Cerebral interneurons controlling fictive feeding in Limax maximus

Summary Initiation and modulation of fictive feeding by cerebral to buccal interneurons (CBs) was examined in an isolated CNS preparation of Limax maximus. Three CBs which are phasically active during fictive feeding, CB₁, CB₃ and CB₄, will reliably trigger bouts of fictive feeding when activated alone or in pairs. Another phasic CB, CB_EC, is not effective for triggering feeding. One CB which is tonically active during fictive feeding, CB_ST, drives fictive feeding in 50% of preparations when activated alone and enhances triggering of feeding when co-activated with phasic CBs. The metacerebral giant cell (MGC) was found to be capable of triggering fictive feeding in preparations with an intact subcerebral commissure. The MGC was especially effective at increasing the effectiveness of other CBs for initiation of feeding. Short high-frequency bursts of phasic CB or MGC action potentials are capable of resetting ongoing fictive feeding. Resetting effects of CB action potentials are relatively independent of the phase of the bite-cycle in which they are activated. CB₄ phase-advances the bite-cycle while the other phasic CBs phase-delay the bite cycle. Moderate frequency stimulation of CB₄ speeds up the bite rate while moderate frequency stimulation of CB₃ slows biting. All CBs, except the tonic CB, CB_DL, increase the intensity of buccal motor neuron bursting during feeding. The excitatory effects of phasic CBs and the tonic CB, CB_EPSP, on fictive feeding persist for many seconds after the offset of stimulation. CBs form both monosynaptic excitatory and monosynaptic inhibitory connections with different BG motor neurons.Abbreviations BG buccal ganglion - BR buccal root - CB cerebral-buccal interneuron - CBC cerebral-buccal connective - CPG central pattern generator - FB fast burster neuron - FMP feeding motor program - IBI interbite interval - MGC metacerebral giant cell     

Anandamide modulates carotid sinus nerve afferent activity via TRPV1 receptors increasing responses to heat

Abnormal respiratory chemosensitivity is implicated in recurrent apnea syndromes, with the peripheral chemoreceptors, the carotid bodies, playing a particularly important role. Previous work suggests that supraphysiological concentrations of the endocannabinoid endovanilloid and TASK channel blocker anandamide (ANA) excite carotid bodies, but the mechanism(s) and physiological significance are unknown. Given that carotid body output is temperature-sensitive, we hypothesized that ANA stimulates carotid body chemosensory afferents via temperature-sensitive vanilloid (TRPV1) receptors. To test this hypothesis, we used the dual-perfused in situ rat preparation to confirm that independent perfusion of carotid arteries with supraphysiological concentrations of ANA strongly excites carotid sinus nerve afferents and that this activity is sufficient to increase phrenic activity. Next, using ex vivo carotid body preparations, we demonstrate that these effects are mediated by TRPV1 receptors, not CB1 receptors or TASK channels: in CB1-null mouse preparations, ANA increased afferent activity across all levels of Po(2), whereas in TRPV1-null mouse preparations, the stimulatory effect of ANA was absent. In rat ex vivo preparations, ANA's stimulatory effects were mimicked by olvanil, a nonpungent TRPV1 agonist, and suppressed by the TRPV1 antagonist AMG-9810. The specific CB1 agonist oleamide had no effect. Physiological levels of ANA had no effect alone but increased sensitivity to mild hyperthermia. AMG-9810 blocked ANA's effect on the temperature response. Immunolabeling and RT-PCR demonstrated that TRPV1 receptors are not expressed in carotid body glomus cells but reside in petrosal sensory afferents. Together, these results suggest that ANA plays a physiological role in augmenting afferent responses to mild hyperthermia by activating TRPV1 receptors on petrosal afferents.     

Cell block as an adjunct to conventional Papanicolaou smear for diagnosis of cervical cancer in resource-limited settings

