Primitive roles for inhibitory interneurons in developing frog spinal cord

Abstract

Understanding the neuronal networks in the mammal spinal cord is hampered by the diversity of neurons and their connections. The simpler networks in developing lower vertebrates may offer insights into basic organization. To investigate the function of spinal inhibitory interneurons in Xenopus tadpoles, paired whole-cell recordings were used. We show directly that one class of interneuron, with distinctive anatomy, produces glycinergic, negative feedback inhibition that can limit firing in motoneurons and interneurons of the central pattern generator during swimming. These same neurons also produce inhibitory gating of sensory pathways during swimming. This discovery raises the possibility that some classes of interneuron, with distinct functions later in development, may differentiate from an earlier class in which these functions are shared. Preliminary evidence suggests that these inhibitory interneurons express the transcription factor engrailed, supporting a probable homology with interneurons in developing zebrafish that also express engrailed and have very similar anatomy and functions.

Figure 1.

The tadpole and its spinal neurons. The Xenopus tadpole at the time of hatching (stage 37/38), with its head to the left, showing the eye, the border between midbrain and hindbrain (m/h), and the spinal cord lying under segmented swimming muscles. Below is a diagram of a length of spinal cord viewed from the side to show the neurons. Glycinergic aINs (purple) have ascending and descending axons that could contact and inhibit: dorsolateral commissural INs (dlc) and dorsolateral ascending INs (dla). These are excitatory sensory pathway interneurons, excited by skin sensory, touch-sensitive Rohon-Beard neurons (RB) that have no dendrites. aINs could also inhibit neurons that are active during swimming: mns, excitatory dINs, glycinergic reciprocal inhibitory cINs, and other aINs (data not shown). For each neuron class, the circle/oval is the soma; oblique lines indicate the dorsoventral extent of the dendrites; and the thin line shows the axon projection (asterisk indicates that the axon crosses ventrally to the opposite side). Each class of neuron actually forms a longitudinal column of 50-150 neurons on each side of the cord.

Figure 2.

Diagram of the tadpole preparation and paired whole-cell recordings to show that aINs produce direct inhibition of spinal CPG neurons. A, Scale diagram of the CNS viewed from the right showing the location of two neurobiotin-filled neurons with a synaptic connection, shown in more detail in the micrographs below. Each neuron has a spherical soma and a ventral process with dendrites. The aIN has an ascending axon (a) and a descending axon (d) with possible synaptic contact onto a cIN dendrite (see arrow). The ventral cIN axon goes out of focus (asterisk). B, inset, the dissected tadpole with its spinal cord exposed is held with four micropins. Patch electrodes record two spinal neurons. Swimming can be initiated by a current pulse to excite sensory innervation of the tail skin (stim tail) and recorded from a ventral root (VR). Current-clamp recordings show impulses evoked by current injection into the aIN in A lead to constant latency, variable amplitude IPSPs in the cIN that was deploarized to increase IPSP amplitude (7 traces overlapped). The aIN impulse causes a cross-talk artifact in the cIN (arrowhead). C, Tracings to show the anatomy of two neurobiotin-filled aINs with a possible synaptic contact (arrow) from the axon of the presynaptic aIN (black) onto the postsynaptic aIN (gray), the descending axon of which is not shown beyond the asterisk. D, Three current-evoked impulses in the caudal aIN in C (dots mark its resting potential) lead to short latency IPSPs in the other aIN. The postsynaptic aIN was deploarized to increase IPSP amplitude. E, The same neurons as in C, where superimposed records show small constant latency IPSPs, with some failures, in the rostral aIN after impulses in the caudal aIN. F, Tracings to show the anatomy of an aIN (black) and a dIN (gray) with possible synaptic contact (arrow). The dIN descending axon is not shown further than the asterisk. G, Current-evoked impulses in the aIN in F lead to IPSPs in the dIN that can be reversed at near -40 mV by current injection into the dIN. a, Ascending aIN axon; d, descending aIN axon; h/m, hindbrain/midbrain border.

