Modulation of ion channels in rod photoreceptors by nitric oxide.

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K+ or nonspecific cation currents. Given the steep relation between voltage-dependent Ca2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.

Temperature dependence of T-type calcium channel gating.

T-type calcium channel isoforms are expressed in a multitude of tissues and have a key role in a variety of physiological processes. To fully appreciate the physiological role of distinct channel isoforms it is essential to determine their kinetic properties under physiologically relevant conditions. We therefore characterized the gating behavior of expressed rat voltage-dependent calcium channels (Ca(v)) 3.1, Ca(v)3.2, and Ca(v)3.3, as well as human Ca(v)3.3 at 21 degrees C and 37 degrees C in saline that approximates physiological conditions. Exposure to 37 degrees C caused significant increases in the rates of activation, inactivation, and recovery from inactivation, increased the current amplitudes, and induced a hyperpolarizing shift of half-activation for Ca(v)3.1 and Ca(v)3.2. At 37 degrees C the half-inactivation showed a hyperpolarizing shift for Ca(v)3.1 and Ca(v)3.2 and human Ca(v)3.3, but not rat Ca(v)3.3. The observed changes in the kinetics were significant but not identical for the three isoforms, showing that the ability of T-type channels to conduct calcium varies with both channel isoform and temperature.

The contribution of dendritic Kv3 K+ channels to burst threshold in a sensory neuron.

Voltage-gated ion channels localized to dendritic membranes can shape signal processing in central neurons. This study describes the distribution and functional role of a high voltage-activating K(+) channel in the electrosensory lobe (ELL) of an apteronotid weakly electric fish. We identify a homolog of the Kv3.3 K(+) channel, AptKv3.3, that exhibits a high density of mRNA expression and immunolabel that is distributed over the entire soma-dendritic axis of ELL pyramidal cells. The kinetics and pharmacology of native K(+) channels recorded in pyramidal cell somata and apical dendrites match those of AptKv3.3 channels expressed in a heterologous expression system. The functional role of AptKv3.3 channels was assessed using focal drug ejections in somatic and dendritic regions of an in vitro slice preparation. Local blockade of AptKv3.3 channels slows the repolarization of spikes in pyramidal cell somata as well as spikes backpropagating into apical dendrites. The resulting increase in dendritic spike duration lowers the threshold for a gamma-frequency burst discharge that is driven by inward current associated with backpropagating dendritic spikes. Thus, dendritic AptKv3.3 K(+) channels influence the threshold for a form of burst discharge that has an established role in feature extraction of sensory input.

Anxiety and depression in a primary care clinic. Comparison of Diagnostic Interview Schedule, General Health Questionnaire, and practitioner assessments.

Over one half of all persons seen in a primary care clinic were identified as having anxiety or depressive disorder by the primary care provider, the General Health Questionnaire (GHQ), or the Diagnostic Interview Schedule (DIS). In only about 5% of all patients were findings positive on all three assessments concurrently. Both the GHQ and the practitioners identified over 30% of all patients as having a disorder, while about 8% had one or more of five DIS anxiety or depressive disorders (major depression, dysthymia, panic disorder, generalized anxiety disorder, or obsessive-compulsive disorder). Of the patients with DIS disorders 83% had positive GHQ scores, and 73% were identified by the practitioner as having a mental disorder.

Progressive multifocal leukoencephalopathy diagnosed by amplification of JC virus-specific DNA from cerebrospinal fluid.

OBJECTIVE: To study the diagnostic sensitivity and specificity of polymerase chain reaction (PCR) for the non-invasive diagnosis of progressive multifocal leukoencephalopathy (PML) in HIV-1-infected individuals. DESIGN: Retrospective analysis of stored cerebrospinal fluid (CSF) samples by PCR of HIV-1-infected patients. METHODS: Results of the PCR analysis of the CSF of three AIDS patients with autopsy-proven PML were compared with the results in 15 neurologically asymptomatic HIV-1-infected patients and with 15 AIDS patients with other opportunistic infections of the central nervous system (CNS). A polyclonal antiserum to simian virus 40 (SV40) cross-reacting with JC virus (JCV) late antigens was used for immunocytochemical confirmation of the diagnosis. Two different primer pairs, one taken from the VP1/large T gene and the other from the large T gene, were used to amplify JCV-specific DNA sequences from CSF. RESULTS: Five CSF samples were analysed and JCV-specific DNA found in three patients with autopsy-proven PML. No JCV-specific DNA was detected in 47 CSF samples, including serial samples from 14 of the 30 non-PML patients. The diagnosis of PML was confirmed in all three cases by immunocytochemistry. CONCLUSION: PML can be diagnosed by PCR analysis of CSF. The sensitivity and specificity of the method depends on the sensitivity of the primers used for amplification. Using a primer pair from the large T gene, JCV-specific DNA was amplified in three cases with PML as early as the day of presentation with the first neurological symptom of PML.

