Article Figures & Data
Figures
- Fig. 1.
Anatomy of B2 and B7nor neurons in the buccal ganglia. A, NADPH-diaphorase staining of the buccal ganglia. The pair of large (cell body, ∼50 μm in diameter) diaphorase-positive cell bodies (arrows) are theleft and right B2 neurons (B2L , B2R ), identified as NOS-expressing by Moroz et al. (1994a,b). There are also other unidentified positively stained neurons in the ganglia.B, A schematic diagram of the buccal ganglia and the positions of the cell bodies of neurons referred to in this article together with their main projections. The main axon branch of B2 leaves the ganglion via the dorsobuccal nerve (DBN), whereas the axon of the B7nor neuron (cell body, filled circle) projects to the posterior jugalis nerve (PJN). The overlapping aborizations shown by the double-dye fills in D are near the midline of the ganglion in the area covered by the shaded rectangle.A, Anterior; BC, buccal commissure;CBC, cerebral-buccal connective; P, posterior; and SO, slow oscillator. C, Double-dye injection of the B2 and the identified follower neuron B7nor. In this experiment, the B2 neuron was injected with MPTS (blue), and the B7nor neuron was injected with 5-CF (yellow). Here the two cell bodies are apposed, but the position of the B7nor neuron is quite variable and can lie as much as 100 μm from the presynaptic B2 cell body. D, Detail of the overlapping fields of arborization of the B2 (blue) and B7nor (yellow) neurons whose cell bodies are shown in C.
- Fig. 2.
Transmission between B2 and B7nor and the activity of B7nor during fictive feeding. A, In physiological saline, brief bursts of B2 activity, but not single action potentials, typically elicit a slow depolarization in the follower neuron (upper) that can reach spiking threshold (lower). B, In HiDi saline, B2 action potentials produce constant latency, one-to-one unitary EPSPs in the B7nor neuron. The oscilloscope was triggered from the rising phase of the B2 action potentials, and five sweeps are superimposed.C, Simultaneous recordings from the B2, B7nor, and SO neurons are shown. The feeding CPG is activated by depolarization of the SO. Note that rhythmic activity in B7nor is entrained to the feeding CPG. A brief burst of spikes occurs during the protraction phase (P); the neuron is inhibited during rasping (R) and recovers in the swallowing phase (S). In this example, the B2 neuron is only weakly active. Although single B2 action potentials occur at irregular intervals throughout the record, note that bursts of two to four B2 action potentials always occur at the end or just after the rasp phase.
- Fig. 3.
Effect of NOS inhibitors on transmission from B2 to B7nor in the intact CNS. A, Treatment withl-NNA (1 mm; shaded box) causes a rapid decline in the amplitude of the EPSP that in the experiment illustrated is completely eliminated in ∼15 min and did not recover during the 1 hr experiment. Sample intracellular recordings (insets) from the two neurons are shown before and at the end of the l-NNA application. The current injected into the presynaptic B2 neuron was the same in each test and produced approximately equivalent bursts of B2 action potentials.B, l-NAME (1 mm) treatment also reduces the amplitude of the B2-induced EPSP, but in comparison withl-NNA, the effect is slower and incomplete, declining in this example to approximately one-third of the control value in 25 min. C, A 27 min exposure to d-NAME (1 mm) has no effect on EPSP amplitude in this experiment. Note that the data in C is continuous with and precedes data taken from the same preparation shown in B. Thus the d-NAME treatment was followed by the superfusion of l-NAME. D, A summary of the effects of NOS inhibitors expressed as a percentage of control (pretreatment EPSP amplitude) ± SEM is shown. On average,l-NNA (1 mm) causes a decrease of the EPSP amplitude to 2 ± 1% (n = 3) of the control value. l-NAME (1 mm) reduced the EPSP amplitude to 51 ± 8% (n = 4) of the control.d-NAME has no significant effect (94 ± 4%;n = 3).
- Fig. 4.
The NO scavenger PTIO produces a reversible suppression of the B2-induced EPSP in B7nor in the intact CNS.A, A sample treatment with PTIO (0.25 mm) in which PTIO causes a rapid decrease in the EPSP amplitude and completely abolishes the depolarization. The block is readily reversed after removal of PTIO from the bath. B, Summary of the PTIO experiments with average changes given as percentages ± SEM. PTIO treatment blocks 99 ± 1% (n = 3) of the transmission, but the EPSP amplitude shows an almost complete recovery after washout of PTIO and returns to 93 ± 16% (n = 3) of the control value.
- Fig. 5.