OBJECTIVE: To assess the utility of indigenously prepared cell blocks (CBs) as an adjunct to a conventional smear test in providing a reliable diagnosis of clinically suspicious cervical cancer in resource-limited settings. METHODS: Eighty-six clinically suspicious cervical cancer cases underwent a conventional smear test, CB preparation from residual cellular samples and biopsies at the same sitting. Correlations were performed between these modalities in order to derive the sensitivity and specificity of the CB technique to diagnose cervical cancer. OBSERVATION & RESULTS: Out of 86 clinically suspicious cervical cancers, 72 (83.7%), 70 (81.4%) and 67 (77.9%) cases were diagnosed as malignant on tissue biopsies, CBs and smears respectively. CB-biopsy agreement in the diagnosis of malignancy was feasible in 87.5% of the cases while CB-Pap smear agreement was feasible in 92.5% of the cases. Sensitivity and specificity of CB preparation to diagnose malignancy was 92.5% and 100%, respectively, when the smear was taken as the reference test (excluding the unsatisfactory smears). When biopsy was taken as the gold standard, the sensitivity and specificity of CBs were 87.5% and 100% respectively (excluding the unsatisfactory biopsies). In 8/19 cases where the smear diagnoses were either unsatisfactory or atypical squamous cells/atypical glandular cells, CBs picked up malignant lesions. CONCLUSION: CBs prepared from the residual cellular sample of conventional cervical scrapes augment the sensitivity of the smear test. When used as an adjunct to the smear, CBs aid in providing a reliable diagnosis of cervical cancer in the majority of the clinically suspected cases and thus the biopsy load can be reduced significantly in resource-poor settings.     

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.     

The cranial sensory ganglia in culture: Differences in the response of placode-derived and neural crest-derived neurons to nerve growth factor

Explants of cranial sensory ganglia and dorsal root ganglia from embryonic chicks of 4 to 16 days incubation (E4 to E16) were grown for 24 hr in collagen gels with and without nerve growth factor (NGF) in the culture medium. NGF elicited marked neurite outgrowth from neural crest-derived explants, i.e., dorsal root ganglia, the dorsomedial part of the trigeminal ganglion, and the jugular ganglion. This response was first observed in ganglia taken from E6 embryos, reached a maximum between E8 and E11, and gradually declined through E16. Explants in which the neurons were of placodal origin varied in their response to NGF. There was negligible neurite outgrowth from explants of the ventrolateral part of the trigeminal ganglion and the vestibular ganglion grown in the presence of NGF. The geniculate, petrosal, and nodose ganglia exhibited an early moderate response to NGF. This was first evident in ganglia taken from E5 embryos, reached a maximum by E6, and declined through later ages, becoming negligible by E13. Dissociated neuron-enriched cultures of vestibular, petrosal, jugular, and dorsal root ganglia were established from embryos taken at E6 and E9. At both ages NGF elicited neurite outgrowth from a substantial proportion of neural crest-derived neurons (jugular and dorsal root ganglia) but did not promote the growth of placode-derived neurons (vestibular and petrosal ganglia). Our findings demonstrate a marked difference in the response of neural crest and placode-derived sensory neurones to NGF. The data from dissociated neuron-enriched cultures suggest that NGF promotes survival and growth of sensory ganglionic neurons of neural crest origin but not of placodal origin. The data from explant cultures suggest that NGF promotes neurite outgrowth from placodal neurons of the geniculate, petrosal, and nodose ganglia early in their ontogeny. However, we argue that this fibre outgrowth emanates not from the placodal neurons but from neural crest-derived cells which normally give rise only to satellite cells of these ganglia.     

Tim50a,a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

Background  

The Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation.     

Primary explants as a model of the hypothalamus in situ

Anatomical and electrophysiological studies with neurotensin and somatostatin were performed to assess the integrity of primary explants of the preoptic anterior hypothalamus (POAH) maintained in organ culture as a model to study peptidergic neurons in vitro. POAH neurons maintained in vitro were found to be morphologically similar to those visualized histologically in situ. Somatostatin- and neurotensin-immunoreactive neurons were also present in both preparations. Additionally, electrophysiological responses of neurons in vitro to neurotensin and somatostatin were qualitatively similar to neuronal responses in situ. The striking morphological and pharmacological similarity between POAH neurons in vitro and in situ suggests that primary explants of the POAH are organotypic.     

In vitro explant culture of mantle epithelium of freshwater pearl mussel

The in vitro culture of nacre secreting pallial mantle explants of freshwater pearl producing mussel, Lamellidens marginalis (Lamarck) included depuration of pearl mussels with different physical and chemical agents to eradicate various commensals, removal of pallial mantle ribbon, aseptic preparation of explants from the ribbon and transfer of those explants into tissue culture petri dishes. Special synthetic tissue culture media enriched with additives viz., inactivated calf fetal serum and antibiotics were poured into plates with explants. The culture plates were incubated at 30 degrees C in a CO2 incubator at 5%, CO2. The cultures could be maintained for 42-45 days without any contamination. After 12 hr epithelial like cells began to migrate out and formed a complete cell sheet surrounding the explant within 12-15 days. The epithelial cells in the culture indicated functional viability as subsequently after 38-40 days of culture, typical aragonitic 'nacre' crystals of CaCO3 could be observed throughout the culture plates.     

