Changes in cerebellar intrinsic neuronal excitability and synaptic plasticity result from eyeblink conditioning

Neurobiology of Learning and Memory 166 (2019) 107094

Contents lists available at ScienceDirect

Neurobiology of Learning and Memory journal homepage: www.elsevier.com/locate/ynlme

Changes in cerebellar intrinsic neuronal excitability and synaptic plasticity result from eyeblink conditioning

T

Bernard G. Schreurs Department of Neuroscience, West Virginia University, Morgantown, WV, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Intrinsic membrane excitability Eyeblink conditioning Synaptic plasticity Cerebellum Purkinje cell Rabbit

There is a long history of research documenting plasticity in the cerebellum as well as the role of the cerebellum in learning and memory. Recordings in slices of cerebellum have provided evidence of long-term depression and long-term potentiation at several excitatory and inhibitory synapses. Lesions and recordings show the cerebellum is crucial for eyeblink conditioning and it appears changes in both synaptic and membrane plasticity are involved. In addition to its role in fine motor control, there is growing consensus that the cerebellum is crucial for perceptual, cognitive, and emotional functions. In the current review, we explore the evidence that eyeblink conditioning results in significant changes in intrinsic membrane excitability as well as synaptic plasticity in Purkinje cells of the cerebellar cortex in rabbits and changes in intrinsic membrane excitability in principal neurons of the deep cerebellar nuclei in rats.

1. Introduction Eyeblink conditioning Ernest Hilgard was the first to report eyeblink conditioning (EBC) in the 1930s with experiments in dogs (Hilgard & Marquis, 1935) and subsequent studies in humans (Hilgard & Campbell, 1936) and rats (Hughes & Schlosberg, 1938). Although there continued to be considerable theoretical and experimental interest in human eyeblink conditioning well into the 1960s (Spence & Spence, 1966), methodological issues and a search for the neural substrates of learning and memory returned focus to animal models (Coleman & Webster, 1988; Coleman, 1985; Steinmetz & Woodruff-Pak, 2000; Woodruff-Pak & Steinmetz, 2000). This focus was intensified when rabbits were added to the list of mammals capable of showing eyeblink conditioning (Gormezano, Kehoe, & Marshall, 1983; Kehoe & Macrae, 2002; Schneiderman, Fuentes, & Gormezano, 1962) and the neural substrates of rabbit eyeblink conditioning began to be explored in the 1970s with reports of the role of the hippocampus (Berger, Alger, & Thompson, 1976; Solomon & Moore, 1975) and in the 1980s with descriptions of the crucial role played by the cerebellum (McCormick & Thompson, 1984a,1984b; McCormick, Clark, Lavond, & Thompson, 1982; Yeo, Hardiman, & Glickstein, 1984, 1985a) – reports that were not without controversy (Delgado-Garcia & Gruart, 2002, 2006; Kelly, Zuo, & Bloedel, 1990; Welsh & Harvey, 1989). Publication by R.F. Thompson of a circuit diagram of the essential input and output pathways of the

rabbit eyblink (Thompson, 1986) that included the cerebellum (reimagined in Fig. 1) suggested that, like the hippocampus (Coulter et al., 1989; Schwartzkroin, 1975), brain slices might be cut through the cerebellum (Llinas & Sugimori, 1980a,1980b) to record changes in cells involved in the eyeblink conditioning circuit. In the 1990s we developed and began to record from Purkinje cells in slices of cerebellar cortex following eyeblink conditioning (Schreurs & Alkon, 1993; Schreurs, Sanchez-Andres, & Alkon, 1991, 1992). Because of technical issues, most notably the unusually dense perineuronal net surrounding over 90% of neurons in the deep cerebellar nuclei (Blosa et al., 2016; Carulli, Rhodes, & Fawcett, 2007; Mueller, Davis, Sovich, Carlson, & Robinson, 2016), it would be another 15 years before we were able to successfully use whole-cell patch clamp recording techniques to record from neurons in the deep cerebellar nuclei of young rats (Wang & Schreurs, 2014). Intrinsic membrane excitability The discovery of learning-specific changes in intrinsic membrane excitability has been described elsewhere (Debanne, Inglebert, & Russier, 2019; Llinas, 2014; Mozzachiodi & Byrne, 2010; Oh & Disterhoft, 2015; Shim, Lee, & Kim, 2018; Titley, Brunel, & Hansel, 2017; Zhang & Linden, 2003) including other papers in this special issue. Briefly, changes in membrane excitability that resulted from learning were reported in invertebrates, particularly the marine mollusks Aplysia californica (Cleary, Lee, & Byrne, 1998) and Hermissenda