Figure 3.

Inhibition from aINs is glycinergic and can produce IPSPs in CPG neurons during swimming. Recordings from an aIN and a cIN. A, Tracings to show the anatomy of an aIN (black) and a cIN (gray) with possible synaptic contacts (arrows) from the ascending (a) axon of the aIN. The aIN also has a descending axon branch (d). The cIN axon is not shown beyond the asterisk, where it crosses the cord ventrally. B, Impulses evoked in the aIN by current injection produce IPSPs in the cIN that are blocked by 5 μm strychnine, with partial recovery on wash. Each trace is the average of seven traces. C, Six cycles of swimming initiated by a current pulse to the skin and monitored from a ventral root on the right side (Rvr). The cIN fires a single impulse and receives IPSPs on most swimming cycles, revealed by injection of depolarizing current (asterisk, mid-cycle IPSP). The aIN fires less reliably. Two early-cycle IPSPs (arrowheads) in the cIN follow spikes in the aIN; another early-cycle IPSP does not (open arrowhead). D, Synchronizing records to the aIN spikes shows short latency IPSPs in the cIN follow most aIN spikes. Note that these IPSPs and the aIN spikes are not similarly time-locked to the preceding cIN spikes. E, IPSPs in the cIN follow spikes evoked in the aIN by current injection at the same latency as those during swimming in D. The IPSPs are smaller than in D because the cIN is less depolarized.

Figure 5.

A single aIN inhibits both a CPG and a sensory pathway dlc interneuron. A, Tracings to show the anatomy of an aIN (black) and two interneurons that it contacts: an unidentified CPG interneuron (gray) with possible synaptic contacts (arrow) from the ascending axon (a) and a dlc interneuron with possible contacts (arrow) from the descending axon branch (d). The CPG neuron staining was weak, so its detailed features and axon could not be resolved. B, When swimming (see vr; recorded ∼5 segments more caudal) is initiated by a skin stimulus (at arrow), the aIN is depolarized from the resting potential (dotted line) and fires on two cycles. C, During swimming, the CPG interneuron is also depolarized but fires on each cycle of swimming. D, In the first paired recording from this aIN, injecting current to evoke action potentials leads to variable amplitude IPSPs and some failures (gray traces) in the CPG interneuron. E, During swimming, the sensory pathway dlc interneuron receives some early-cycle (arrowheads) and some mid-cycle IPSPs, made larger by injection of depolarizing current. F, When the same aIN is recorded again with this dlc interneuron, current-evoked action potentials leads to variable amplitude IPSPs.

Figure 4.

Pattern of early-cycle IPSCs during swimming. A, Voltage-clamp records made with a patch electrode (p) at positive holding potentials show IPSCs during swimming in a mn, cIN, dIN, and aIN. Swimming activity recorded from a ventral root (vr). Here, and in B and C, some early-cycle IPSCs are indicated by arrowheads and mid-cycle IPSCs by an asterisk. B, Waterfall plots showing the timing of IPSCs during swimming for the same recordings as in A. Each line shows a consecutive pair of cycles, normalized individually to a cycle phase of 1.0. Cycles are defined by the start of consecutive ventral root bursts and corrected for the longitudinal spacing between the neuron and vr recording position (which would otherwise add a timing error) (see Li et al., 2002). The second cycle on each line becomes the first cycle of the line below (n = ∼15 cycles per plot). C, Phase histograms to show the timing of IPSCs during swimming in CPG interneurons and motoneurons. Data for each cycle are plotted twice (cycle phase, 0-2), and the peak of early-cycle IPSCs is indicated (arrowhead). The numbers in each histogram bin were normalized to the mid-cycle peak value for each type of neuron (100%). In calculating correlations (see Results), the values between phase 0.35 and 0.7 were omitted to minimize the influence of mid-cycle IPSCs (asterisks), which are from cINs on the opposite side, not from aINs. D, The timing of early-cycle IPSCs during swimming (shaded bars are combined data from all plots in C) correlates well with the firing phase of aINs (solid line; total of 668 spikes in 16 aINs). The aIN spike data have been normalized to give the same total number of spikes as IPSCs between cycle phases 0 and 0.35. The dotted line shows the timing of IPSPs in sensory pathway dlc interneurons (data from Li et al., 2002). The peak in CPG IPSCs at phases of ∼0.5 (mid-cycle) goes off scale. E, Early-cycle IPSCs in swimming, measured under voltage clamp in an aIN, are blocked by 2 μm strychnine with partial reversal but are not affected by 20 μm bicuculline. The averages are of ∼15 cycles each. Traces are aligned to the onset of the cycle, indicated by the start of the ventral root burst (vr).

