Giant interneurons mediating equilibrium reception in an insect.

In the burrowing cockroach Arenivaga, two giant interneurons in each connective of the ventral nerve cord provide gravity orientation information. The interneurons receive input from plumb bob-like equilibrium receptors on the ventral surface of the cerci. Ouir results support the theory that the cerci of cockroaches are specialized equilibrium organs.

Segmental differentiation in the leech nervous system: the genesis of cell number in the segmental ganglia of Haemopis marmorata.

In hirudinid leeches, the segmental ganglia associated with the sexual organs contain several hundred more neurons than other midbody ganglia. To determine whether this difference arises by differential cell addition or by differential cell death, cell counts were made in several segmental ganglia during the course of embryonic and postembryonic development. The results show that all ganglia behave equally in early development. In each case, at least 10-20% more cells than will make up the adult complement of about 400 neurons is generated, and by about 20 days of embryonic development cell loss brings the number down to about 400 cells. By about 30 days, when animals emerge from their cocoons, additional cells have begun to appear in the sex ganglia. The number of extra cells continue to increase gradually over the next several months, until the adult number of 600-700 neurons is attained. These observations indicate that at least some segmental differences in the size of neuronal populations are due to differential cell proliferation and that these differences can arise quite late in the maturation of an animal.

Segmental differentiation in the leech central nervous system: proposed segmental homologs of the heart accessory neurons.

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.

Alcohols inhibit N-methyl-D-aspartate receptors via a site exposed to the extracellular environment.

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.

Membrane properties of microglial cells isolated from the leech central nervous system.

Membrane potentials and channel properties of microglial cells isolated from the leech central nervous system and maintained in culture on different substrates were investigated by using the patch-clamp technique. As expected, microglia on concanavalin A (con-A) were round and stationary, whereas those on extract of extracellular matrix (ECM) were spindle shaped and mobile. The mean membrane capacitance was 9 +/- 1 pF (s.e., n = 46), and the input resistance ranged from 0.135 G omega to 21 G omega with a mean of 4.2 +/- 1.6 G omega (n = 19). On-cell patches exhibited no single-channel activity. Voltage-dependent Na+, K+ and Ca2+ currents typical of neurons were absent. Currents evoked in response to voltage ramps from -100 mV to +100 mV or to steps of 4 s duration reversed in sign at or near 0 mV and exhibited single-channel activity of increasing amplitude for incrementally larger positive and negative voltage steps. No differences between the membrane properties of microglial cells on con-A and on ECM were evident. Currents were increased in fluid in which Na+ was substituted with K+, and were decreased when Na+ was substituted with N-methyl-D-glucamine. Varying external [Cl-] was without effect, as was addition to the fluid of 100 microM anthracene-9-carboxylate, a Cl- channel blocker. Together these characteristics indicated a cation channel. Bath application of 100 microM serotonin reversibly increased both inward and outward currents as well as single-channel activity. It is concluded that cultured microglial cells isolated from the adult leech have high membrane resistance and cation channel activity influenced by serotonin.

Regulation of GABAB receptors by histamine and neuronal activity in the isolated spinal cord of neonatal opossum in culture.

The aim of these experiments has been to analyse the properties of receptors for the transmitter gamma-aminobutyric acid (GABA) in developing mammalian nervous system. Changes in responses of GABAB receptors have been measured after alterations of the chemical environment and the level of electrical activity. We have previously shown that when the central nervous system (CNS) of the new-born opossum, Monodelphis domestica, is cultured for three to five days in the presence of histidine, inhibition by baclofen, a GABAB agonist, disappears (Stewart et al. 1991). We have now investigated whether histidine acts indirectly by way of conversion to histamine. As with histidine, culture with 150 microM histamine for five days virtually abolished the inhibition by baclofen. The effects of histidine, as well as histamine, were blocked by mepyramine, a histamine H1-receptor antagonist, and by ranitidine, an H2-antagonist. Tetrodotoxin (TTX), which blocks all electrical activity, protected preparations from the action of histidine but not histamine. Our results suggest that histidine is converted to histamine, which reduces the efficacy of GABAB agonists. We conclude that, in the developing mammalian CNS, transmitter levels and electrical activity can selectively influence the properties of receptors.

Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7-5-HT3 receptors expressed in Xenopus oocytes.

The effect of halothane and isoflurane was studied on the function of recombinant neurotransmitter receptors expressed in Xenopus oocytes. Both anaesthetics inhibited nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptor-mediated responses, potentiated 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses at low agonist concentrations, and inhibited the function of a chimeric receptor (with the N-terminal domain from the nACh alpha 7 receptor and the transmembrane and C-terminal domains from the 5-HT3 receptor) in a manner similar to that of the nACh alpha 7 receptor. Since the N-terminal domain of the chimeric receptor was from the nACh alpha 7 receptor, the observations suggest that the inhibition involves the N-terminal domain of the receptor.