E-mail address: [email protected]. https://doi.org/10.1016/j.nlm.2019.107094 Received 28 June 2019; Received in revised form 27 August 2019; Accepted 16 September 2019 Available online 19 September 2019 1074-7427/ © 2019 Elsevier Inc. All rights reserved.

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

Fig. 1. Schematic of the neural pathways involved in eyeblink conditioning. A simplified diagram of the minimal circuitry necessary for eyeblink conditioning (EBC). The tone-conditioned stimulus (CS) pathway is shown in blue. The air puff (AP) unconditioned stimulus (US) pathway is shown in red. The unconditioned response (UR) and conditioned response (CR) pathways are shown in black. Arrows indicate direction of inputs and outputs. Auditory information from the tone-CS is conveyed to the cochlear nucleus (CN) which in turn send projections to the pontine nuclei (PN) and medial auditory thalamic nuclei (MATN). The medial auditory thalamic nuclei project to PN neurons that give rise to mossy fibers (MF) that contact neurons in the anterior interpositus nucleus (AIN) of the deep cerebellar nuclei and granule cells (GC) in the cerebellar cortex. The axons of GCs form parallel fibers (PF) that contact Purkinje cells (PC) as well as inhibitory interneurons (not shown). Sensory information from the air puff-US is conveyed to the trigeminal nucleus (5 N) which in turn sends projections to the inferior olive (IO). Neurons of the IO give rise to climbing fibers (CF) which contact neurons in the AIN and PCs. Information from the tone-CS and air puff-US converged in AIN neurons and PCs. In addition, PCs convey information processed in the cerebellar cortex (interneurons are omitted for simplicity) to their target nuclear neurons in the AIN which are the final output from the cerebellum. The CR is executed by AIN neurons that project to the red nucleus (RN) which in turn activates motor neurons in the facial nucleus (7 N) and other brain stem nuclei that together elicit an eyeblink CR. Modified from (Gonzalez-Joekes, 2014). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

memory (Debanne, Inglebert, & Russier, 2019; Jang & Kim, 2019; Koppensteiner, Galvin, & Ninan, 2019; Lisman, Cooper, Sehgal, & Silva, 2018; Meadows et al., 2016; Mozzachiodi & Byrne, 2010; Ross et al., 2019; Sehgal, Song, Ehlers, & Moyer, 2013; Shim, Lee, & Kim, 2018; Titley, Brunel, & Hansel, 2017).