Compartmentation of NADPH-diaphorase activity in the mouse cerebellar cortex.

The mammalian cerebellum is built around an array of parasagittal bands of Purkinje cells that can be demonstrated by immunocytochemical staining for the differentiation antigen zebrin II. Climbing and mossy fiber afferents also terminate in bands, and the afferent terminal fields and the Purkinje cell bands are aligned. The convergence of mossy and climbing fiber pathways onto the Purkinje cells, which are the sole output of the cerebellar cortex, is a characteristic feature of cerebellar circuitry. Previous studies showed that when both afferent pathways are activated synchronously there develops a long-term depression of synaptic efficacy at the parallel fiber-Purkinje cell synapse. Two second messenger pathways mediate long-term depression: one involves diacylglycerol and protein kinase C, and the other involves nitric oxide that is generated by a nitric oxide synthase. We have studied the distribution of nitric oxide synthase in the adult mouse cerebellum by using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. NADPH-diaphorase activity is found mainly in the granule and basket cells. Within the granular layer NADPH-diaphorase activity is expressed nonuniformly by patches of granular cells and synaptic glomeruli. The patches are seen in all lobules, are reproducible from individual to individual, and are topographically ordered with respect to the Purkinje cell compartments as revealed by using anti-zebrin II immunocytochemistry. These data imply that nitric oxide-dependent, long-term depression may only involve a subset of mossy fiber/granule cell projections, and that one role for nitric oxide may be to refine cerebellar receptive fields.

Acutely isolated and cultured cells from the electrosensory lateral line lobe of a gymnotiform teleost.

The present study established the morphological and immunocytochemical criteria necessary to identify neuronal and nonneuronal cells after dissociating select regions of the medullary electrosensory lateral line lobe of adult weakly electric fish (Apteronotus leptorhynchus). Cells dissociated from the pyramidal cell body layers of the centromedial and lateral segments exhibited similar characteristics in the acutely dissociated preparation and up to 14 days in culture. Basilar and nonbasilar pyramidal cells were tentatively identified according to a bipolar or monopolar process extension, and polymorphic cells by the extension of three or more processes and positive immunoreactivity for gamma-aminobutyric acid. Nonneuronal cells were identified by the pattern of process arborization and positive immunolabel for gamma-aminobutyric acid or glial fibrillary acidic protein. Neuronal cells increased in total number over the first 4 days and could appear for the first time on any day in culture. Individual pyramidal cells could maintain their morphology from the time of dissociation and over several days in culture. Pyramidal cell processes were phenotypically similar to apical and basal dendrites found in situ but were reduced in size and in the degree of process branching. These results indicate that dissociated adult apteronotid neurons can maintain a morphology sufficiently similar to that found in situ as to allow tentative identification, opening up a wide range of possibilities for studying the electrophysiological and regenerative properties of electrosensory neurons.

Localization of nicotinamide adenine dinucleotide phosphate-diaphorase activity in electrosensory and electromotor systems of a gymnotiform teleost, Apteronotus leptorhynchus.

The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity was determined in electrosensory and electromotor systems of the weakly electric gymnotiform teleost Apteronotus leptorhynchus as an indicator of putative nitric oxide synthase-containing cells. NADPH-d activity was detected in electroreceptors and in afferent nerves of both ampullary and type I and type II tuberous organs. All cell bodies within the anterior lateral line nerve ganglion were positive for NADPH-d activity, as were the primary afferent axons and termination fields in the medullary electrosensory lateral line lobe. In the corpus cerebelli and valvula cerebelli, NADPH-d label was present in Purkinje cell somata, mossy fiber synaptic glomeruli, granule cells, and parallel fibers. In the midbrain, NADPH-d activity was apparent in layer VIIIB of the torus semicircularis dorsalis and in electrosensory laminae of the optic tectum. NADPH-d was particularly associated with diencephalic electrosensory and electromotor nuclei, including the prepacemaker nucleus, the nucleus subelectrosensorius, and the central posterior nucleus of the thalamus. Intense NADPH-d activity was present in pacemaker and relay cells of the medullary pacemaker nucleus but was absent from a novel class of smaller cells in this structure. Relay cell axons and spinal electromotor neurons and their axons within the electric organ were positive for NADPH-d activity. These results indicate that putative nitric oxide synthase-containing neurons in Apteronotus are localized preferentially to electrosensory and electromotor structures, suggesting a role for nitric oxide in determining the activity of cells involved in detecting or generating weakly electric fields.

