Disruption of perineuronal nets increases the frequency of sharp wave ripple events

Abstract

Hippocampal sharp wave ripples (SWRs) represent irregularly occurring synchronous neuronal population events that are observed during phases of rest and slow wave sleep. SWR activity that follows learning involves sequential replay of training-associated neuronal assemblies and is critical for systems level memory consolidation. SWRs are initiated by CA2 or CA3 pyramidal cells (PCs) and require initial excitation of CA1 PCs as well as participation of parvalbumin (PV) expressing fast spiking (FS) inhibitory interneurons. These interneurons are relatively unique in that they represent the major neuronal cell type known to be surrounded by perineuronal nets (PNNs), lattice like structures composed of a hyaluronin backbone that surround the cell soma and proximal dendrites. Though the function of the PNN is not completely understood, previous studies suggest it may serve to localize glutamatergic input to synaptic contacts and thus influence the activity of ensheathed cells. Noting that FS PV interneurons impact the activity of PCs thought to initiate SWRs, and that their activity is critical to ripple expression, we examine the effects of PNN integrity on SWR activity in the hippocampus. Extracellular recordings from the stratum radiatum of horizontal murine hippocampal hemisections demonstrate SWRs that occur spontaneously in CA1. As compared with vehicle, pre-treatment (120 min) of paired hemislices with hyaluronidase, which cleaves the hyaluronin backbone of the PNN, decreases PNN integrity and increases SWR frequency. Pre-treatment with chondroitinase, which cleaves PNN side chains, also increases SWR frequency. Together, these data contribute to an emerging appreciation of extracellular matrix as a regulator of neuronal plasticity and suggest that one function of mature perineuronal nets could be to modulate the frequency of SWR events.

Keywords: PV interneuron; chondrotinase; hippocampus; hyaluronidase; protease.

Figure 1

Spontaneous SWRs in 490 mM horizontal hippocampal slices. Top: raw LFP traces (red and black) showing sharp waves from two recording locations in CA1 (filtered; 0.1 to 1500 Hz). Bottom: examples of ripples from the top trace are displayed on an expanded time scale (filtered; 30 to 1500 Hz). Note that in this 15 second section of recordings there are 26 SWRs, some large events (note 1,4,8) and some small events (note 6,7,9).

Figure 2

Horizontal slice preparation and staining. Horizontal slices were prepared and bisected, with matched hemisections placed in one of two recovery solutions (ACSF or ACSF with hyaluronidase). Following a 2h recovery period, hemislices were used for additional studies. A schematic of the set up is shown in 2A, and representative PV fluorescence in cells from control (n=1392; 6 fields) and hyaluronidase (n=927; 5 fields) treated slices, prepared from PVtd-Tomato mice, is shown in 2B. No significant difference was detected. In 2C and D, we show representative immunostaining of a hippocampal slice from a PVtd-Tomato mouse with WFA. Layers of CA1, CA2, and CA3 areas indicated by yellow rectangles are shown magnified in c1–c3 for control slices, and d1–d3 for hyaluronidase treated slices, respectively. WFA positive PV expressing cells can be appreciated in control but not hyaluronidase treatment conditions. Scale bars: 100 µm (C, D), 50 µm (c1–c3, d1–d3), and 20 µm for high magnification single neurons.

Figure 3

SWR event frequency in hyaluronidase treated slices. A: 1^st trace: Control slice 15s recording of LFP (filtered 0.5–1500Hz). Blue bars indicate detected SWR event, defined as a coincident SW and ripple event. 2^nd trace: lower frequency SW component filtered 1–30Hz. 3^rd trace: Root mean square of SW filtered from 1–30Hz. Dashed lines indicate 2 and 4 SD above baseline for event detection. Red bars below indicate detected SW event. 4^th trace: high frequency ripple component filtered from 80–250Hz. 5^th trace: Root mean square of ripple filtered 80–250Hz. Dashed lines indicate 2 and 4 SD above baseline for event detection. Green bars below indicate detected ripple event. B shows the same features for Hyaluronidase treatment. Zoomed in views of indicated SWR events are shown, as are spectrogram averages for the indicated frequency ranges in all detected SWR events from 8 control and 8 hyaluronidase pretreated slices (time-locked to the SW peak; n= 743 control events and 1114 hyaluronidase events). C shows the fold change in average SWR event frequency for two to three paired slices from each of 3 different animals (n=8 slices per group), and overall SWR inter-event interval results from the 8 control and 8 treated slices is shown in D. The difference between control and treatment SWR frequency in 3C and D is significant (p < 0.05). A frequency histogram of inter-event intervals is shown in E. In 3F, we show that in contrast to SWR event frequency, SWR duration is unchanged by hyaluronidase treatment.

Figure 4

SWR event frequency in chondroitinase treated slices. Representative traces are shown in 4A and B, with SW events again in red and ripples in green. The fold change in average SWR event frequency results for 4 control and 4 treated slices is shown in 4C. The difference between control and treatment SWR frequency in 4C is significant (p < 0.05). In 4D, we show that in contrast to SWR event frequency, SWR duration is unchanged by chondroitinase treatment.

Figure 5

Hypothetical model by which PNN disruption influences the frequency of SWR events. In figure 6 we show a hypothetical model. A PV neuron with an intact PNN is shown at the top left. This neuron receives spatially localized glutamatergic input, allowing it to in turn inhibit pyramidal cell excitability. In contrast, a PV neuron with a disrupted PNN is shown at bottom left. In this scenario, glutamate may more easily diffuse from the synaptic cleft and/or glutamate receptors may demonstrate enhanced lateral mobility. A potential result is that this PV is less activated and thus less able to inhibit pyramidal cell excitability.