64 C
ganglia. The OC interneurons are also intrinsic neuro- 2. Materials and methods
modulators of the feeding system, because they receive regular inhibitory inputs in the N1 protraction and N2
Pond snails, Lymnaea stagnalis were obtained from rasp phases of the feeding rhythm [21].
dealers Blades Biological, Kent, UK and fed on lettuce. There are just three OC octopamine immunoreactive
The circumoesophageal and buccal ganglia were isolated interneurons in the buccal ganglia [2]. They are weakly
and neurons impaled as described previously [7,21]. The electrically coupled together and do not fire 1:1 action
large buccal motoneurons were visually identified [1] and potentials when one is stimulated. During fictive feeding,
the feeding interneurons, SO, N1L, N3p and OC were they tend to fire in the third, swallowing, phase of the
identified by probing in their usual positions, and checking rhythm. In some preparations the OC cells rarely fire
their identity by firing pattern and interneuronal con- action potentials, but usually their firing rate increases as
nections with the motoneurons [4,5,21,24]. The preparation fictive feeding is driven more strongly, with a maximum
was bathed in normal Lymnaea saline [6]. Octopamine was firing rate in the isolated central nervous system CNS
obtained from Sigma Poole, Dorset, UK. being 15 Hz. They are always robustly inhibited in the first
two phases of the rhythm protraction and rasp and these inputs are always present [20,21].
3. Experimental design
We have previously demonstrated rapidly activating, short-term intra-cyclic connections to buccal interneurons
3.1. Repetitive OC stimulation and motoneurons. The OC interneurons affect all the cells
tested with the overall effect of these transient connections We used repetitive intracellular stimulation of the OC
being to enhance the retraction of the radula and the neurons in these experiments to mimic the way it fires in
swallowing movement [20,21]. The inhibitory connections the feeding pattern. Intact Lymnaea rasp at up to 20
to the B3 motoneurons, N2 and N3p interneurons are bites min one cycle every 3–4 s with the third, swallow-
known to be octopaminergic since they are blocked by ing, phase lasting about 1 s. Passing suprathreshold
antagonists including phentolamine and epinastine [19]. depolarising current pulses at a similar rate and duration
The OC interneurons also make electrical connections to into an OC interneuron Fig. 1A produces synaptic inputs
buccal neurons, including the B4 motoneurons and the an alternating pattern of inhibition and excitation in the
N3p interneurons which participate in rhythm generation. N1L and SO neurons. These inputs increase in size with
Like the connections between the OC interneurons, these each repetition and eventually the burst of spikes in the
electrical synapses are weak and the neurons do not N1L evokes a single cycle of the feeding pattern, as shown
normally fire 1:1 action potentials. Although they are by the deep inhibitory N2 rasp phase inputs on both the
electrically coupled, the OC and N3p interneurons make N1L and SO interneurons.
different short-lasting connections to many of their follow- ers. For example the B3 motoneuron is excited by N3p [4]
but inhibited by OC [20] suggesting these interneurons do not release the same neurotransmitter.
In our experiments, we demonstrate long-term modula- tion by pre-stimulating an OC interneuron and showing
that this facilitates the ability of the SO modulatory interneuron to drive fictive feeding. The SO [16] is an
identified buccal interneuron which is excited by sucrose solutions and mechanical stimuli applied to the buccal
cavity [11]. In the isolated CNS, when stimulated by steady current, the SO will drive fictive feeding up to the
normal feeding rate mean 16, maximum 20 bites min of
Fig. 1. Comparison of pulse and tonic stimulation of OC interneuron. A
intact Lymnaea [11,21]. Small currents produce a weak
Suprathreshold pulses of stimuli five in 10 s into the OC interneuron
rhythm, with the intensity of the synaptic inputs and the
produce short-latency inputs onto the protraction phase interneurons N1L
feeding rate increasing with stimulating current. The SO is
and SO. With repetition, the size of the potential grows and eventually the
electrically coupled to another protraction phase neuro-
N1L reaches threshold and fires a burst of action potentials. This activates
modulator, the N1L interneuron [24]. Both of these
the feeding system and a single feeding cycle occurs, with a strong rasp N2 input. this is marked by an asterisk. B Tonic stimulation of the OC
neurons are strongly and reliably inhibited in the rasp N2
interneuron with the same current produces maintained, short latency
phase, so that their pattern can be used to count the feeding
inputs on the N1L and SO interneurons, but no activation of the feeding
cycles in the isolated CNS. Each inhibitory input would
system. Note that the OC firing rate over the first second of stimulation
correspond to a rasp bite performed by the intact animal
was 28 Hz in A and 30 Hz in B. Similar results were found in two
during feeding.
other preparations.
C .J.H. Elliott, A. Vehovszky Brain Research 887 2000 63 –69
65
The effects of tonically stimulating the OC is much min. In the isolated CNS, the fictive feeding pattern was
smaller than the repetitive stimulation Fig. 1B. This monitored and each N2 rasp phase input on the SO
could be because the OC →
SO and OC →
N1L connections counted as 1 bite. The N2 inputs are the only central
are biphasic, with both inhibitory and excitatory com- pattern generator inputs that inhibit the SO interneuron
ponents [20]. In Fig. 1B, the inputs to the SO and N1L do strongly enough to stop it firing.
not lead to activation of the feeding pattern. Note that the firing rate of the OC in the tonic and repetitive stimuli
Fig. 1A and b are nearly the same 25–30 spikes s.
4. Results