Focal application of NO-containing solutions to the B7nor neuron in the intact CNS. A, Effect of NO-saturated saline (pipette concentration, <2 mm; see Materials and Methods) ejected by a train of pressure pulses of 200 msec duration at 1 Hz for the period indicated by the horizontal line (upper trace). The pipette tip was placed ∼50 μm from the cell body of the B7nor neuron. The NO-saturated saline caused a depolarization accompanied by a prolonged burst of B7nor action potentials. The lower trace shows that the ejection of saline without NO has no effect. B, Effect of a single pulse of fresh SNC at 100 mm (upper trace) or of a degassed and nitrogen-purged solution of SNC (lower trace). Fresh SNC caused a strong depolarization of B7nor that triggered the generation of a burst of activity. In contrast, degassed SNC had no consistent effect. Ci, Effect of single pressure pulses of SNAP and NAP. SNAP (50 mm; 40 msec) but not NAP (50 mm; 40 msec) caused a consistent depolarization in B7nor. Cii, Effect of a brief burst of B2 action potentials. In the same preparation shown in Ci, this burst causes the expected depolarizing response in B7nor that is directly comparable with the effect of SNAP (also see Fig. 11 for the effect of SNAP on isolated cultured neurons).
- Fig. 6.
Reconstitution of the B2–B7nor interaction in cell culture. A, Photomicrograph of identified B2 and B7nor neurons after 1 d in cell culture is shown. Both neurons have regenerated extensive new processes and have already established contact after their isolation from the buccal ganglion. Note the relatively large growth cones at the tips of the processes.B, In the intact CNS before their isolation, a burst of five action potentials in the B2 neuron (∼10 Hz) triggered by current injection caused a slow depolarization of the B7nor neuron that began ∼200 msec after the start of B2 activity and peaked at ∼0.7 sec.C, Two days after the isolation of the same two neurons, an excitatory connection was restored in cell culture. The injection of a depolarizing current into the isolated B2 neuron caused a burst of five action potentials but at a lower frequency (∼2.5 Hz). The B2 activity produced a slow depolarization in the B7nor neuron that peaked ∼2.5 sec after the start of B2 activity. Weak, brief depolarizing events (indicated by small arrows) are caused by electrotonic coupling between B2 and B7nor and are seen in approximately one-half of the cocultured preparations examined (see also Figs. 7, 9-11). The start and the end of the current injection are indicated by up and down arrowheads, respectively, inB and C. The records in the intact CNS and in cell culture are shown at the same scale. The vertical bar is equivalent to 5 and 40 mV for the B7nor and B2 recordings, respectively.
- Fig. 7.
Relationship between the number of B2 action potentials and the size of the B7nor response in cell culture.Ai–Aiv, Incremental length, constant amplitude current pulses were injected into a B2 neuron to trigger one, two, three, or four action potentials. The start and the end of each current pulse are indicated by the up and down arrowheads, respectively. B2 activation caused a slow depolarization in B7nor that summates with increasing numbers of B2 action potentials. In addition to the slow depolarization, each individual B2 action potential also caused a brief depolarization of the B7nor membrane potential because of electrotonic coupling. All records are shown at the same scale.B, Plotting the amplitude of the B7nor EPSP against the number of B2 action potentials showed a linear increase in EPSP amplitude.
- Fig. 8.
Anomalous cholinergic transmission from B2 to B7nor neurons in culture. A, Biphasic response of a B7nor neuron to B2 activity observed in a pair of neurons that had established soma–soma contact in cell culture is shown. Each of the two B2 action potentials that were triggered by the injection of a constant positive current pulse elicited a fast unitary IPSP in B7nor followed by a slower, delayed depolarization. B, Perfusing the bath with d-TC (0.1 mm) completely abolished the fast IPSPs but had no significant effect on the amplitude of the slow depolarization. C, The fast IPSP was restored when the d-TC was exchanged for l-NAME (1 mm), which blocked the slow depolarization. The start and end of the current pulses that triggered B2 activity are indicated by upand down arrowheads, respectively.
- Fig. 9.
