PMC

Middle spike phases of the HN(15) (top) and HN(16) (bottom) interneurons are constant over a broad range of periods in the peristaltic and synchronous coordination mode. Phases are relative to the HN(4) interneuron on the peristaltic side (assigned 0 phase). Data are from the same preparations as in .

Rear HN interneurons elicit excitatory responses (dashed lines) in their contralateral homologs with a short latency indicative of electrical coupling. A: extracellular recording from the HN(L,15) interneuron during simultaneous intracellular recording from the contralateral HN(R,15) interneuron. Spikes in the HN(L,15) interneuron elicit EPSPs or spikes in the HN(R,15) with short latencies as seen in the spike-triggered averages (B). C: extracellular recording from the HN(L,15) interneuron the contralateral HN(R,15) interneuron is in voltage clamp (different preparation than in A). Holding potential: −45 mV. Spikes in the HN(L,15) interneuron elicit EPSCs or spikes in the HN(R,15), again with short latencies (D). Dashed lines indicate some of the HN(L,15)-mediated postsynaptic responses in the HN(R,15) interneuron.

Connections between the rear and the middle premotor HN interneurons. A: extracellular recording from the HN(L,7) interneuron during simultaneous intracellular recording from the rear HN(L,16) interneuron. The barrages of excitatory postsynaptic potentials (EPSPs) seen during bursting in the rear HN interneuron coincide with spikes in the HN(7) interneuron as seen in the spike-triggered averages (B). C: same preparation as in A with the HN(16) now in voltage clamp. Holding potential: −50 mV. The barrages of excitatory postsynaptic currents (EPSCs) in the rear HN interneuron coincide with spikes in the HN(7) interneuron as seen in the spike-triggered averages (D). The long latencies (B and D) reflect the conduction delays over the distance of 9 segments.

Bursting activity of the rear HN(L,15) interneuron is time-locked with the front premotor HN(L,4) interneuron and coincides with postsynaptic events in the rear HE(L,17) motor neuron as illustrated in simultaneous extracellular recordings from the HN interneurons during intracellular recording from the HE motor neuron. Inhibitory postsynaptic potentials (IPSPs) in the HE(L,17) motor neuron (A) and inhibitory postsynaptic currents (IPSCs) in the subsequent single-electrode voltage-clamp recording (B) coincide with bursts of action potentials in the HN(15) interneuron. Note that burst activity in the HN(15) interneuron is time-locked and, as seen by comparing A and B, switches phase relations with the HN(4) interneuron (slanted lines). Dotted lines in the HE motor neuron recording refer to −40-mV membrane potential (A) and 0-nA holding current (B). Holding potential: −45 mV (B).

Synaptic connections between the rear HN interneurons and the HE motor neurons. A: spikes from several bursts from a rear HN(R,15) interneuron (burgundy) were used to generate spike-triggered averages to determine the size of the IPSCs elicited in ipsilateral heart motor neurons of midbody segments 16, 17, and 18 (top). Spikes from the rear HN(L,16) interneuron (teal) were used to generate spike-triggered averages to determine the size of the IPSCs elicited in ipsilateral heart motor neurons of midbody segments 16, 17, and 18 (bottom). Recordings are from the same experiment. Arrows mark the time of the triggering spike. Latencies (in ms) of the peak average IPSC from the triggering event are noted for each heart motor neuron. The holding potentials were −45 and −40 mV in the HE(R,16) motor neuron (gray trace). B: the hemilateral circuit diagram shows the connections identified between the rear HN interneurons and the rear HE motor neurons. Symbols are as defined in .