Figure 6.

Early-cycle IPSCs occur more reliably at higher swimming frequencies. A, Recording from a ventral root (vr) and a cIN at the start of a swimming episode initiated by a skin stimulus (open arrowhead). Voltage-clamp recordings in the cIN (at a positive holding potential close to EPSC reversal level) show early-cycle IPSCs (arrowheads) when the swimming frequency is highest. These become less reliable as the swimming frequency subsequently falls (asterisk marks a mid-cycle IPSP.) B, Early-cycle IPSCs (arrowheads) in a cIN are more common when the swimming frequency rises after a brief tail-skin stimulus (open arrowhead) applied during swimming. They are absent immediately before the stimulus (shaded region) when the frequency is low. C, Swimming frequency and the probability of recording early cycle IPSCs, plotted for 10 neurons after the start of an episode of swimming (left, as in A) and after a stimulus applied during swimming (right, as in B). D, The probability of recording early-cycle IPSCs increases with swimming frequency. Points represent mean ± SEM for frequency and the proportion of cycles with early-cycle IPSCs for data grouped into consecutive 1 Hz bins (8-10 Hz measurements combined). The regression line was fitted to the raw data (n = 393 cycles; equation: y = 0.06 x - 0.40).

Figure 7.

Early-cycle inhibition and control of multiple firing in CPG neurons. A, Voltage-clamp record of a cIN, clamped at a positive potential, to illustrate a typical pattern of inward (excitatory) and outward (inhibitory) PSCs during swimming. The record shows 16 overlapped cycles of swimming (mean period, 69.5 ± 2.1 msec), each normalized to a cycle phase of 1 and plotted twice. Synchronous, “on-cycle” EPSCs (open arrowhead) just precede the start of the ventral root burst (vr; timing adjusted for longitudinal spacing of electrodes). Early-cycle IPSCs from aINs (filled arrowheads) are delayed relative to the on-cycle excitation and are less synchronized than on-cycle EPSCs or mid-cycle IPSCs from contralateral cINs (asterisk). The shaded region shows the window for impulse firing before early-cycle inhibition. B, Examples of firing activity in two cINs during swimming shown with ventral root activity (vr). Early-cycle IPSPs are present on some cycles (e.g., arrowheads). However, the neurons fire more impulses on cycles where these are absent (open arrowheads).

Figure 8.

The distribution of En-1-positive neurons. A, En1-positive nuclei of neurons on one side of the whole spinal cord seen in an optical section at 1.5 mm caudal to the midbrain. Dark stained nuclei (e.g., at white arrow) form a column in a middle dorsoventral position. B, The distribution of spinal En1-positive neurons as a function of distance from the midbrain. The plot shows the mean number of neurons (aINs are marked nuclei) on each side for consecutive 0.1 mm blocks in four tadpoles (different symbol for each; regression line: y = 9.37 - 2.25x). C, An aIN with the ascending axon (arrow) clearly visible is labeled by GFP. The neuron is unipolar with dendrites near the point at which the main axonal process turns rostrally. Scale bar, 20 μm. D, aIN in C with ascending axon marked in yellow to show descending branch (at asterisk). E-G, Images of the aIN in C to show the En1-positive nucleus (red) lies within the aIN cell body.