Sensory regulation of network components underlying ciliary locomotion in Hermissenda.

Ciliary locomotion in the nudibranch mollusk Hermissenda is modulated by the visual and graviceptive systems. Components of the neural network mediating ciliary locomotion have been identified including aggregates of polysensory interneurons that receive monosynaptic input from identified photoreceptors and efferent neurons that activate cilia. Illumination produces an inhibition of type I(i) (off-cell) spike activity, excitation of type I(e) (on-cell) spike activity, decreased spike activity in type III(i) inhibitory interneurons, and increased spike activity of ciliary efferent neurons. Here we show that pairs of type I(i) interneurons and pairs of type I(e) interneurons are electrically coupled. Neither electrical coupling or synaptic connections were observed between I(e) and I(i) interneurons. Coupling is effective in synchronizing dark-adapted spontaneous firing between pairs of I(e) and pairs of I(i) interneurons. Out-of-phase burst activity, occasionally observed in dark-adapted and light-adapted pairs of I(e) and I(i) interneurons, suggests that they receive synaptic input from a common presynaptic source or sources. Rhythmic activity is typically not a characteristic of dark-adapted, light-adapted, or light-evoked firing of type I interneurons. However, burst activity in I(e) and I(i) interneurons may be elicited by electrical stimulation of pedal nerves or generated at the offset of light. Our results indicate that type I interneurons can support the generation of both rhythmic activity and changes in tonic firing depending on sensory input. This suggests that the neural network supporting ciliary locomotion may be multifunctional. However, consistent with the nonmuscular and nonrhythmic characteristics of visually modulated ciliary locomotion, type I interneurons exhibit changes in tonic activity evoked by illumination.

Neocortical hyperexcitability defect in a mutant mouse model of spike-wave epilepsy, {\it stargazer}

Single-locus mutations in mice can express epileptic phenotypes and provide critical insights into the naturally occurring defects that alter excitability and mediate synchronization in the central nervous system (CNS). One such recessive mutation (on chromosome (Chr) 15), stargazer(stg/stg) expresses frequent bilateral 6-7 cycles per second (c/sec) spike-wave seizures associated with behavioral arrest, and provides a valuable opportunity to examine the inherited lesion associated with spike-wave synchronization.^ The existence of distinct and heterogeneous defects mediating spike-wave discharge (SWD) generation has been demonstrated by the presence of multiple genetic loci expressing generalized spike-wave activity and the differential effects of pharmacological agents on SWDs in different spike-wave epilepsy models. Attempts at understanding the different basic mechanisms underlying spike-wave synchronization have focused on $\gamma$-aminobutyric acid (GABA) receptor-, low threshold T-type Ca$\sp{2+}$ channel-, and N-methyl-D-aspartate receptor (NMDA-R)-mediated transmission. It is believed that defects in these modes of transmission can mediate the conversion of normal oscillations in a trisynaptic circuit, which includes the neocortex, reticular nucleus and thalamus, into spike-wave activity. However, the underlying lesions involved in spike-wave synchronization have not been clearly identified.^ The purpose of this research project was to locate and characterize a distinct neuronal hyperexcitability defect favoring spike-wave synchronization in the stargazer brain. One experimental approach for anatomically locating areas of synchronization and hyperexcitability involved an attempt to map patterns of hypersynchronous activity with antibodies to activity-induced proteins.^ A second approach to characterizing the neuronal defect involved examining the neuronal responses in the mutant following application of pharmacological agents with well known sites of action.^ In order to test the hypothesis that an NMDA receptor mediated hyperexcitability defect exists in stargazer neocortex, extracellular field recordings were used to examine the effects of CPP and MK-801 on coronal neocortical brain slices of stargazer and wild type perfused with 0 Mg$\sp{2+}$ artificial cerebral spinal fluid (aCSF).^ To study how NMDA receptor antagonists might promote increased excitability in stargazer neocortex, two basic hypotheses were tested: (1) NMDA receptor antagonists directly activate deep layer principal pyramidal cells in the neocortex of stargazer, presumably by opening NMDA receptor channels altered by the stg mutation; and (2) NMDA receptor antagonists disinhibit the neocortical network by blocking recurrent excitatory synaptic inputs onto inhibitory interneurons in the deep layers of stargazer neocortex.^ In order to test whether CPP might disinhibit the 0 Mg$\sp{2+}$ bursting network in the mutant by acting on inhibitory interneurons, the inhibitory inputs were pharmacologically removed by application of GABA receptor antagonists to the cortical network, and the effects of CPP under 0 Mg$\sp{2+}$aCSF perfusion in layer V of stg/stg were then compared with those found in +/+ neocortex using in vitro extracellular field recordings. (Abstract shortened by UMI.) ^

5-HT and GABA modulate intrinsic excitability of type I interneurons in Hermissenda.