crassicornis (Alkon, 1984), and in the mammalian cortex (Woody, 1970) and hippocampus (Disterhoft, Coulter, & Alkon, 1986). These changes occurred in a number of different measures including an increase in input resistance, the number of action potentials resulting from a depolarizing injection of current and a reduction in the amplitude of the action potential after-hyperpolarization – changes that were mediated by alterations in a range of calcium cannels and calcium- and voltagedependent potassium channels (Alkon, 1984; Debanne et al., 2019; Disterhoft & Oh, 2006; Misonou et al., 2004; Nolan et al., 2003; Oh & Disterhoft, 2015; Xu & Kang, 2005; Zhang & Linden, 2003; Zheng & Raman, 2010). However, despite the widespread nature of learningspecific changes in intrinsic membrane excitability across both invertebrate and vertebrate species, in a synapto-centric world where long-term potentiation (LTP) and long term-depression (LTD) predominate, changes in intrinsic membrane excitability have not garnered the same level of attention as synaptic plasticity. One reason membrane excitability may not be emphasized as much as synaptic plasticity is that LTP and LTD are thought to be synapse-specific, whereas increased excitability affects large areas of the membrane. The former may enable a neuron to encode a larger amount of information than the latter. Nevertheless, the ubiquitous nature of learning-related changes in intrinsic membrane excitability, also known as intrinsic plasticity, continues to draw interest and a consensus seems to be forming that both synaptic and membrane plasticity may be necessary for learning and

Slices of lobule HVI Lesions specific to cerebellar lobule HVI that were shown to abolish eyeblink conditioning in the rabbit (Yeo, Hardiman, & Glickstein, 1984, 1985b,1985c) prompted us to develop a parasagittal slice through rabbit lobule HVI that preserved the essential crystalline architecture of the cerebellar cortex and allowed stimulation of climbing fiber and parallel fiber inputs, and recordings from Purkinje cells. Fig. 2A depicts the anatomy of the right cerebellar hemisphere and Fig. 2B shows the outline and Purkinje cell layer of parasagittal slices through the right HVI ipsilateral to the side of rabbit eyeblink conditioning. Our first recordings were with intracellular, thick-walled, glass electrodes that were advanced blindly through the molecular where Purkinje cell dendrites were impaled. Fig. 3 shows a biocytin-filled Purkinje cell with an extensive dendritic arbor that encompasses the entire extent of the molecular layer right to the pial surface and an axon exiting the Purkinje cell layer and entering the white matter below. 2

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

Fig. 2. Slices through Lobule HVI of the rabbit cerebellar cortex. The right cerebellar cortex of a rabbit with dashed lines showing the plane of section of parasagittal slices cut with a Vibratome 1000 vibrating slicer is shown on the left. On the right are tracings of each of six 350-µm slices cut through lobule HVI showing the location of the Purkinje cell layer depicted but dots.

generate a current-voltage relationship for each cell and determine the somatic spike and dendritic spike thresholds. Threshold measurements were based on the specific current step required to reach somatic spike and then dendritic spike threshold. The major finding of these experiments (Table 1), and for our subsequent experiments (Schreurs, Gusev, Tomsic, Alkon, & Shi, 1998; Schreurs, Tomsic, Gusev, & Alkon, 1997), was a conditioning-specific increase in the excitability of Purkinje cell dendrites measured as a decrease in the amount of current required to elicit dendritic spikes (Ids) without significant changes in dendritic membrane potential (Vm) or input resistance (Rm). Importantly, the increase in intrinsic membrane excitability was also subsequently observed in both a transient and after-hyperpolarization shown in Fig. 4, was correlated with the strength of conditioning, and was shown to last for up to a month following eyeblink conditioning (Schreurs et al., 1998). We went on to determine the nature of the conditioning-specific decrease in the current required to elicit dendritic spikes by manipulating potassium channels with a range of channel blockers including 4-AP, TEA, and Iberiotoxin (Schreurs et al., 1997, 1998) in slices from naïve rabbits and measured their effects on a transient hyperpolarization and the after-hyperpolarization which decreased as a result of eyeblink conditioning (Schreurs et al., 1998). Although all blockers were able to produce a significant reduction in the threshold for Purkinje cell dendritic spiking, only 4-AP, a relatively specific blocker of the transient voltage-dependent potassium channel (IA) at low concentrations, was able to reduce the transient- and after-hyperpolarization (Schreurs et al., 1998). As a result, we concluded that eyeblink conditioning-induced changes in dendritic excitability were mediated by changes in an IA-like potassium current – a finding consistent with a conditioning-specific role for potassium channels in the marine mollusk Hermissenda (Farley & Alkon, 1985) and in the rabbit hippocampus (Disterhoft et al., 1986). Finally, we determined the potential molecular pathways mediating learning-specific changes in cerebellar potassium channel by examining the role of the enzyme Protein Kinase C (Freeman, Scharenberg, Olds, & Schreurs, 1998) previously implicated in rabbit eyeblink conditioning (Olds, Anderson, McPhie, Staten, & Alkon, 1989; Scharenberg, Olds, Schreurs, Craig, & Alkon, 1991). PKC is enriched in the nervous system and has been implicated in, among other things, the phosphorylation of