Bilateral phase diagram of all premotor HN interneurons is shown for the peristaltic (top) and synchronous (bottom) coordination mode. Each box represents the average duty cycle of a neuron's activity with the vertical line indicating the middle spike (see for details). Absolute phase reference (phase 0) is the HN(4) interneuron on the peristaltic side. The HN(15) and HN(16) interneurons fire in phase in both coordination modes. They contribute to the rear-to-front progression in the peristaltic mode and to the front-to-rear progression in the synchronous mode. Standard colors are used for the HN interneurons (see methods). Data for the front and middle heart premotor interneurons and for the HN(X) interneuron are from . To illustrate the variability in phasing, the middle spike phases of individual rear premotor HN interneurons are shown at bottom. Middle spike phases were determined in both coordination modes for each rear HN interneuron.

Synaptic weight (expressed as conductance; see text for details) of all the premotor HN interneurons connected to the rear HE motor neurons in midbody segments 15 to 18. Data for the front and middle heart premotor interneurons are from . Standard colors are used for the HN interneurons (see methods). A: on average, synaptic weight from the front premotor HN(3) and HN(4) interneurons was weak in all 4 HE motor neurons. When the synaptic strength profiles of the HE(15) and HE(16) are compared with those of the HE(17) and HE(18) motor neurons, synaptic strengths from the middle premotor HN(6) and HN(7) interneurons fall off sharply, whereas the strengths of the rear premotor HN(15) and HN(16) interneurons increase in the HE(17) and (18) motor neurons. SD are omitted for clarity. B: synaptic weight varies in individual heart motor neurons. Each dot represents the spike-triggered average of 1 connection tested. The transparent squares are the means shown in A.

Rear premotor interneurons are coordinated with the heartbeat CPG and switch phase relations with the other HN interneurons. A: extracellular recordings from the front premotor HN(L,4) interneuron, middle premotor HN(L,6) interneuron, and both rear premotor HN interneurons, also on the left side. Dashed lines mark the position of the middle spike in the HN(4) (top trace), the reference, and are provided to facilitate observation of relative phasing. Color-coded different symbols above each burst denote the middle spikes of the other interneurons. Standard colors are used for the HN interneurons (see methods). The recording starts in the synchronous mode with the HN(4) interneuron leading, followed by the HN(6) interneuron and then by the HN(15) and HN(16) interneurons. After the switch into the peristaltic mode (arrow), the rear HN(15) and HN(16) interneurons are leading, followed by the HN(6) interneuron and, after a delay, by the HN(4) interneuron. B: the actogram demonstrates the timing relationship for the middle spike of 38 consecutive bursts across the switch between the 2 coordination modes (same symbols as in A). Note that the rear HN interneurons fire nearly in phase in both coordination modes. The shaded box represents the bursts shown in A. Time 0 corresponds to the cycle period (7.1 s).

Identification and neuroanatomy of additional HN interneurons. A: extracellular recordings from the HN(L,4) interneuron and from a neuron in segment 15 on the contralateral side [HN(R,15) candidate]. Note that burst activity in these 2 neurons is time-locked. B and C: ventral aspect of a dye-injected HN(R,16) interneuron. Anterior is up. B: the confocal image shows rhodamine dextran in the injected cell. The HN(R,16) interneuron has a main neurite that loops and gives rise to many secondary neurites. The main neurite tapers in diameter to form a rearward-going axon leaving the ganglion. The anterior-going neurites do not leave the ganglion. Note that some processes cross the ventral midline. C: superimposed confocal image shows both rhodamine dextran (red fluorescence) and the Alexa fluorophore coupled to Neurobiotin (green fluorescence). The color combination makes the injected cell appear yellow because it contains both rhodamine dextran and Neurobiotin. Its contralateral homolog, the HN(L,16) interneuron, appears green due to dye coupling. Note the 2 neurites in the anterior right connective (arrows). D: same preparation, ganglion 15. Two cell bodies in the position of the HN(15) interneurons (posterior lateral packet) are labeled with Neurobiotin (asterisks).

