Rhythmicity without synchrony in the electrically uncoupled inferior olive

Abstract

Neurons of the inferior olivary nucleus (IO) form the climbing fibers that excite Purkinje cells of the cerebellar cortex. IO neurons are electrically coupled through gap junctions, and they generate synchronous, subthreshold oscillations of membrane potential at approximately 5-10 Hz. Experimental and theoretical studies have suggested that both the rhythmicity and synchrony of IO neurons require electrical coupling. We recorded from pairs of IO neurons in slices of mouse brainstem in vitro. Most pairs of neurons from wild-type (WT) mice were electrically coupled, but coupling was rare and weak between neurons of knock-out (KO) mice for connexin36, a neuronal gap junction protein. IO cells in both WT and KO mice generated rhythmic membrane fluctuations of similar frequency and amplitude. Oscillations in neighboring pairs of WT neurons were strongly synchronized, whereas the oscillations of KO pairs were uncorrelated. Spontaneous oscillations in KO neurons were not blocked by tetrodotoxin. Spontaneously oscillating neurons of both WT and KO mice generated occasional action potentials in phase with their membrane rhythms, but only the action potentials of WT neuron pairs were synchronous. Harmaline, a beta-carboline derivative thought to induce tremor by facilitating rhythmogenesis in the IO, was injected systemically into WT and KO mice. Harmaline-induced tremors were robust and indistinguishable in the two genotypes, suggesting that gap junction-mediated synchrony does not play a role in harmaline-induced tremor. We conclude that electrical coupling is not necessary for the generation of spontaneous subthreshold oscillations in single IO neurons, but that coupling can serve to synchronize rhythmic activity among IO neurons.

Fig. 1.

IO neurons from KO mice. A, β-Galactosidase histochemistry of a parasagittal section from aCx36 KO mouse shows many strongly stained cells in the IO nucleus. B, IR-DIC image of two very closely spaced neurons during paired whole-cell recordings from the IO of a KO mouse. Despite their proximity, these cells were not electrically coupled to each other.

Fig. 2.

Electrical coupling is greatly diminished in theCx36 KO mouse. A, Paired whole-cell recordings from coupled WT cells show synchronized subthreshold oscillations, occasional action potentials in cell 1, and correlated electrical coupling potentials in cell 2. Note that the gain for cell 2 is higher than that from cell 1. B, Expanded view of a spontaneous action potential in cell 1 and its associated electrotonically propagated spikelet in cell 2 (same recordings shown in A). Dashed lines show where traces inA are expanded in B. C, Electrical coupling in a pair of WT neurons. A hyperpolarizing current step (−1 nA) injected into cell 1 induced an electrotonically propagated hyperpolarization in cell 2. D, E, Electrical coupling was absent in a representative pair of IO cells from a Cx36KO mouse. To remove the effects of spontaneous membrane oscillations, voltage traces in C–E are the averages of 10 trials, with the exception of the representative action potential in the KO cell shown in D.

Fig. 3.

The frequency of spontaneous subthreshold oscillations in WT cells is determined by the electrically coupled network. Membrane potentials were altered by injecting steady currents, either hyperpolarizing or depolarizing, into WT (A) and KO (B) cells.C, Amplitudes of subthreshold oscillations were a strong function of membrane potential in both WT and KO neurons: summary of data from nine WT and nine KO cells. Mean amplitudes at various membrane potentials were scaled to largest responses and summed across 5-mV-wide bins, along with SE bars. The only significant difference was at the most hyperpolarized level (−66 to −70 mV), where the WT response was greater than the KO response (p< 0.05; t test). D, Oscillation frequency was a strong function of membrane polarization in neurons from the KO but was invariant with membrane potential in WT neurons. The graph shows data obtained from cells illustrated inA (WT) and B (KO). E, To summarize the voltage dependence of oscillation frequency, the best-fit line was calculated for each set of data points (as inD) (9 WT and 9 KO cells). The graph in Eplots the slopes of the linear fits, in Hertz per millivolts.Dashed line shows slope of zero.

Fig. 4.

Electrical synapses are required for synchrony of spontaneous rhythms. Left, Data derived from one WT pair; right, data derived from one KO pair.A, Rhythms recorded simultaneously from two closely spaced WT (left) and KO (right) neurons appear similar in amplitude and frequency, although the phase of the two KO recordings obviously varies. B, Data from the same cell pairs are shown in Lissajous figures, which plot the membrane potential of IO₁ versus IO₂. Recordings from the WT neurons are nearly identical (left), but those from KO neurons show no correlation (right).C, Cross-correlograms (Cross Corr) of both pairs derived from >60 sec of data quantify the high degree of rhythmic correlation between WT cells (left) and the absence of correlation between KO cells (right).Dashed lines mark zero time. D, Power spectra calculated from the same data show that the frequencies (Freq) of paired WT neurons were identical, whereas KO cell frequencies differed.

Fig. 5.

Rhythms of electrically uncoupled IO neurons are asynchronous. The graph plots the peak correlations obtained from cross-correlograms of 12 WT and KO neuron pairs, obtained as described in Figure 3C. Filled triangles are from cell pairs that were measurably, electrically coupled; open triangles are from pairs that were not coupled.Horizontal lines are mean values. The shaded area shows the 95% confidence interval, derived from shuffled data of 10 WT neurons.

Fig. 6.

Action potentials are synchronized by electrical coupling. A, Spontaneous spiking from two WT neurons during a 4-sec-long epoch of spontaneous subthreshold rhythms shows simultaneous spiking. The traces below show magnified views of an action potential in the top cell, a subthreshold spikelet in the bottom cell (left), and two nearly simultaneous spikes (right). Dashed lines show wheretop traces are expanded in bottom traces.B, A similar 4 sec epoch of rhythmic activity in two KO neurons shows only asynchronous spiking. C, When 22 spiking epochs from the WT pair are aligned on the spikes of one cell, it is evident that spikes in the second cell most often occur with a brief lag or lead. Dashed line is aligned with peaks of spikes in cell 1. D, Spike cross-correlogram (5 msec bin width) taken from 200 sec of spontaneous spiking from the pairs illustrated above. WT cells show a strong peak at 0 msec, whereas spikes from KO cells were uncorrelated. E, When the same WT spiking data were cross-correlated with finer temporal resolution (0.5 msec bin width), it is clear that the periods of highest spiking probability occurred before and after spikes in the reference neuron.

Fig. 7.

Harmaline-induced tremor does not require electrical coupling in IO. A, Force transducer output from representative spontaneous tremor in a WT mouse before (thick line) and after (thin line) harmaline administration. Transducer output units are arbitrary but fixed across all measurements. B, Power spectra from force measurements in control (thick) and harmaline (thin) states; harmaline tremor is evident as a sharp, strong peak at ∼14 Hz. C, Similar force measurements in a representative KO mouse before and after harmaline (same scale as in A). D, The power spectrum from the KO mouse tremor is similar to that of the WT mouse.