Learning-specific changes in Purkinje cell dendrite membrane excitability In 1991, using the blind impalement technique, we first made sharp electrode recordings from Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI following eyeblink conditioning. For these and all other rabbit eyeblink conditioning experiments, the subjects were adult, male, albino rabbits (Oryctolagus cuniculus) allocated randomly to one of three groups in which they received either: (1) paired stimulus presentations (Paired); (2) unpaired stimulus presentations (Unpaired); or (3) no stimulus presentations (Naïve or Sit). Paired and Unpaired subjects received 3 consecutive days of stimulus presentation. For paired subjects, stimulus presentations consisted of 80 presentations of a 400-ms, 1000-Hz, 82-dB tone conditioned stimulus (CS) that coterminated with a 100-ms, 60-Hz, 2-mA electrical pulse unconditioned stimulus (US). For unpaired subjects, stimulus presentations consisted of 80 CS-alone and 80 US-alone trials which occurred in an explicitly unpaired manner. When included in the experiment, naive subjects remained in their home cages before slice recordings and Sit control subjects were restrained in the training chamber without stimulus presentations. When stable dendritic recordings were obtained, the majority of rabbit Purkinje-cell dendrites revealed autorhythmic spontaneous activity that had previously been described in the guinea pig (Llinas & Sugimori, 1980a) and comprised two or more phases: (1) a hyperpolarized quiescent phase; (2) a more depolarized somatic spiking phase; and (3) a still more depolarized dendritic spiking phase. The spontaneous activity of the Purkinje-cell dendrites necessitated some standardization of the electrophysiological measures we collected. First, spontaneous activity precluded the measurement of a ‘resting’ membrane potential so that comparisons of membrane potential for all cells were based on values observed during a phase common to all cells, the somatic activity phase. Second, somatic and dendritic spike amplitudes were measured during the somatic and dendritic phases of spontaneous activity rather than after they were elicited by depolarizing current steps. Third, because input resistance values changed during the different phases of spontaneous activity, input resistance measures were based on the 600-ms current step necessary to hyperpolarize the dendrite 20 mV from the membrane potential. Fourth, the same current value used to measure input resistance was then applied to the cell to hyperpolarize it below the spontaneous activity level (−20 mV) to 3