The sensory neurons (photoreceptors) in the visual system of Hermissenda are one site of plasticity produced by Pavlovian conditioning. A second site of plasticity produced by conditioning is the type I interneurons in the cerebropleural ganglia. Both photoreceptors and statocyst hair cells of the graviceptive system form monosynaptic connections with identified type I interneurons. Two proposed neurotransmitters in the graviceptive system, serotonin (5-HT) and gamma-aminobutyric acid (GABA), have been shown to modify synaptic strength and intrinsic neuronal excitability in identified photoreceptors. However, the potential role of 5-HT and GABA in plasticity of type I interneurons has not been investigated. Here we show that 5-HT increased the peak amplitude of light-evoked complex excitatory postsynaptic potentials (EPSPs), enhanced intrinsic excitability, and increased spike activity of identified type I(e(A)) interneurons. In contrast, 5-HT decreased spike activity and intrinsic excitability of type I(e(B)) interneurons. The classification of two categories of type I(e) interneurons was also supported by the observation that 5-HT produced opposite effects on whole cell steady-state outward currents in type I(e) interneurons. Serotonin produced a reduction in the amplitude of light-evoked complex inhibitory PSPs (IPSPs), increased spontaneous spike activity, decreased intrinsic excitability, and depolarized the resting membrane potential of identified type I(i) interneurons. In contrast to the effects of 5-HT, GABA produced inhibition in both types of I(e) interneurons and type I(i) interneurons. These results show that 5-HT and GABA can modulate the intrinsic excitability of type I interneurons independent of the presynaptic effects of the same transmitters on excitability and synaptic efficacy of photoreceptors.

Convergent and divergent modulatory pathways in {\it Aplysia\/}: Cellular and network studies

Neuromodulation is essential to many functions of the nervous system. In the simple gastropod mollusk Aplysia californica, neuromodulation of the circuits for the defensive withdrawal reflexes has been associated with several forms of learning. In the present work, the neurotransmitters and neural circuitry which contribute to the modulation of the tail-siphon withdrawal reflex were examined.^ A recently-identified neuropeptide transmitter, buccalin A was found to modulate the biophysical properties of the sensory neurons that mediate the reflex. The actions of buccalin A on the sensory neurons were compared with those of the well-characterized modulatory transmitter serotonin, and convergence and divergence in the actions of these two transmitters were evaluated. Buccalin A dramatically increased the excitability of sensory neurons and occluded further enhancement of excitability by serotonin. Buccalin A produced no significant change in spike duration, and it did not block serotonin-induced spike broadening. Voltage-clamp analysis revealed the currents that may be involved in the effects on spike duration and excitability. Buccalin A decreased an outward current similar to the S-K$\sp+$ current (I$\sb{\rm K,S}$). Buccalin A appeared to occlude further modulation of I$\sb{\rm K,S}$ by serotonin, but did not block serotonin-induced modulation of the voltage-dependent delayed rectifier K$\sp+$ current (I$\sb{\rm K,V}$). These results suggest that buccalin A converges on some, but not all, of the same subcellular modulatory pathways as serotonin.^ In order to begin to understand neuromodulation in a more physiological context for the tail-siphon withdrawal reflex, the modulatory circuitry for the tail-withdrawal circuit was examined. Mechanoafferent neurons in the J cluster of the cerebral ganglion were identified as elements of a modulatory circuit for the reflex. Excitatory and inhibitory connections were observed between the J cells and the pleural sensory neurons, the tail motor neurons, and several classes of interneurons for the tail-siphon withdrawal circuit. The J cells produced both fast and slow PSPs in these neurons. Of particular interest was the ability of the J cells to produce slow EPSPs in the pleural sensory neurons. These slow EPSPs were associated with an increase in the excitability of the sensory neurons. The J cells appear to mediate both sensory and modulatory inputs to the circuit for the tail-siphon withdrawal reflex from the anterior part of the animal. ^

THE INHIBITORY EFFECTS OF VSV INFECTION ON MULV PRODUCTION OCCUR BETWEEN SYNTHESIS AND RELEASE OF MULV GENOMIC RNA

The inhibitory effects of VSV infection on MuLV production were investigated using the VSV temperature-sensitive mutants t1B17(I & V), tsT1026(I), tsG22(II), and ts052(II). At the permissive temperature, all four mutants suppressed the release of virion-associated MuLV gRNA by approximately 98% within 0.5 to 2.5 hr post infection. At the restrictive temperature and in the absence of cell killing, infection with t1B17(I & V) inhibited the release of MuLV gRNA, while tsT1026(I) and tsG22(II) did not. In contrast, ts052(II) inhibited the release of MuLV gRNA and induced cell killing. During the same time period and at either temperature, all four mutants did not suppress either MuLV-associated protein release or intracellular MuLV sRNA synthesis. These results indicate that VSV inhibits MULV gRNA release at a level somewhere between the synthesis and release of newly synthesized gRNA.^

Thermo -modulation of MuSVts110 splicing by inhibitory RNA sequences

Viral systems have contributed tremendously to the understanding of eukaryotic molecular biology. The proportional pattern of retroviral RNA expression offers many clues into the alternative splicing of cellular transcripts. The MuSVts110 virus presents an unusual expression system, where the mechanistic combination of RNA splicing and cellular transformation can be physiologically manipulated. Splicing of MuSVts110 pre-mRNA occurs inefficiently (30%-50%) at 33$\sp\circ$C or below and is subdued at 39$\sp\circ$C ($<$5%). Like most alternatively spliced cellular and retroviral transcripts, the MuSVts110 pre-mRNA contains cis-acting intron and exon sequences that attenuate splicing. These include a splicing inhibitory sequence at the 3$\prime$ end of the MuSVts110 v-mos exon, called the E2 Distal Element (E2DE), and a sub-optimal 3$\prime$ splice site. The E2DE directly inhibits MuSVts110 RNA splicing in a sequence-specific fashion at 39$\sp\circ$C but not at 28$\sp\circ$C, potentially through the association of cellular factors. Inefficient MuSVts110 splicing is pre-dominantly attributed to the utilization of multiple weak branchpoint sequences located between $-113$ and $-34$ nucleotides upstream of the 3$\prime$ splice site. The molecular control of MuSVts110 splicing, represented primarily by scattered multiple inefficient branchpoint sequences that are conditionally modulated by the E2DE at higher growth temperatures, is discussed. ^