Involvement of non-NMDA receptors in the rescue of weaver cerebellar granule neurons and sensitivity to ethanol of cerebellar AMPA receptors in oocytes.

The cellular mechanism responsible for the death of cerebellar granule neurons in the weaver mutant mouse is still being intensely investigated. To determine if alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors are involved in producing the weaver phenotype or are altered by the weaver gene, we used (1) reverse transcription and polymerase chain reaction (RT-PCR) to detect transcripts of glutamate receptors (GluR1-4) from wild-type and mutant cerebella; (2) immunocytochemistry to establish the types of glutamate receptors present in granule neurons cultured from normal and homozygous weaver postnatal day 5-6 (P5-6) cerebella; (3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a blocker of glutamate (AMPA/Kainate/NMDA) receptors, and 6,7-dinitroquinoxaline-2,3-dione (NBQX), a blocker of AMPA and kainate receptors, to assess the number of neurons and the number of neurons with long neurites in cultures of homozygous weaver granule neurons; (4) two-electrode voltage clamp recordings to study AMPA glutamate receptor expression in Xenopus oocytes after injection of mRNA isolated from cerebella of normal and weaver P5-6, postnatal day 10 (P10) and postnatal day 23 (P23) mice; and (5) ethanol, which at low 1-10 mM concentrations had been shown previously to rescue homozygous weaver granule neurons in culture [Liesi et al., J. Neurosci. Res. 48 (1997) 571-579], to examine its effect on modulation of AMPA receptors expressed from mRNA. By RT-PCR, the mRNA coding for AMPA receptor subunits GluR1-4 were detected from +/+ and wv/wv cerebella, and by immunocytochemistry, GluR1, GluR2/3 and GluR4 were observed to be expressed in cultured +/+ and wv/wv granule cells. CNQX at 10 microM or NBQX at 10 microM significantly increased the number of surviving neurons and the number with long neurites as compared to wv/wv controls. In addition, CNQX was significantly more effective than NBQX. In oocytes injected with mRNA from P10 normal or weaver cerebella, the amplitudes of the responses to kainate were about equal. In contrast, the amplitudes of the kainate-activated currents in oocytes injected with weaver P23 mRNA were about twice as large as the currents observed in oocytes injected with mRNA from normal P23 cerebella, and both were larger than kainate-activated currents observed after injection of P10 normal and weaver mRNA. Kainate-activated AMPA receptor currents in oocytes injected with mRNA from P10 and P23 normal and homozygous weaver cerebella were inhibited by ethanol. There were no significant differences in the inhibition produced by ethanol on currents from P10 or P23 normal and wv/wv mRNA. Thus, P23 weaver cerebellar mRNA expressed more kainate-activated current in oocytes than P23 normal cerebellar mRNA; both normal and weaver cerebellar granule neurons express mRNA coding for functional AMPA receptors that are susceptible to ethanol inhibition.

Characteristics of endothelial cells derived from the blood-brain barrier and of astrocytes in culture.

In this study, cultures of astrocytes and capillary endothelial cells from the blood-brain barrier (BBB) of the postnatal (P1) mouse cerebral cortex were analyzed with the aim of acquiring information on the distinguishing characteristics of each cell type. For isolation and purification of astrocyte cells, the methods of McCarthy and DeVellis [J. Cell Biol. 85 (1980) 890] were employed. The methods of Chen et al. [Lab. Invest. 78 (1998) 353], Duport et al. [Proc. Natl. Acad. Sci. USA 95 (1998) 1840], Rubin et al. [J Cell Biol. 115 (1991) 1725] and Tontsch and Bauer [Microvasc. Res. 37 (1989) 148] were utilized for culturing of cells from the BBB. A simple protocol was also created for isolating and purifying brain endothelial cells with 10 mM sodium cyanide. The vascular system of the cerebral cortex is derived from the leptomeningeal blood vessels [Qin and Sato, Dev. Dyn. 202 (1995) 172; Risau et al., EMBO J. 5 (1986) 3179]. With this in mind, cultures of the P1 mouse meninges were used as a comparative cell type in order to differentiate between BBB cells and astrocytes. In this regard, the expression of a number of markers were correlated, and an antibody double labeling technique was employed. The staining of these markers was then compared to cells cultured from leptomeninges and to two other types of endothelial cells, human umbilical vein and bovine aortic. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from adult mouse brain, cells cultured from P1 mouse cortex or meninges, bovine aortic endothelial cells and human umbilical vein endothelial cells (HUV-EC) to detect the expression of glial fibrillary acidic protein (GFAP), Von Willebrand factor (factor VIII-related antigen) and fibronectin. These analyses revealed the presence of GFAP mRNA in the cultures of cortical and leptomeningeal cells and of protein in all cell types; Von Willebrand factor mRNA was detectable in HUV-EC cells but undetectable in cortical, leptomeningeal and bovine aortic endothelial cells. Fibronectin mRNA and protein were present in all of the cell types. Given the results of our investigations we conclude that in culture, astrocytes are actually brain endothelial cells.