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

granule cell layer, white matter, or the deep nuclei (Shimohama, Saitoh, & Gage, 1990). We found a learning-specific increase in membrane-bound PKC in the molecular layer of cerebellar lobule HVI. Specifically, we found that PKC activation measured by quantitative film autoradiography of [3H]phorbol 12,13-dibutyrate binding in the molecular layer of rabbit cerebellar lobule HVI ipsilateral to the side of eyeblink conditioning shown in Fig. 5 was higher than in the molecular layer of lobule HVI of rabbits given unpaired stimulus presentations or those that were restrained in the training chamber with no stimulus presentations. There were no significant changes in PKC activation in the granule cell layer of lobule HVI, the cerebellar vermis, lobule crus I, or the deep cerebellar nuclei among the three groups (Freeman, Scharenberg, et al., 1998). Synaptic plasticity in the cerebellar cortex There is a long history of studying cerebellar synaptic plasticity in the form of long-term depression that has been reviewed extensively elsewhere (D’Angelo et al., 2015; Hesslow, Jirenhed, Rasmusson, & Johansson, 2013; Hirano, 2018; Hoxha, Tempia, Lippiello, & Miniaci, 2016; Ito, Yamaguchi, Nagao, & Yamzaki, 2014; Jorntell, 2016; Shim et al., 2017) but which is germane to the current review. In brief, stimulation of the climbing fibers and parallel fibers synapsing onto Purkinje cells produced a long-lasting reduction in the parallel fiber synaptic potentials recorded in Purkinje cells – a form of cerebellar plasticity first proposed by Marr (1969) and Albus (1971) and later confirmed experimentally by Ito and Kano (1982), Ito, Sakurai, and Tongroach (1982) and Gilbert and Thach (1977). Although originally used to describe experience-dependent adaptation of the vestibuloocular reflex (Ito, 1998), LTD had also been used in efforts to account for eyeblink conditioning – with varying degrees of success (Carey & Lisberger, 2002; Hesslow, Jirenhed, Rasmusson, & Johansson, 2013; Ito, 2001; Johansson, Jirenhed, Rasmussen, Zucca, & Hesslow, 2018; Koekkoek et al., 2003; Mauk, Garcia, Medina, & Steele, 1998; TakeharaNishiuchi, 2018; Thompson & Steinmetz, 2009). In these accounts, information about a CS such as a tone reaches the cerebellum via mossy fibers whose axons contact the deep cerebellar nuclei via collaterals and then travel up to the cerebellar cortex to synapse onto granule cells whose axons form parallel fibers onto Purkinje cell dendrites and inhibitory interneurons (Cajal, 1894; Sotelo, 2008; Yuste & Tank, 1996). Information about the US such as a puff of air or periorbital shock reaches the cerebellum via climbing fibers whose axons also contact the deep cerebellar nuclei via collaterals and then travel up to the cerebellar cortex to entwine a single Purkinje cell dendritic tree [see Fig. 1]. It has been argued that in the case of delay eyeblink conditioning where the CS and US overlap and coterminate, parallel and climbing fiber inputs do occur together. However, eyeblink conditioning in several species is readily achieved using a trace conditioning paradigm where the CS is terminated several hundred milliseconds before the onset of US (Clark & Zola, 1998; McEchron, Bouwmeester, Tseng, Weiss, & Disterhoft, 1998; McEchron, Tseng, & Disterhoft, 2003; McGlincheyBerroth, Carillo, Gabrieli, Brawn, & Disterhoft, 1997; Moye & Rudy, 1987; Schneiderman, 1966; Solomon, Vander Schaaf, Thompson, &

Fig. 3. Biocytin-labeled and reacted Purkinje cell in a slice of lobule HVI. A patch-clamp electrode with 0.2% biocytin in the intracellular recording solution was used to form a seal on a Purkinje cell soma and the biocytin allowed to passively defuse from the electrode over the course of the recording. The tissue was subsequently post-fixed in 4% paraformaldehyde, re-sectioned, and the biotin was visualized with diaminobenzidine-based processing according to the manufacturer’s instructions. The low-magnification image was recorded with a 4x objective on a Zeiss LSM 510 confocal microscope and shows the entire dendritic tree through to the top of the molecular layer and the axon exiting down into the white matter. (Unpublished data contributed by D. Wang).

potassium channels (Freeman, Scharenberg, et al., 1998; Scharenberg et al., 1991). lmmunocytochemical studies have previously demonstrated that PKC isoforms alpha and gamma are primarily localized within the Purkinje cell layer and molecular layers of the cerebellar cortex and very little PKC immunoreactivity was found in the cerebellar

Table 1 Electrophysiological measures from the Purkinje-cell dendrites in lobule HVI of subjects from Paired, Unpaired, and Naive groups.