In vitro perfused-superfused cat carotid body for physiological and pharmacological studies

An in vitro perfused carotid body preparation was developed to study its chemosensory responses to physiological and pharmacological stimuli. The carotid bifurcation with the carotid body was vascularly isolated and excised from pentobarbital sodium-anesthetized cats. The CB was perfused in a chamber by gravity (80 Torr) with modified Tyrode's solution (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-NaOH at pH 7.40) equilibrated at a given Po2 and superfused with the same medium at (Po2 of 20 Torr). The temperature was maintained at 35.5 +/- 0.5 degrees C. The frequency of chemosensory discharges (CD) was recorded from the whole carotid sinus nerve (n = 24), and the responses were tested by repeated interruptions of perfusate flow (SF), perfusion with hypoxic medium, and injections of nicotine and cyanide (0.1 nmol to 1 mumol) and hypercapnic medium. During hyperoxic perfusion, SF resulted in a sigmoidal increase in CD, reaching a maximum that was 23.6 +/- 4.4-fold greater than the basal activity. Restoration of flow returned CD promptly to basal values. After normoxic perfusion, SF led to a similar maximal activity more rapidly, but the duration was shorter. Reduction of the perfusate PO2 (Po2 from 450 Torr to 150, 30, and less than 10 Torr) caused a nonlinear increase in CD. CO2 stimuli (PCo2 38-110 Torr) resulted in a linear increase in CD. Nicotine or cyanide increased CD in a dose-dependent manner. The preparation retained its initial responsiveness for 2-3 h, making extensive experimental studies feasible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autonomic microganglion cells: a source of acetylcholine in the rat carotid body.

Hypoxic chemosensitivity of peripheral arterial chemoreceptors and the ventilatory response to O2 deprivation increases with postnatal development. Multiple putative neurotransmitters, which are synthesized in the carotid body (CB), are thought to mediate signals generated by hypoxia. Acetylcholine (ACh) is believed to be a major excitatory neurotransmitter participating in hypoxic chemosensitivity. However, it is not known whether ACh originates from type I cells in the CB. In these studies, we tested the hypothesis that choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) mRNAs are expressed in the CB and that mRNA levels would increase with postnatal maturation or exposure to hypoxia. Semiquantitative in situ hybridization histochemistry and immunohistochemistry were used to localize cholinergic markers within neurons and cells of the rat CB, the nodose-petrosal-jugular ganglion complex, and the superior cervical ganglion up to postnatal day 28. We show that the pattern of distribution, in tissue sections, is similar for both ACh markers; however, the level of VAChT mRNA is uniformly greater than that of ChAT. VAChT mRNA and immunoreactivity are detected abundantly in the nodose-petrosal-jugular ganglion complex in a number of microganglion cells embedded in nerve fibers innervating the CB for all postnatal groups, whereas ChAT mRNA is detected in only a few of these cells. Contrary to our hypothesis, postnatal maturation caused a reduction in ACh trait expression, whereas hypoxic exposure did not induce the upregulation of VAChT and ChAT mRNA levels in the CB, microganglion, or within the ganglion complex. The present findings indicate that the source of ACh in the CB is likely within autonomic microganglion cells and cholinergic nerve terminals.     

Cell blocks in cytopathology: a review of preparative methods,utility in diagnosis and role in ancillary studies

The cell block (CB) is a routine procedure in cytopathology that has gained importance because of its pivotal role in diagnosis and ancillary studies. There is no precise review in the published literature that deals with the various methods of preparation of CB, its utility in diagnosis, immunocytochemistry (ICC) or molecular testing, and its drawbacks. An extensive literature search on CB in cytology using internet search engines was performed for this review employing the following keywords: cell block, cytoblock, cytology, cytopathology, methods, preparation, fixatives, diagnostic yield, ancillary and molecular studies. Ever since its introduction more than a century ago, the CB technique has undergone numerous modifications to improve the quality of the procedure; however, the overall principle remains the same in each method. CBs can be prepared from virtually all varieties of cytological samples. In today's era of personalized medicine, cytological specimens, including CBs, augment the utility of cytological samples in analysing the molecular alterations as effectively as surgical biopsies or resection specimens. With the availability of molecular targeted therapy for many cancers, a large number of recent studies have used cytological material or CBs for molecular characterization. The various techniques of CB preparation with different fixatives, their advantages and limitations, and issues of diagnostic yield are discussed in this review.     

Intracellular Recording,Sensory Field Mapping,and Culturing Identified Neurons in the Leech,Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.     

Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.     