The embryonic development of peripheral neurons in the body wall of the leech Haemopis marmorata.

The appearance of peripheral neurons within the skin during embryonic development of the leech is described. These neurons were labeled using a monoclonal antibody, Lan3-6, which recognizes antigens in both the cell body and the axons of these cells. Within the 5 annuli that are found in each midbody segment, peripheral neurons first label in the middle and last in the most anterior and posterior ones. In each annulus, the number of cells labeled is initially 4 and increases as development proceeds. By the end of embryogenesis, all annuli show approximately equal numbers of Lan3-6 labeled neurons. The development of peripheral neurons in the skin of the rear sucker is also described.

Expression of surface antigens recognized by the monoclonal antibody lan 3-2 during embryonic development in the leech.

The monoclonal antibody lan 3-2 was used as a marker for the developmental expression of surface antigens which are specific for the two pairs of nociceptive neurons and a subset of axons in the adult CNS of the leech. In Haemopis embryos labeling of both nerve fibers and cell bodies with the antibody appears as expected for a metameric animal in a rostrocaudal temporal gradient from about day 5-6. Surprisingly, all central cell bodies are stained by the antibody in early development. However, later in embryogenesis around day 17 the staining intensity of most cells decreases except for the nociceptive cells, which remain antibody-positive, and the adult staining pattern gradually emerges. In addition to describing the central staining pattern, we show that specific peripheral neurons associated with the segmental sensilla also are antibody-positive during development. The distribution and developmental expression of the lan 3-2-positive antigens are compared between two phylogenetically different species of leeches and the diversity of the staining pattern of the monoclonal antibody is discussed.

Phosphorylation of the GABAA receptor gamma2L subunit in rat sensory neurons may not be necessary for ethanol sensitivity.

The effect of ethanol on the current activated by 2.5 to 40 microM gamma-aminobutyric acid (GABA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons under voltage clamp in the whole-cell and perforated-patch recording configurations. Our results confirmed that GABAA-activated current in these neurons was insensitive to ethanol at concentrations from 2.5 to 100 mM [G. White, D.M. Lovinger, F.F. Weight, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507 (1990) 332-336.]. In addition, the ethanol sensitivity of GABA receptors was studied under conditions that promote phosphorylation of the PKC site on the gamma2L subunit. The presence of the gamma2L and other subunit mRNAs was detected by reverse transcription (RT) of total RNA purified from adult DRG followed by polymerase chain reaction (PCR) using subunit specific primer sets. We found that the GABA response remained insensitive to 2.5-100 mM ethanol despite: (i) the extracellular preapplication of 5, 20 or 500 nM phorbol 12-myristate 13-acetate (PMA); (ii) raising free intracellular Ca2+ ([Ca2+]i) from 7 to 100 or 600 nM by altering the intracellular Ca2+/EGTA ratio; (iii) intracellular application of PKC (0.247 U ml-1 ); and (iv) combining the intracellular application of 1 microM okadaic acid and 30 microM peptide 3 with the extracellular application of 20 nM PMA. These results suggest that phosphorylation of the gamma2L subunit is not the only requirement for ethanol sensitivity of GABAA receptors.

Cytomegalic inclusion disease: intrauterine sonographic diagnosis using findings involving the brain.

Two second-trimester cases and one third-trimester case of intrauterine cytomegalic inclusion disease (CID) are presented, each having a different intracranial sonographic presentation. The findings are correlated with radiographic studies and the known pathophysiology. Sonographic evidence of intrauterine cerebral necrosis or calcification should alert one to the possibility of CID, particularly if other signs of in utero infection are present. A pattern of bilateral periventricular calcifications, which may be preceded by hypoechoic periventricular ringlike zones, seems to be specific for intrauterine CID. However, CID also may result in widespread cerebral destruction. If the sonographic study produces an uncertain diagnosis, sonography can still aid in the prenatal diagnosis of CID by guiding percutaneous umbilical cord blood sampling for serology or by directing amniocentesis for cytomegalovirus culture. The ability of sonography to demonstrate specific characteristics of CID in utero enables prenatal diagnosis of this disease.