Distribution of omega-Conotoxin GVIA binding sites in teleost cerebellar and electrosensory neurons.

The distribution of omega-Conotoxin GVIA (CgTx) binding sites was used to localize putative N-type Ca2+ channels in an electrosensory cerebellar lobule, the eminentia granularis pars posterior, and in the electrosensory lateral line lobe of a gymnotiform teleost (Apteronotus leptorhynchus). The binding sites for CgTx revealed by an anti-CgTx antibody had a consistent distribution on somatic and dendritic membranes of specific cell types in both structures. The distribution of CgTx binding was unaffected by co-incubation with nifedipine or AgaToxin IVA, blocking agents for L- and P-type Ca2+ channels, respectively. Incubation with CgTx in the presence of varying levels of extracellular Ca2+ altered the number but not the cell types exhibiting immunolabel. A punctate immunolabel was detected on somatic membranes of granule and stellate cell interneurons in both the eminentia granularis pars posterior and the electrosensory lateral line lobe. Punctate CgTx binding sites were also present on spherical cell somata and on the large presynaptic terminals of primary afferents that terminate on spherical cells in the electrosensory lateral line lobe. No label was detected in association with distal dendritic membranes of any cell class, or with parallel fibers in the respective molecular layers. Binding sites for CgTx in the eminentia granularis are consistent with the established role for N-type Ca2+ channels in cell migrations, an activity which is known to persist in this layer in adult Apteronotus. The distribution of labeled stellate cells with respect to topographic maps in the electrosensory lateral line lobe further suggest that N-type Ca2+ channels are expressed in relation to functional activity across these sensory maps.

A prominent soma-dendritic distribution of Kv3.3 K+ channels in electrosensory and cerebellar neurons.

The expression pattern and subcellular distribution of a teleost homologue of the mammalian Kv3.3 potassium channel, AptKv3.3, was examined in the electrosensory lateral line lobe (ELL) and two cerebellar lobes in the hindbrain of the weakly electric gymnotiform Apteronotus leptorhynchus. AptKv3.3 expression was brain specific, with the highest level of expression in the cerebellum and 56% relative expression in the ELL. In situ hybridization revealed that AptKv3.3 mRNA was present in virtually all cell classes in the ELL as well as in the cerebellar lobes eminentia granularis pars posterior (EGp) and corpus cerebellum (CCb). Immunocytochemistry indicated a distribution of AptKv3.3 channels over the entire soma-dendritic axis of ELL pyramidal, granule, and polymorphic cells and over the soma and at least proximal dendrites (100 microm) of multipolar cells and neurons of the ventral molecular layer. AptKv3.3 immunolabel was present at the soma of cerebellar granule, golgi, eurydendroid, and CCb Purkinje cells, with an equally intense label throughout the dendrites of CCb Purkinje cells and EGp eurydendroid cells. Immunolabel was virtually absent in afferent or efferent axon tracts of the ELL but was detected on climbing fiber axons and on the axons and putative terminal boutons of CCb Purkinje cells. These data reveal a prominent soma-dendritic distribution of AptKv3.3 K+ channels in both principal output and local circuit neurons, a pattern that is distinct from the soma-axonal distribution that characterizes all other Kv3 K+ channels examined to date. The widespread distribution of AptKv3.3 immunolabel in electrosensory cells implies an important role in several aspects of signal processing.

Tissue printed cells from teleost electrosensory and cerebellar structures.