Paired (n = 13) Unpaired (n = 12) Naïve (n = 13)

Vm (mV)

Rm (MΩ)

Ihyper (nA)

ss (mV)

Ds (mV)

Iss (nA)

Ids (nA)**

57.9 ± 2.2 53.9 ± 2.0 59.3 ± 1.7

29.9 ± 1.7 25.5 ± 1.6 29.0 ± 1.9

0.7 ± 0.1 0.8 ± 0.1 0.7 ± 0.1

6.3 ± 1.2 5.7 ± 0.8 9.8 ± 2.3

39.8 ± 2.9 34.8 ± 3.5 35.5 ± 3.2

0.4 ± 0.1 0.5 ± 0.1 0.5 ± 0.1

1.2 ± 0.1 1.9 ± 0.2 2.0 ± 0.2

Values are mean ± S.E.M. Vm is the membrane potential during the phase of activity shown by all Purkinje-cell dendrites, i.e., the somatic activity phase. Rm is the input resistance based on the value of the 600-ms current step (hyper) necessary to hyperpolarize the membrane 20 mV below the level of somatic activity. Somatic and dendritic spike amplitude is denoted by ss and, ds, respectively. Threshold current required to elicit somatic and dendritic spikes is denoted by lss and, Ids, respectively. Table from Schreurs et al. (1991), in the public domain. ** p < 0.005, one-way ANOVA. 4

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

Paired

A

Unpaired Transient Hyperpolarization

Transient Hyperpolarization 10 mV

500 ms

After Hyperpolarization

After Hyperpolarization

B Paired

Unpaired

Paired

Unpaired

-0.5

-1.0

-1.5

*** -2.0

-2.5

After Hyperpolarization (mV)

Transient Hyperpolarization (mV)

0.0

-0.5

-1.0

-1.5

-2.0

** -2.5

-3.0

-3.0

Fig. 4. Changes in transient and after hyperpolarization after eyeblink conditioning. A, Example of depolarizing current steps showing smaller transient and after hyperpolarization in a cell from a paired rabbit than in a cell from an unpaired rabbit. B, Mean transient and after hyperpolarization for all cells from paired and unpaired animals. ***p < 0.001; **p < 0.01. Figure from Schreurs et al. (1998), in the public domain.

sequence of stimulation used in slice or intact preparations requires climbing fiber stimulation before parallel fiber stimulation (Crepel & Jaillard, 1991; Ekerot & Kano, 1985; Hansel, Linden, & D’Angelo, 2001; Ito & Kano, 1982; Linden & Connor, 1993; Lisberger, 1998; Raymond, Lisberger, & Mauk, 1996; Sakurai, 1987). Based on intra-dendritic Purkinje cell recordings obtained from rabbit cerebellar slices, we reported that repeated stimulation of climbing fibers and then parallel fibers in the presence of the GABA antagonist, bicuculline, also produced significant depression of parallel fiber excitatory post synaptic potential (epsp) amplitude that continued to increase for at least 20 min after stimulation (Schreurs & Alkon, 1993). However, application of the same stimulation protocol without a GABA antagonist produced a brief depression of parallel fiber epsps that disappeared within minutes. Activation of parallel fibers and then climbing fibers in an order opposite to the LTD-producing sequence (i.e., a classical conditioning-like order of parallel fiber before climbing fiber) produced a brief depression that dissipated quickly. Stimulation of parallel fibers alone produced a small, slowly developing potentiation, but stimulation of parallel fibers during depolarization-induced local dendritic calcium spikes produced significant depression almost immediately which then declined slowly to more modest levels suggesting plasticity may occur in the presence of an influx of calcium (Cormier, Greenwood, & Connor,

