Reduction of high-frequency network oscillations (ripples) and pathological network discharges in hippocampal slices from connexin 36-deficient mice

Abstract

Recent evidence suggests that electrotonic coupling is an important mechanism for neuronal synchronisation in the mammalian cortex and hippocampus. Various types of network oscillations have been shown to depend on, or be sharpened by, gap junctions between inhibitory interneurones or excitatory projection cells. Here we made use of a targeted disruption of the gene coding for Cx36, a recently discovered neuronal gap junction subunit, to analyse its role in hippocampal network behaviour. Mice lacking Cx36 are viable and lack obvious morphological or behavioural abnormalities. Stimulation of afferent and efferent fibre pathways in hippocampal slices revealed a largely normal function of the synaptic circuitry, including tetanically evoked network oscillations. Spontaneous sharp waves and ripple (approximately 200 Hz) oscillations, however, occurred less frequently in slices from Cx36 -/- mice, and ripples were slightly slower than in littermate controls. Moreover, epileptiform discharges elicited by 4-aminopyridine were attenuated in slices from Cx36 -/- mice. Our findings indicate that Cx36 plays a role in the generation of certain forms of network synchronisation in the hippocampus, namely sharp wave-ripple complexes and hypersynchronous epileptiform discharges.

Figure 3. Decreased occurrence of sharp waves and ripple oscillations in CA1 of Cx36 -/- mice

A, raw data traces from a Cx36 +/+ slice (top) and a Cx36-/- slice (bottom). Spontaneous sharp waves are visible as distinct positive peaks above baseline. B, separation of sharp waves and ‘ripples’ from the sharp wave-ripple complex marked by * in A. Top: raw data; middle: low-pass filtered data (50 Hz) showing sharp wave; bottom: band-pass filtered data (135-500 Hz) showing ripple. Detection threshold for sharp waves (5 × s.d. of baseline noise; middle) and ripples (4 × s.d. of baseline noise; bottom) indicated by the horizontal line. C, frequency of occurrence of sharp waves in CA1 is reduced in Cx36 -/- mice (n = 42 control and 53 KO slices; P < 0.001). D, frequency of occurrence of 200 Hz oscillation bursts is reduced in Cx36 -/- slices (n = 42/54 samples; P < 0.005). E, mean frequency within ripples is lower in slices from Cx36 -/- mice (n = 42/46 samples; P < 0.05). F, mean number of spikes per ripple event is not different between both groups.

Figure 4. Altered epileptiform activity patterns in slices from Cx36 -/- mice

A, distribution of different patterns of pathological activity evoked by 100 μM 4-aminopyridine (4-AP) in Cx36 +/+ (black bars) and Cx36 -/- mice (grey bars). Ongoing seizure-like activity is more frequent in wild-type animals (P < 0.05, χ² test). Absence of discharges was only observed in Cx36 -/- slices (4/25). Data are expressed as the percentage of slices showing the respective pattern. B, examples of the four distinct patterns of activity. Bottom traces show examples from top traces at higher time resolution (marked by *). Left: ongoing seizure-like activity; second trace: complex bursts; third trace: interictal-like events; right: no activity in a slice from a Cx36 -/- mouse exposed to 4-AP.

Figure 1. Unaltered excitability and unchanged stimulation-induced network oscillations in Cx36 -/- mice

A, examples of paired orthodromically evoked field EPSPs and population spikes in CA1 pyramidal layer slices from Cx36 +/+ (left) and Cx36 -/- (right) mice (20 ms stimulus interval). B, paired-pulse ratios of population spike amplitudes in Cx36 +/+ (○, n = 8) and Cx36 -/- slices (♦, n = 8). Potentiation at short intervals is similar, while paired-pulse depression at 600 ms is absent in Cx36 -/- mice (P < 0.05). C, tetanically evoked oscillations in Cx36 +/+ (left) and Cx36 -/- (right) slices (stimulation artifacts truncated). Note prominent peaks around 10 ms in the autocorrelation functions (below) from the initial 200 ms of the oscillation. D, leading frequencies of the oscillation are not different between both groups.

Figure 2. Simultaneous sharp wave-ripple complexes in CA3 and CA1

A, sharp waves recorded in area CA1 are preceded by sharp wave events in CA3 in slices from control and Cx36 -/- mice. B, original recordings (upper traces) and band-pass (135-500 Hz) filtered derivatives (lower traces) of the event marked by * in A. C, cross-correlation functions of the recordings shown in A. Lateral shift of the peaks indicates time lag between sharp waves occurring in CA3 and CA1. Coherence of superimposed fast ripples is not evident from the cross-correlation.