A modification of the tissue printing technique was used to acutely isolate and culture cells from the electrosensory lateral line lobe (ELL), corpus cerebelli (CCb), and eminentia granularis pars posterior (EGp) of the adult weakly electric fish, Apteronotus leptorhynchus. Cells were isolated without the use of proteolytic enzymes and tissue printed as a monolayer onto glass coverslips through centrifugation in the presence of a medium designed to preserve cell structure. Tissue printed cells were reliably distributed in an organotypic fashion that allowed for the identification of anatomical boundaries between the ELL and cerebellar regions, distinct sensory maps in the ELL, and specific cell laminae. Many cells were isolated with an excellent preservation of soma-dendritic structure, permitting direct identification of all electrosensory cell classes according to morphological or immunocytochemical criteria. Several classes of glial cells were isolated, including small diameter microglia and the complex arborizations of oligodendrocytes. A plexus of fine processes were often isolated in conjunction with cell somata and dendrites, potentially preserving synaptic contacts in vitro. In particular, immunolabel for gamma-aminobutyric acid (GABA) revealed a previously unrecognized network of GABAergic axonal processes in the CCb and EGp granule cell body and molecular layers. Tissue printed cells were readily maintained with an organotypic distribution of glial and neuronal elements for up to 27 days in culture. This procedure will allow for the isolation of electrosensory cells from adult central nervous system for electrophysiological analyses of membrane properties or synaptic interactions between identified cells.

Nitric oxide synthase in tiger salamander retina.

Previous studies have indicated that nitric oxide, a labile freely diffusible biological messenger synthesized by nitric oxide synthase, may modulate light transduction and signal transmission in the retina. In the present work, the large size of retinal cells in tiger salamander (Ambystoma tigrinum) allowed the utilization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and nitric oxide synthase immunocytochemistry to delineate the cell-specific intracellular localization of nitric oxide synthase. NADPH-diaphorase activity was highly concentrated in the outer retina, in rod and cone inner segment ellipsoids, and between and adjacent to the photoreceptor cell bodies in the outer nuclear layer. Examination of enzymatically isolated retinal cells indicated that outer nuclear layer NADPH-diaphorase activity was localized to the distal processes of the retinal glial (Müller) cells and to putative bipolar cell Landolt clubs. Less intense NADPH-diaphorase activity was seen in the photoreceptor inner segment myoid region, in a small number of inner nuclear layer cells, in cap-like configurations at the distal poles of cells in the ganglion cell layer and surrounding ganglion cell layer somata, and in punctate form within both plexiform layers, the pigment epithelium, and the optic nerve. Nitric oxide synthase-like immunoreactivity was similarly localized, but was also concentrated along a thin sublamina centered within the inner plexiform layer. The potential for nitric oxide generation at multiple retinal sites suggests that this molecule may play a number of roles in the processing of visual information in the retina.

2-Aryloxymethyl-2,3,5,6-tetrahydro-1,4-oxazines, a new class of antidepressants.

Some 2-aryloxymethyl-2,3,5,6-tetrahydro-1,4-oxazines have been shown to possess marked antidepressant activity. The 1,4-oxazines were synthesized by lithium aluminum hydride reduction of the readily available 6-aryloxymethyl-2,3,5,6-tetrahydro-1,4-oxazin-3-ones. High antidepressant activity was associated with ortho substitution of the 2-phenoxymethyl group and with 1,4-oxazines devoid of 4-substituents.

Apical dendritic depolarizations and field interactions evoked by stimulation of afferent inputs to rat hippocampal CA1 pyramidal cells.

The relationship between orthodromic extracellular field potentials and intradendritic depolarizations in apical dendrites of CA1 pyramidal neurons was investigated using the in vitro slice preparation of rat hippocampus. Orthodromic synaptic field potentials evoked by stimulation of afferent inputs in stratum radiatum or stratum oriens were used to measure extracellular voltage gradients generated over the pyramidal cell axis. Extracellular gradients were of opposite polarity over the region of pyramidal cell apical dendrites in stratum radiatum. The stratum radiatum-evoked gradient was negative towards the apical dendrites and the stratum oriens-evoked gradient negative towards the cell body layer, with gradients reaching values of up to 50 mV/mm over the apical dendritic axis. Intradendritic recordings obtained greater than 150 microns from stratum pyramidale directly measured the subthreshold apical dendritic excitatory postsynaptic potentials evoked by stratum radiatum or stratum oriens stimulation. These ground-referenced recordings were then compared to the transmembrane potential calculated by subtraction of the corresponding extradendritic field potential. Both stratum radiatum and stratum oriens stimulation evoked graded excitatory postsynaptic potentials that could be recorded in apical dendritic impalements up to 265 microns from stratum pyramidale. The calculated transmembrane potential of the stratum radiatum-evoked excitatory postsynaptic potential had a significantly greater rate of rise, peak amplitude, and rate of decay than that of the ground-referenced excitatory postsynaptic potential. In contrast, the rates of rise and decay of the transmembrane potential of the stratum oriens-evoked excitatory postsynaptic potential were reduced with respect to the ground-referenced recording. The peak amplitude of the stratum oriens-evoked transmembrane potential, however, varied according to the polarity of the corresponding extradendritic population spike response recorded in stratum radiatum. These data reveal that synaptic activation of either basal or apical dendrites of CA1 pyramidal cells evokes a depolarization that can be recorded over a substantial region of the apical dendritic arbor. Furthermore, extradendritic field potentials evoked by stimulation of these inputs produce opposite effects on the transmembrane potential of apical dendrites. The magnitude of the accompanying extracellular voltage gradients suggest that these shifts in transmembrane potential reflect ephaptic interactions at the apical dendritic level of pyramidal cells.