Weisz, 1986; Tseng, Guan, Disterhoft, & Weiss, 2004; Weiss et al., 1999). In such cases, delay lines in the mossy/parallel fiber circuit have been proposed which modify the timing of the CS input so that it is coincident with the US input to the cerebellar cortex (Thompson, 1986). However, Huang and Liu have shown that peak latencies of evoked auditory potentials recorded in lobule VI and VII of the cat cerebellum are an order of magnitude shorter (range 8–18 ms) than those required by a delay-line proposal (Huang & Liu, 1985). Moreover, Gould and colleagues have shown that stimulation of pontine nuclei and the inferior olive - areas where stimulation has been substituted for tone and air puff to classically condition the rabbit eyeblink - produce cerebellar population and single unit potentials with latencies of 1–5 ms suggesting there is no significant delay in activation of mossy or climbing fibers (Gould, Sears, & Steinmetz, 1993). More recently, research and theorizing suggest that trace intervals may be bridged by activity in the medial prefrontal cortex which then activates mossy fibers. Thus, the temporal activation of parallel and climbing fibers could fit within the window for pairing-specific LTD (Hattori, Yoon, Disterhoft, & Weiss, 2014; Siegel & Mauk, 2013). LTD in the cerebellum has usually been obtained reliably when: (1) cerebellar slices are bathed in GABA antagonists which abolish disynaptic inhibitory post synaptic potentials; and (2) the temporal 5

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

fiber test pulses. Intra-dendritically recorded Purkinje cell epsps underwent a long-term (> 20 min) reduction in peak amplitude (30%) following paired stimulation of the parallel and climbing fibers but not following unpaired or parallel fiber alone stimulation. Facilitation of the peak amplitude of a second epsp elicited by a parallel fiber train occurred both before and after paired stimulation, suggesting that the locus of depression was not presynaptic. Depression of the peak amplitude of a depolarizing response to focal application of glutamate following pairings of parallel and climbing fiber stimulation added support to a suggested postsynaptic locus of the pairing-specific longterm depression effect. The application of the gamma-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor agonist aniracetam potentiated EPSP peak amplitude by 40% but these values returned to baseline as a result of pairings. With the removal of aniracetam from the bath 20 min following pairings, normal levels of pairing-specific epsp depression were observed indicating that the effect did not result from direct desensitization of AMPA receptors. Incubation of slices in the protein kinase inhibitor H-7 potentiated epsp peak amplitudes slightly (9%) but peak amplitudes returned to baseline levels following pairings. The net reduction in epsp peak amplitude of less than 10% following pairings suggested that H-7 partially blocked pairing-specific long-term depression and that, in turn, this form of long-term depression involved protein kinases. Pretreatment of slices with the protein kinase C inhibitor calphostin C or iontophoretically injected with the calcium chelator EGTA prevented pairing-specific long-term depression (Freeman, Shi, & Schreurs, 1998). The means of induction and specificity of induction suggested that the phenomenology of pairing-specific long-term depression was different from that of traditional LTD because (1) it only occurred with pairings of trains of parallel fiber and climbing fiber stimulation, (2) it occurred without the need for bicuculline, and (3) it overcame the blocking effects of aniracetam. Nevertheless, the involvement of protein kinases and the potential role of calcium suggested the mechanisms involved in the induction of pairing-specific long-term depression and LTD had several features in common. Due to the pairing-specific nature of the long-term synaptic depression observed in these experiments, pairing-specific long-term depression provides a mechanism that may contribute to the role of the cerebellar cortex in classical conditioning. To assess the effects of eyeblink conditioning on synaptic and membrane plasticity further, we again made intra-dendritic recordings in Purkinje cells from parasagittal slices of cerebellar lobule HVI obtained from rabbits given either paired presentations of tone and periorbital electrical stimulation or explicitly unpaired presentations of tone and periorbital electrical stimulation (Schreurs et al., 1997). As before, Purkinje cell dendritic membrane excitability, assessed by the current required to elicit local dendritic calcium spikes, was found to be increased significantly in slices from animals that received eyeblink conditioning whereas membrane potential, input resistance and amplitude of somatic and dendritic spikes were not different in slices from animals given paired or explicitly unpaired stimulus presentations. The location of cells with low thresholds for local dendritic calcium spikes suggested that there are specific sites within lobule HVI where learningrelated changes took place (Schreurs et al., 1997). These areas may correspond to learning “microzones” and are consistent with the locations of some learning-related changes in Purkinje cell activity recorded in vivo (Berthier & Moore, 1986). In cells where thresholds for eliciting parallel fiber-stimulated Purkinje cell epsps were measured, the level of parallel fiber stimulation required to elicit a 6-mV epsp, as well as a 4mV epsp, and a Purkinje cell spike was found to be significantly lower in slices from animals given eyeblink conditioning than those given unpaired stimulus presentations – suggesting conditioning-specific synaptic plasticity. Following a pairing-specific long-term depression protocol shown in Fig. 6, Purkinje cell epsps underwent a long-term (> 20 min) reduction in peak amplitude (−24%) in cells from animals given unpaired stimulus presentations but to a far less extent (−9%) in cells from animals given eyeblink conditioning. In fact, whereas 92% of