Effect of d- and l-NAME on the interaction between B2 and B7nor neurons in culture.Ai–Aiv, The injection of a constant current 1 sec pulse into a B2 neuron triggered a reproducible burst of three presynaptic action potentials. Electrotonic coupling between B2 and B7nor produced the initial fast one-to-one depolarizations of the B7nor membrane potential that were followed by a slowly developing depolarization.Aii, The amplitude of the slow EPSP was not effected by the application of d-NAME (0.1 mm) for 35 min.Aiii, The application of l-NAME (0.1 mm) for 45 min reduced the slow EPSP amplitude from 6.9 to 4.3 mV. Aiv, After a 40 min washout ofl-NAME, the EPSP amplitude had returned to 6.2 mV. All records are shown at the same scale. Up and down arrowheads indicate the start and end of the current pulse.B, Plot of the amplitude of B7nor EPSPs against time for the experiment shown in Ai–Aiv illustrates the time course of the effects of d- and l-NAME applications. Each data point represents the B7nor response to a single burst of three B2 action potentials. The individual bursts were triggered at 5 min intervals. The period of d- andl-NAME application is indicated by a shaded box. C, Summary of three experiments showing the average changes in B7nor amplitude caused by applications ofd- and l-NAME (both at 0.1 mm) is shown. The average changes are given as percentages (± SEM) of the control response measured before the application of any drug.
- Fig. 10.
Effect of PTIO on the interaction between B2 and B7nor neurons in culture. Ai–Aiii, The injection of a constant current 2 sec pulse into a B2 neuron triggered a reproducible burst of four action potentials. Electrotonic coupling between B2 and B7nor produced fast one-to-one depolarizations of the B7nor neuron that are followed by a slowly developing depolarization that triggered a burst of action potentials in B7nor. Aii, The slow EPSP was abolished by the application of PTIO (0.25 mm) for 5 min. Aiii, After a 13 min washout of PTIO, the amplitude of the slow EPSP had recovered sufficiently to trigger another burst of action potentials in B7nor. All records are shown at the same scale.Up and down arrowheads indicate the start and finish of the current pulse. B, Plot of the amplitude of B7nor EPSPs against time for the experiment shown inA is presented. Note the rapid onset of the PTIO block and its ready reversal. Each data point represents the B7nor response to a single burst of four action potentials in B2. The individual bursts were triggered at 1 min intervals. The period of PTIO application is indicated by the shaded box.C, Summary of three experiments showing the average changes in B7nor amplitude caused by the applications of PTIO (0.25 mm) is presented. The average changes are given as percentages (± SEM) of the control response measured before the application of PTIO.
- Fig. 11.
Effect of SNAP and NAP on an isolated B7nor neuron and comparison with reconstituted B2–B7nor synaptic interaction. A, Focal application (horizontal line) of a pulse of SNAP (1 mm; 1 sec) caused a depolarization of the B7nor membrane potential that triggered the generation of a burst of action potentials. B, The application of a similar pulse of NAP (1 mm; 1 sec) had no effect on the membrane potential of the same B7nor. C, Activity in a B2 neuron that had grown overlapping processes with a B7nor neuron in cell culture depolarized the B7nor neuron and triggered a burst of action potentials. The time course of the depolarization is comparable with the effect of SNAP shown in A. B2 activity was triggered by the injection of a depolarizing current (start and end indicated by up and down arrowheads, respectively). D, Hyperpolarization of the membrane potential of an isolated B7nor caused a reduction in the response to the application of SNAP (1 mm; 1 sec). A linear relationship existed between the amplitude of the SNAP response and the membrane potential (data not shown). E, Recordings from a pair of B2 and B7nor neurons that had established physical contact in cell culture are shown. A series of B2 action potentials was triggered by the injection of a constant depolarizing current pulse, the start and end of which are indicated by theup and down arrowheads, respectively. Each B2 action potential was followed by a fast PSP in B7nor that was caused by electrotonic coupling between the two neurons. (Electrotonic coupling between the neurons was tested for by injection of a series of constant negative current pulses in either of the two neurons; data not shown.) The fast electrotonic PSPs were followed by a slow depolarization that decreased in amplitude when the membrane potential was hyperpolarized.
- Fig. 12.
Nonsynaptic interaction between B2 and B7nor neurons in culture. A–C, Strong bursts of action potentials were triggered in a B2 neuron by injecting a constant, strong 10 sec current pulse. All records are shown at the same scale.Up and down arrowheads indicate the start and finish of the current pulse. The inserts show a schematic representation of the recording configuration (not drawn to scale). A, At a distance of ∼40 μm between the B2 and B7nor soma, the burst of B2 action potentials caused a weak depolarization with a peak amplitude of 2.2 mV. B, Manipulating the same B2 neuron with the recording electrode into a position ∼10 μm from the B7nor soma resulted in an increase of the depolarization, which was caused by the B2 burst, to 7.2 mV.C, Approaching the B2 neuron further, so that it was in direct contact with the B7nor soma, increased the EPSP amplitude to 10.5 mV.
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