Expression of inwardly rectifying K+ channels in the carotid body of rat

The inwardly rectifying K+ channels, Kir1.1, Kir2.3, Kir4.1-Kir5.1, and Kir4.2-Kir5.1, are candidate chemosensory molecules for CO2/H+. Here, we determined the mRNA expression and immunohistochemical localization of these channels in the carotid body (CB) and petrosal ganglion (PG) of the rat. RT-PCR analysis revealed mRNA expression of Kir4.1 and Kir5.1 in CB, and Kir1.1, Kir4.1, and Kir5.1 in PG. Immunohistochemistry identified the glomus cells in CB to express both Kir4.1 and Kir5.1 protein, while the nerve fibers in CB were immunoreactive for Kir1.1, Kir4.1, and Kir5.1. In the PG, immunoreactivity for Kir1.1, Kir4.1, and Kir5.1 was observed in some ganglion cells. Our findings suggest that Kir channels in the peripheral chemoreceptors play a role in sensing hypercapnic acidosis and maintaining the resting membrane potentials.     

Differences in glucose transport rates between perfused and in vitro incubated muscles

In vitro incubated muscles are a convenient preparation for glucose transport studies, but it is not known how closely they reflect the in vivo condition. Perfused muscle preparations more closely resemble the in vivo condition, and thus to validate the use of in vitro incubated muscles, we have compared glucose transport rates in the two preparations. 3-O-Methylglucose transport rates in incubated soleus (SOL) and extensor digitorum longus (EDL) muscle strips were compared to transport rates obtained in SOL and EDL muscles removed from perfused hindquarters. Male Sprague-Dawley rats (250 g) were used for both procedures. SOL muscles showed an average 25% higher transport rate than EDL muscles at all insulin concentrations examined (0-100 nM) in the perfused system. This difference was diminished in the incubated muscles, SOL being 15% greater than EDL, but the relationship between the two muscles was maintained. Basal transport was lower and maximal transport was higher in the perfused muscles compared to the incubated muscles. This resulted in significantly higher fold stimulation in the perfused vs. incubated muscles (15 vs. 2.5 in the SOL, and 9.8 vs. 2.3 in the EDL). We conclude that in vitro muscle preparations may be convenient for showing relative differences between experimental treatments, but absolute transport rates and insulin stimulation must be interpreted with caution.     

Coilin methylation regulates nuclear body formation

Cajal bodies (CBs) are nuclear suborganelles involved in biogenesis of small RNAs. Twin structures, called gems, contain high concentrations of the survival motor neurons (SMN) protein complex. CBs and gems often colocalize, and communication between these subdomains is mediated by coilin, the CB marker. Coilin contains symmetrical dimethylarginines that modulate its affinity for SMN, and, thus, localization of SMN complexes to CBs. Inhibition of methylation or mutation of the coilin RG box dramatically decreases binding of coilin to SMN, resulting in gem formation. Coilin is hypomethylated in cells that display gems, but not in those that primarily contain CBs. Likewise, extracts prepared from cells that display gems are less efficient in methylating coilin and Sm constructs in vitro. These results demonstrate that alterations in protein methylation status can affect nuclear organization.     

Subcellular fractionation and morphology of calf aortic smooth muscle cells: studies on whole aorta, aortic explants, and subcultures grown under

A comparative biochemical and morphological study was made of calf aortic smooth muscle cells found in situ and grown in vitro under various conditions. Striking alterations in enzyme contents, physical properties, and morphological appearances of lysosomes, endoplasmic reticulum, plasma membranes and, to a lesser extent, mitochondria were observed upon culturing of calf aortic smooth muscle cells. These changes first appeared in cells growing out of tissue explants. They developed further upon subculturing of the cells and depended greatly on the culture conditions used. The alterations included increases in specific activities of some 5- to 25-fold of four acid hydrolases, an average ninefold increase in 5' -nucleotidase, sevenfold increase in cytochrome oxidase, and fourfold increase in neutral α-glucosidase in subcultured smooth muscle cells compared to aortic cells in situ. Cell fractionation studies showed significant shifts in the equilibrium densities of plasma membranes, microsomes, and lysosomes, but not of mitochondria, in smooth muscle cells growing out from explants and in subcultured cells, compared to cells isolated from intact aortas. Although the cells grown in vitro exhibited typical phenotypic features of smooth muscle cells such as abundant myofilaments and surface vesicles, alterations in the morphological appearance of the endoplasmic reticulum, Golgi apparatus, and, especially, lysosomes were observed. These results demonstrate significant differences in specific cellular characteristics and functions of aortic smooth muscle cells grown in vitro compared to aortic cells in situ.