Calcium-induced long-term potentiation in the hippocampus.

The effect of a transient increase in extracellular calcium concentration on the Schaffer collateral-commissural evoked excitatory postsynaptic potential and population spike responses of CAI pyramidal neurons was investigated using the rat in vitro hippocampal slice preparation. Brief exposure of slices (5-10 min) to twice the normal concentration of calcium (4 mM) induced a marked potentiation of both the excitatory postsynaptic potential and population spike that could persist for at least 3 h. No long-term changes were observed in either the presynaptic fiber volley of antidromically evoked CAI population spike, indicating that the potentiation could not be attributed to an increase in the number of fibers activated or a generalized increase in cellular excitability. The response of CAI pyramidal neurons to the iontophoretic application of L-glutamate in the apical dendritic zone was also unaffected after exposure to high calcium perfusate, suggesting a lack of alteration in membrane excitability or receptor sensitivity restricted to the region of synaptic input. In addition, total intracellular calcium content of individual slices, measured by atomic absorption spectrophotometry, was significantly increased for at least 1 h following return to the control medium. These data indicate that brief exposure of in vitro hippocampal slices to a high extracellular calcium concentration results in a long-term increase in synaptic efficacy which is similar in many respects to long-term potentiation induced by tetanic stimulation of hippocampal excitatory afferents. The results further suggest that the mechanisms underlying calcium-induced long-term potentiation may reside in presynaptic components and involve an enhanced transmitter release.

Fast pre-potential generation in rat hippocampal CA1 pyramidal neurons.

Small all-or-none pre-potentials have been shown under some conditions to underlie antidromic and orthodromic spike discharge in somatic recordings of hippocampal pyramidal neurons [Andersen P. and Lomo T. (1966) Expl Brain Res. 2, 247-260; Kandel E. R. et al. (1961) J. Neurophysiol. 24, 225-242; Schwartzkroin P. A. (1977) Brain Res. 128, 53-68; Spencer W. A. and Kandel E. R. (1961) J. Neurophysiol. 24, 272-285]. These potentials are taken to reflect spike discharge in distant regions of the cell (axonal or dendritic) [Andersen P. and Lomo T. (1966) Expl Brain Res. 2, 247-260; Kandel E. R. and Spencer W. A. (1961) Ann. N. Y. Acad. Sci. 94, 570-603; Schwartzkroin P. A. (1977) Brain Res. 128, 53-68] or electronic spike conduction across a gap junction between neighboring pyramidal cells [Dudek F. E. et al. (1983) In Basic Mechanisms of Neural Hyperexcitability, pp. 31-73]. The present study compared pre-potentials recorded at the somatic and dendritic levels and used restricted applications of tetrodotoxin to examine the relationship between pre-potentials and Na+ spike discharge. Intrasomatic and intradendritic recordings were obtained from CA1 pyramidal neurons of rat hippocampal slices maintained in vitro. Orthodromic and antidromic spike discharge was evoked by stimulation of afferent fibers in stratum radiatum and pyramidal cell axons in the alveus, respectively. Focal pressure application of tetrodotoxin in the immediate vicinity of somatic or dendritic recordings uncovered pre-potentials following blockade of antidromic spike discharge. Blockade of these pre-potentials required the diffusion of tetrodotoxin to a location remote from the recording site. Focal application of tetrodotoxin in the cell body layer reliably uncovered orthodromic pre-potentials at the soma only when stimulus intensity was raised beyond threshold for somatic spike discharge; e.g. to intensities shown to initiate spike discharge in apical dendritic locations [Turner R. W. et al. (1991) J. Neurosci. 11, 2270-2280]. These data provide evidence that propagation of a Na+ spike over the pyramidal cell axis is preceded by a depolarization in the form of a pre-potential. The uncovering of orthodromic somatic pre-potentials by tetrodotoxin during suprathreshold activation further supports the proposal [Spencer W. A. and Kandel E. R. (1961) J. Neurophysiol. 24, 272-285] that dendritic spike discharge [Turner R. W. et al. (1991) J. Neurosci. 11, 2270-2280] can underlie fast pre-potential generation in pyramidal cell somata.