Mechanisms of isoniazid release from poly(d,l-lactide-co-glycolide) matrices prepared by dry-mixing and low density polymeric foam methods.

The release mechanisms of a small molecular drug from biodegradable poly(d,l-lactide-co-glycolide) (PLGA) cylindrical matrices were investigated. Isoniazid (INH), one of the most effective drugs against tuberculosis (TB), was selected as the model drug. Controlled-release matrices consisting of the drug and polymer were fabricated by two methods. The first of these, the dry-mixing method, involved the extrusion of a mixture of micronized drug and polymer particles as rods. In the second technique, the low density polymeric foam method, drug particles were enclosed in the cells of porous polymeric foams prior to extrusion. In vitro, the dry-mixed matrices released INH more rapidly than the polymeric foam matrices. The Roseman-Higuchi diffusion model, which had previously been found to be effective in analyzing the release kinetics of INH from the dry-mixed matrices, also fit the kinetics of INH released from matrices prepared from polymeric foams. This indicated that the release was still diffusion-controlled rather than degradation-controlled. The release mechanisms were further investigated, and two diffusion mechanisms, pore diffusion and lattice diffusion, were proposed for the INH controlled-release matrices according to the way in which they were prepared. Matrices prepared by the dry-mixing method appear to segregate drug particles along polymer grain boundaries and thus have a pore diffusion mechanism, while matrices prepared by the foam method entrap drug within the porous structure of foams and thus display a lattice diffusion mechanism. Theoretically, these two diffusion mechanisms can be identified by their activation energies for diffusion. With varying in vitro temperature, the activation energies were calculated from plots of ln (DIT) vs T-1 and in D vs T-1, where D is the diffusivity and T is the in vitro temperature in K. According to the results, we concluded that the INH from the dry-mixed matrices diffused through the drug channels filled with the medium, while the INH from the foam matrices diffused through the polymer lattice.

Metorchis conjunctus (Cobbold, 1860) infection in wolves (Canis lupus), with pancreatic involvement in two animals.

The trematode Metorchis conjunctus (Cobbold, 1860) was found in seven of 211 wolves from Saskatchewan which were examined between 1976 and 1983. The parasite caused cholangiohepatitis with periductular fibrosis in the liver of all the wolves, and chronic inflammation and fibrosis of the pancreas in two animals. The wolves with pancreatic involvement were emaciated. Five of the seven infected wolves were from one local area, and three of these were from a pack known to consume fish.

Capture myopathy in a moose.

Of 18 moose captured by dart immobilization from a helicopter, 1 became recumbent after release. Blood samples were collected at the time of capture and on the following 2 days. Serum creatine phosphokinase and glutamic-oxalacetic transaminase activities increased, and serum potassium concentrations decreased. Necropsy revealed extensive myopathy.

Clinical investigation of medical devices.

Clinical investigations are required for implantable and invasive devices in Class IIa or IIb, and all Class III devices. This article describes how to successfully perform a clinical trial according to the standards set in EN 540, Clinical Investigation of Medical Devices for Human Subjects.

Cell death during gangliogenesis in the leech: competition leading to the death of PMS neurons has both random and nonrandom components.

The posteromedial, serotonin-containing (PMS) neurons are found in the ventral aspect of certain anterior segmental ganglia of adult leeches. With one exception, these cells are unpaired in all the ganglia where they are found. During early embryogenesis in Hirudo medicinalis, however, a bilateral pair of PMS neurons appears and differentiates in each of the 21 segmental ganglia (SG1-SG21). Over the next several days, one of the pair dies in SG3-SG21. Examination of the PMS neuron in any one of these segments reveals that either the right or the left cell remains, with equal probability, suggesting that the elimination of one of the pair is a random process. When unpaired PMS neurons are examined in pairs of adjacent ganglia, however, the cells are from opposite sides in the majority of cases (approximately 88%). This observation implies that the death of a PMS neuron in 1 ganglion strongly biases which member of the pair of PMS neurons degenerates in adjacent ganglia. Detailed examination of the sequence of degeneration shows that it begins at several separate loci in the nerve cord. We propose that the mechanism responsible for the death of one of the pair of PMS neurons in a segmental ganglion is competition between these 2 cells for some trophic factor, but that the outcome of this competition is predetermined if one of the PMS neurons in an adjacent ganglion has already begun to degenerate.