Circuit diagrams show the synaptic connections among all known interneurons of the heartbeat CPG in its 2 coordination modes: left synchronous/right peristaltic and left peristaltic/right synchronous. Standard colors are used for the HN interneurons (see methods). Small vertical lines indicate excitatory synapses, small boxes along the HN(X) interneurons' axons in segments 3 to 6 are spike initiation sites, diodes indicate rectifying electrical synapses, and resistors indicate the electrical connection between the HN(3) and HN(4) interneurons and the anterior spike initiation site of the HN(X) interneuron. For simplicity, cells with similar input and output connections and function are combined. HN(1) and HN(2) interneurons (red circles) coordinate the activity of the HN(3) and (4) interneurons, and together these 4 pairs of interneurons form the beat-timing network. Switches in coordination mode of the CPG are associated with switches in which one HN (5) interneuron (the switch interneuron) is active (orange, synchronous side) and which one is silent (gray, peristaltic side). Dashed processes from the HN(5) interneuron to the posterior initiation site in segment 6 of the HN(X) interneuron indicate an indirect excitatory pathway. The newly identified rear premotor HN(15) and HN(16) interneurons receive excitatory input from both middle premotor interneurons. Diagrams are based on data from and , .

Phase diagrams of the constriction patterns of heart segments 3 to 18 from an individual, intact leech (top) and of the average from 13 different preparations (bottom). Vertical dashed lines are provided to facilitate observation of relative phasing. Both phase diagrams show the start of systole (±SD; systole in short) in the peristaltic (circles) and in the synchronous mode (diamonds). Phase reference is heart segment 14 on the peristaltic side, and its systole is assigned 0 phase. To illustrate the intersegmental phase differences in the rear, the peristaltic side is duplicated and shifted by 1. Top: mean phase (±SD) of systole of 9–19 heart beats was determined in an individual juvenile leech. Filled symbols represent 1 switch cycle (left synchronous/right peristaltic); open symbols represent another switch cycle (right synchronous/left peristaltic). Phases could not always be determined in all 32 segments in both switch cycles; for example, segment 16 in the peristaltic mode was obtained once in this animal. Note that patterns are similar on both body sides. Cycle period: 4.1 s. The number of beats per switch cycle was 20 and 21, respectively. Bottom: average of the start of systole and additionally that of full diastole (left edge of gray bars) is shown. Note that heart segments in the front and in the rear converge in phase and that rear heart segments 16 to 18 on both sides fill (diastole) and empty (systole) from front to rear. Graph shows the average (±SD) of the average of the 2 switch cycles analyzed per preparation. Mean cycle period: 3.9 ± 0.45 s.

Bilateral phase diagram of the timing pattern (A) and hemilateral circuit diagram of the premotor heart (HN) interneurons of the leech heartbeat central pattern generator (CPG) and the postsynaptic heart (HE) motor neurons (B). HE motor neurons and HN interneurons are indexed by the body side and the midbody ganglion number they reside in (see also methods). Standard colors are used for the HN interneurons (see Terminology and color code in methods). A: bilateral phase diagram of the premotor HN interneurons and the entire HE motor neuron ensemble for the peristaltic (top) and synchronous (bottom) coordination mode. Each box represents the average duty cycle of a neuron's activity with the vertical line in the center representing the middle spike (±SD), the left edge the first spike, and the right edge the last spike, respectively (error bars omitted for clarity). Phase reference is the middle spike of the peristaltic HN(4) interneuron and is assigned 0 phase. To illustrate the side-to-side phase relations of the HE motor neurons, the diagram of the peristaltic mode was copied into the diagram of the synchronous mode (translucent pink boxes, no error bars). Note that HE motor neurons come together in phase in both the front and rear segments. B: HE motor neurons of segments 3 to 18 receive staggered input from ipsilateral premotor HN interneurons: from the HN(3) and HN(4) (front premotor HN interneurons), from the HN(6) and HN(7) (middle premotor HN interneurons), and from the unidentified HN(X) interneuron. Large colored circles are cell bodies (indexed by midbody segment number), lines indicate cell processes, and small circles indicate inhibitory chemical synapses. [Adapted from .]