Complex mixtures in industrial workspaces: lessons for indoor air quality evaluations.

Acceptable occupational exposure levels for hundreds of airborne concentrations of dusts, vapors, fumes, and gases have been set by consensus organizations and regulatory bodies for decades. These levels have established tremendous precedent and are tempting reference values in the relatively new field of indoor air quality evaluations where validated criteria are greatly needed. The American Conference of Government Industrial Hygienists (ACGIH) has been the most visible and productive group setting these guidelines for industrial exposure. The ACGIH Chemical Substances Committee has published an annual list of threshold limit values (TLVs) for more than 40 years. Currently the list covers more than 400 substances. In 1989, the Occupational Safety and Health Administration (OSHA) published updated permissible exposure limits (PELs) for approximately 600 substances. Most PELs before this update were adopted from the 1968 ACGIH list of TLVs and consensus standards of the American Standards Association. This OSHA update has resulted in reductions of 212 PELs and the addition of 164 new levels. The magnitude of the problem of protecting workers can be seen by the small fraction that the OSHA PELs represent of the more than 60,000 entries in the National Institute for Occupational Safety and Health's Registry of Toxic Effects of Chemical Substances. None of these levels, whether guidelines or regulatory requirements, are established based on any possible synergistic effect with other chemicals. The only guidance given by the ACGIH for synergistic effects is that such cases must be determined individually. Clearly, there are major drawbacks in using occupational standards and guidelines for evaluating the health effects of chemical agents that can be found in office settings, often in concentrations orders of magnitude less than what is routinely measured in the workplace. These guidelines are even less valuable when the concern is the complex mixing of chemicals in nonoccupational environments.

Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea.

The presence and distribution of nitric oxide synthase (NOS) in the CNS and peripheral organs (buccal muscles, oesophagus, salivary glands, foot, mantle and pneumostome) of the pulmonate mollusc, Lymnaea stagnalis were studied using an antiserum developed against rat cerebellar NOS. NOS-immunopositive neurones in Lymnaea were localized predominantly in the buccal ganglia as well as in distinct areas of the cerebral and suboesophageal ganglia. NOS-immunoreactive terminals were also found on the somata of some central neurones. In the periphery, NOS-immunostaining was detected only in a few neurones in the pneumostome area and in the osphradial ganglion. In addition, approximately 100 NOS-immunopositive cells have been found in the salivary glands. Our data supports other recent reports indicating that NO may be a signal molecule in the CNS of molluscs.

A technique for the primary dissociation of neurons from restricted regions of the vertebrate CNS.

Acute isolation of vertebrate neurons has been used extensively to characterize membrane properties in the absence of circuit connections or extensive dendritic arborizations. We describe a technique that allows cells to be dissociated from anatomically defined regions of a tissue slice at a resolution beyond that attainable by micro-dissection. Dissociation is performed by using a fire-polished electrode with a tip diameter of 40-100 microns connected by tubing to a micrometer syringe that allows graded levels of positive or negative pressure to be applied at the electrode tip. The electrode tip is placed under microscopic observation upon a cell group within an enzymatically treated slice and negative pressure is applied to dissociate cells into the electrode shaft. Positive pressure is used to eject the cells onto the surface of poly-L-lysine-coated glass coverslips. We have used this technique to dissociate and culture cells from specific laminae of separate sensory maps in a medullary nucleus of adult weakly electric fish. Isolated cells were viable, could be identified by morphological criteria, and exhibited process extension within 2 h of plating. This technique greatly increases the probability of isolating morphologically identifiable vertebrate neurons for electrophysiological analysis or for the reconstruction of neural circuits in vitro.

Experience with Devine-Horton dermal patch graft for Peyronie's disease.

Peyronie's disease is a malady frequently seen by the urologist. Many conservative treatment modalities have yeilded successful alleviation of symptoms in a significant number of these patients. A number, however, do not respond and are left with penile curvature and pain on erection such that intercourse is severely impaired. Devine and Horton have proposed resection of the plaque with replacement of the defect with a full-thickness dermal graft. They reported excellent results with minimal complications. Five patients were treated by this technique with successful penile straightening and alleviation of pain. To date, follow-up results have been gratifying, and the Devine-Horton dermal graft technique is suggested for use in treatment of patients with disabling Peyronie's disease.