An updated suite of viral vectors for in vivo calcium imaging using intracerebral and retro-orbital injections in male mice.

Genetically encoded Ca2+ indicators (GECIs) are widely used to measure neural activity. Here, we explore the use of systemically administered PHP.eB AAVs for brain-wide expression of GECIs and compare the expression properties to intracerebrally injected AAVs in male mice. We show that systemic administration is a promising strategy for imaging neural activity. Next, we establish the use of EE-RR- (soma) and RPL10a (Ribo) soma-targeting peptides with the latest jGCaMP and show that EE-RR-tagged jGCaMP8 gives rise to strong expression but limited soma-targeting. In contrast, Ribo-tagged jGCaMP8 lacks neuropil signal, but the expression rate is reduced. To combat this, we modified the linker region of the Ribo-tag (RiboL1-). RiboL1-jGCaMP8 expresses faster than Ribo-jGCaMP8 but remains too dim for reliable use with systemic virus administration. However, intracerebral injections of the RiboL1-tagged jGCaMP8 constructs provide strong Ca2+ signals devoid of neuropil contamination, with remarkable labeling density.

Optogenetic pacing of medial septum parvalbumin-positive cells disrupts temporal but not spatial firing in grid cells.

Grid cells in the medial entorhinal cortex (MEC) exhibit remarkable spatial activity patterns with spikes coordinated by theta oscillations driven by the medial septal area (MSA). Spikes from grid cells progress relative to the theta phase in a phenomenon called phase precession, which is suggested as essential to create the spatial periodicity of grid cells. Here, we show that optogenetic activation of parvalbumin-positive (PV+) cells in the MSA enabled selective pacing of local field potential (LFP) oscillations in MEC. During optogenetic stimulation, the grid cells were locked to the imposed pacing frequency but kept their spatial patterns. Phase precession was abolished, and speed information was no longer reflected in the LFP oscillations but was still carried by rate coding of individual MEC neurons. Together, these results support that theta oscillations are not critical to the spatial pattern of grid cells and do not carry a crucial velocity signal.

Perineuronal nets stabilize the grid cell network.

Grid cells are part of a widespread network which supports navigation and spatial memory. Stable grid patterns appear late in development, in concert with extracellular matrix aggregates termed perineuronal nets (PNNs) that condense around inhibitory neurons. It has been suggested that PNNs stabilize synaptic connections and long-term memories, but their role in the grid cell network remains elusive. We show that removal of PNNs leads to lower inhibitory spiking activity, and reduces grid cells' ability to create stable representations of a novel environment. Furthermore, in animals with disrupted PNNs, exposure to a novel arena corrupted the spatiotemporal relationships within grid cell modules, and the stored representations of a familiar arena. Finally, we show that PNN removal in entorhinal cortex distorted spatial representations in downstream hippocampal neurons. Together this work suggests that PNNs provide a key stabilizing element for the grid cell network.

Removal of perineuronal nets disrupts recall of a remote fear memory.

Throughout life animals learn to recognize cues that signal danger and instantaneously initiate an adequate threat response. Memories of such associations may last a lifetime and far outlast the intracellular molecules currently found to be important for memory processing. The memory engram may be supported by other more stable molecular components, such as the extracellular matrix structure of perineuronal nets (PNNs). Here, we show that recall of remote, but not recent, visual fear memories in rats depend on intact PNNs in the secondary visual cortex (V2L). Supporting our behavioral findings, increased synchronized theta oscillations between V2L and basolateral amygdala, a physiological correlate of successful recall, was absent in rats with degraded PNNs in V2L. Together, our findings suggest a role for PNNs in remote memory processing by stabilizing the neural network of the engram.

Differential Expression and Cell-Type Specificity of Perineuronal Nets in Hippocampus, Medial Entorhinal Cortex, and Visual Cortex Examined in the Rat and Mouse.

Perineuronal nets (PNNs) are specialized extracellular matrix (ECM) structures that condense around the soma and proximal dendrites of subpopulations of neurons. Emerging evidence suggests that they are involved in regulating brain plasticity. However, the expression of PNNs varies between and within brain areas. A lack of quantitative studies describing the distribution and cell-specificity of PNNs makes it difficult to reveal the functional roles of PNNs. In the current study, we examine the distribution of PNNs and the identity of PNN-enwrapped neurons in three brain areas with different cognitive functions: the dorsal hippocampus, medial entorhinal cortex (mEC) and primary visual cortex (V1). We compared rats and mice as knowledge from these species are often intermingled. The most abundant expression of PNNs was found in the mEC and V1, while dorsal hippocampus showed strikingly low levels of PNNs, apart from dense expression in the CA2 region. In hippocampus we also found apparent species differences in expression of PNNs. While we confirm that the PNNs enwrap parvalbumin-expressing (PV+) neurons in V1, we found that they mainly colocalize with excitatory CamKII-expressing neurons in CA2. In mEC, we demonstrate that in addition to PV+ cells, the PNNs colocalize with reelin-expressing stellate cells. We also show that the maturation of PNNs in mEC coincides with the formation of grid cell pattern, while PV+ cells, unlike in other cortical areas, are present from early postnatal development. Finally, we demonstrate considerable effects on the number of PSD-95-gephyrin puncta after enzymatic removal of PNNs.

Removal of Perineuronal Nets Unlocks Juvenile Plasticity Through Network Mechanisms of Decreased Inhibition and Increased Gamma Activity.

Perineuronal nets (PNNs) are extracellular matrix structures mainly enwrapping parvalbumin-expressing inhibitory neurons. The assembly of PNNs coincides with the end of the period of heightened visual cortex plasticity in juveniles, whereas removal of PNNs in adults reopens for plasticity. The mechanisms underlying this phenomenon remain elusive. We have used chronic electrophysiological recordings to investigate accompanying electrophysiological changes to activity-dependent plasticity and we report on novel mechanisms involved in both induced and critical period plasticity. By inducing activity-dependent plasticity in the visual cortex of adult rats while recording single unit and population activity, we demonstrate that PNN removal alters the balance between inhibitory and excitatory spiking activity directly. Without PNNs, inhibitory activity was reduced, whereas spiking variability was increased as predicted in a simulation with a Brunel neural network. Together with a shift in ocular dominance and large effects on unit activity during the first 48 h of monocular deprivation (MD), we show that PNN removal resets the neural network to an immature, juvenile state. Furthermore, in PNN-depleted adults as well as in juveniles, MD caused an immediate potentiation of gamma activity, suggesting a novel mechanism initiating activity-dependent plasticity and driving the rapid changes in unit activity. SIGNIFICANCE STATEMENT: Emerging evidence suggests a role for perineuronal nets (PNNs) in learning and regulation of plasticity, but the underlying mechanisms remain unresolved. Here, we used chronic in vivo extracellular recordings to investigate how removal of PNNs opens for plasticity and how activity-dependent plasticity affects neural activity over time. PNN removal caused reduced inhibitory activity and reset the network to a juvenile state. Experimentally induced activity-dependent plasticity by monocular deprivation caused rapid changes in single unit activity and a remarkable potentiation of gamma oscillations. Our results demonstrate how PNNs may be involved directly in stabilizing the neural network. Moreover, the immediate potentiation of gamma activity after plasticity onset points to potential new mechanisms for the initiation of activity-dependent plasticity.