The Role of Cerebellar Intrinsic Neuronal Excitability, Synaptic Plasticity, and Perineuronal Nets in Eyeblink Conditioning.

Evidence is strong that, in addition to fine motor control, there is an important role for the cerebellum in cognition and emotion. The deep nuclei of the mammalian cerebellum also contain the highest density of perineural nets-mesh-like structures that surround neurons-in the brain, and it appears there may be a connection between these nets and cognitive processes, particularly learning and memory. Here, we review how the cerebellum is involved in eyeblink conditioning-a particularly well-understood form of learning and memory-and focus on the role of perineuronal nets in intrinsic membrane excitability and synaptic plasticity that underlie eyeblink conditioning. We explore the development and role of perineuronal nets and the in vivo and in vitro evidence that manipulations of the perineuronal net in the deep cerebellar nuclei affect eyeblink conditioning. Together, these findings provide evidence of an important role for perineuronal net in learning and memory.

Eyeblink tract tracing with two strains of herpes simplex virus 1.

BACKGROUND: Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre. NEW METHOD: We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability. RESULTS AND COMPARISON WITH EXISTING METHODS: Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity. CONCLUSIONS: Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target.

Disruption of rat deep cerebellar perineuronal net alters eyeblink conditioning and neuronal electrophysiology.

The perineuronal net (PNN) is a specialized type of extracellular matrix found in the central nervous system. The PNN forms on fast spiking neurons during postnatal development but the ontogeny of PNN development has yet to be elucidated. By studying the development and prevalence of the PNN in the juvenile and adult rat brain, we may be able to understand the PNN's role in development and learning and memory. We show that the PNN is fully developed in the deep cerebellar nuclei (DCN) of rats by P18. By using enzymatic digestion of the PNN with chondroitinase ABC (ChABC), we are able to study how digestion of the PNN affects cerebellar-dependent eyeblink conditioning in vivo and perform electrophysiological recordings from DCN neurons in vitro. In vivo degradation of the PNN resulted in significant differences in eyeblink conditioning amplitude and area. Female animals in the vehicle group demonstrated higher levels of conditioning as well as significantly higher post-probe conditioned responses compared to males in that group, differences not present in the ChABC group. In vitro, we found that DCN neurons with a disrupted PNN following exposure to ChABC had altered membrane properties, fewer rebound spikes, and decreased intrinsic excitability. Together, this study further elucidates the role of the PNN in cerebellar learning in the DCN and is the first to demonstrate PNN degradation may erase sex differences in delay conditioning.

Changes in membrane properties of rat deep cerebellar nuclear projection neurons during acquisition of eyeblink conditioning.

Previous studies have shown changes in membrane properties of neurons in rat deep cerebellar nuclei (DCN) as a function of development, but due to technical difficulties in obtaining viable DCN slices from adult animals, it remains unclear whether there are learning-related alterations in the membrane properties of DCN neurons in adult rats. This study was designed to record from identified DCN cells in cerebellar slices from postnatal day 25-26 (P25-26) rats that had a relatively mature sensory nervous system and were able to acquire learning as a result of tone-shock eyeblink conditioning (EBC) and to document resulting changes in electrophysiological properties. After electromyographic electrode implantation at P21 and inoculation with a fluorescent pseudorabies virus (PRV-152) at P22-23, rats received either four sessions of paired delay EBC or unpaired stimulus presentations with a tone conditioned stimulus and a shock unconditioned stimulus or sat in the training chamber without stimulus presentations. Compared with rats given unpaired stimuli or no stimulus presentations, rats given paired EBC showed an increase in conditioned responses across sessions. Whole-cell recordings of both fluorescent and nonfluorescent DCN projection neurons showed that delay EBC induced significant changes in membrane properties of evoked DCN action potentials including a reduced after-hyperpolarization amplitude and shortened latency. Similar findings were obtained in hyperpolarization-induced rebound spikes of DCN neurons. In sum, delay EBC produced significant changes in the membrane properties of juvenile rat DCN projection neurons. These learning-specific changes in DCN excitability have not previously been reported in any species or task.