even after animals were returned to short days (LD 8:16). The results are consistent with the view that 3 to 4 weeks of long-day lightcycles are required to promote irreversibly the release from slug cerebral ganglia of a male-phase gonadotropic factor which directly or indirectly promotes spermatogonial proliferation.

Endocrine control of galactogen synthesis in the albumen gland of the slug, Limax maximus.

An in vitro method for culturing Limax maximus albumen glands is described in which the biosynthetic activity of the slug albumen gland was monitored by measuring the incorporation of [14C]glucose into galactogen. Homogenates of the central nervous system were shown to cause a 3.5- to 12-fold increase in galactogen synthesis in albumen gland explants as compared to controls. The major sources of the galactogen-synthesis stimulating factor (Gal-SF) were found to be the cerebral ganglia and their surrounding connective tissue. Gal-SF was demonstrated to be peptidase sensitive and heat labile suggesting that it is probably a polypeptide. Autoradiographs of semithin araldite sections supported the incorporation data: in albumen gland explants cultured with cerebral ganglion homogenate considerably more label was found over secretory granules than in control-cultured explants. The possible cellular source of Gal-SF is discussed in relation to its possible origin in other investigated pulmonates.

Neurosecretory cells in the central nervous system of the giant garden slug, Limax maximus.

The neurosecretory system of the giant garden slug Limax maximus was studied using the alcian blue/alcian yellow (AB/AY) staining technique for neurosecretion. Stainable cells could be identified in the paired cerebral, pleural, parietal, and buccal ganglia, and in the visceral ganglion. The cells occur as single cells or in groups of up to 100, with diameters ranging between 10 and 70 mu m. Axon tracts could only be traced for a small number of cells; neurohemal areas were not conclusively identified. The morphological similarities of the neurosecretory system of L. maximus is compared with that of other investigated stylommatophoran slugs.

Sleep and activity rhythms in mice: a description of circadian patterns and unexpected disruptions in sleep.

Studies on daily and circadian rhythms in wheel running and electrographically defined wakefulness, NREM sleep, and REM sleep in M. musculus were done to gather data on the temporal distribution of activity and sleep. Generally, peaks in NREM and sleep tended to coincide and to alternate with the coincident peaks of wakefulness and wheel running. However, during the active phase of the circadian wheel running cycle some NREM and REM sleep did occur; conversely, during its rest phase, wakefulness was often present. The most striking finding was that in mice with clearly entrained or free-running activity onsets, the circadian peak-through patterns in wakefulness, NREM, and REM sleep were not always distinct--they could be damped and/or polyphasic. Several explanations of these phenomena are considered.

A circadian rhythm in the locomotive behaviour of the giant garden slug Limax maximus.

The locomotor activity of the garden slug Limax maximus was examined for components of circadian rhythmicity. Behavioural (running wheel) studies clearly demonstrated that the activity satisfies the principal criteria of circadian rhythmicity. In constant darkness at a constant temperature, the locomotor activity freeran with a period of about 24 h (range 23-6-24-6 h). The rhythm was also expressed in constant light with a period for individual slugs that tended to be shorter in LL than in DD. The period of the rhythm was temperature compensated (11-5-21-5 degrees C) with a Q10 approximately equal to 1-00. The locomotor rhythm could be entrained to 24 h LD cycles such that the circadian activity peak occurred during the dark. The phase angle between the onset of activity and lights-off was not fixed, but was a function of the photoperiod of the entraining light cycle.

Localization of the cockroach optic lobe circadian pacemaker with microlesions.

Electrolytic microlesions were made at various sites in optic lobes of cockroaches Leucophaea maderae, and animals were assayed for circadian locomotor rhythms. Lesions placed in the lamina or medulla disrupted rhythmicity in only a few animals (3 out of 55). Lesions placed in or near the lobula produced a greater fraction of arrhythmic roaches (18 out of 45). Over half of these lesions either included or were centered in the cell body regions bordering the second optic chiasm and the lobula. The results suggest that the cell bodies and not the neuropile areas of the lobe are the crucial elements of the clock driving the cockroach's circadian activity rhythm.

Histological evidence for direct connections between the optic lobes of the cockroach Leucophaea maderae.

Heretofore, descriptions of direct interconnections between insect optic lobes have been based on histological examinations of normal brains or on inference from electrophysiological or behavioral data. We present here what we believe to be the first demonstration of such monosynaptic connections by techniques of experimental neuroanatomy. Twenty-four to 39 h after extirpation of the left optic lobe, degenerating axons and axon terminals, as silvered by a modified Nauta technique, were abundant in the central portion of the medulla of the right optic lobe. The periphery of the medulla was free of argyrophilic debris as were the lobula and lamina. The distribution of neuronal somata with processes terminating in the the left optic lobe was established by retrograde axonal transport of horseradish peroxidase injected into the left lobe and by the development of distinctive perinuclear rings of RNA (a 'chromatolytic' reaction) by some cells within 1-2 weeks following amputation of the left lobe. Both techniques revealed distinct clusters of cells in the anteroventral and posterior regions of the right optic lobe, and in the medial portion of the right protocerebrum. The cells which interconnect the two optic lobes may be involved not only in the bilateral representation of visual information, but also in the coordination of optic lobe pacemakers which control a circadian rhythm of locomotory activity.

Analysis of postural motoneuron activity in crayfish abdomen. I. Coordination by premotoneuron connections.

The activity of the crayfish abdominal postural motoneurons and their associated neurons (the accessory neuron(s) and the MRO(1)) were examined with the aid of techniques for the analysis of simultaneously recorded spike trains. A means of reliably identifying the spikes of the individual motoneurons based on their relative axon conduction velocities is presented. The analyses show that: 1) the large, phasically active synergist motoneurons innervating muscles producing the same movement show a marked similarity in their average responses, which is independent of the input source; 2) the small, tonically active and the middle-sized, tonicphasic synergist motoneurons innervating the same muscle and similar synergist motoneurons innervating antagonistic muscles are coordinated entirely by premotoneuron connections; 3) the accessory neuron is coordinated in its activity with the phasically active flexor excitor motoneurons and the extensor inhibitor motoneuron and thereby functions as a flexor synergist; and 4) the simultaneous presentation of flexion-producing and extension-producing inputs to the postural system results in a reciprocal oscillation in flexor-extensor motoneuron output. The functional significance of these results with respect to the operation of the postural system are discussed.

Analysis of postural motoneuron activity in crayfish abdomen. II. Coordination by excitatory and inhibitory connections between motoneurons.

The identified spike trains of the crayfish abdominal postural efferent neurons were recorded simultaneously from one or more segments. The efferent activity was analyzed using cross-correlation histograms, peristimulus time scatter diagrams, and specialized antidromic techniques. The analyses show that the larger, phasically active motoneurons are coordinated in their activity by cross connections made at the motoneuron level. The cross connections are both excitatory and inhibitor in nature and result in significant alterations in spike output. Further, the accessory neuron receives an inhibitory cross connection from a middle-sized extensor excitor motoneuron or motoneurons. In each case, the cross connections appear to be appropriate to the function of the postural system.