Fig. 5. Representative pseudo-color images of [3H]phorbol 12,13-dibutyrate binding in the right lobule HVI for one rabbit from the paired, unpaired and sit groups. The calibrated color bar (nCi/g) is shown for quantitative comparison. There was significantly greater [3H]phorbol 12,13-dibutyrate binding (red) in the section from the animal trained in the paired group than in the two conditions (p < 0.05). Image from Freeman, Scharenberg, et al. (1998), in the public domain. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2000; Finch, Tanaka, & Augustine, 2012; Hartell, 1996; Kohda, Inoue, & Mikoshiba, 1995; Konnerth, Dreessen, & Augustine, 1992; Sakurai, 1990; Sjostrom & Nelson, 2002). Finally, stimulation of parallel fibers at frequencies used in in vivo parallel fiber-climbing fiber stimulation experiments (e.g., 100 Hz) produced an immediate and profound longlasting epsp depression. The depression occurred, however, whether parallel and climbing fibers were stimulated separately (unpaired) or in a classical conditioning-like protocol (paired) where parallel fiber stimulation coterminated with climbing fiber stimulation (10 Hz). The depression observed in both cases was reminiscent of transmitter depletion. Thus, despite obvious differences between in vitro and in vivo preparations, our intra-dendritic Purkinje cell recordings in a rabbit cerebellar slice began to cast some doubt on the hypothesis that traditional LTD functioned as a mechanism underlying eyeblink conditioning. To explore issues of parallel and climbing fiber timing further, we next simulated a classical conditioning procedure by stimulating parallel fiber inputs to Purkinje cells using a brief, high frequency train of eight constant-current pulses 80 ms before climbing fiber inputs to the same Purkinje cell were stimulated using a brief, lower frequency train of three constant-current pulses (Schreurs, Oh, & Alkon, 1996). In these as in other experiments, we assessed the effects of fiber stimulation by measuring the peak amplitude of Purkinje cell epsps to single parallel 6

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

A LTD Pairing

Protocol

5 mV

_

_

20 ms

_

Climbing fiber stimulation

- -- - - - - -

Fig. 6. The effects of eyeblink conditioning on the ability to induce long-term depression. Panel A shows a Purkinje cell intradendritic recording during pairing of a train of parallel fiber pulses (80 ms, 100 Hz, 8 pulses) and a train of climbing fiber pulses (100 ms, 20 Hz, 3 pulses) in a pairing-specific longterm depression protocol. Panel B shows individual epsps in response to single parallel fiber test pulses before (Pre) and 21 min after (Post) the pairing-specific long-term depression protocol for rabbits previously given eyeblink conditioning (Paired) or unpaired stimulus presentations (Unpaired). Panel C documents mean differences in the percent change in EPSP amplitude from Pre to Post for intradendritic recordings from rabbits that had been given paired or unpaired stimulus presentations. Figure from Schreurs et al. (1997), in the public domain.

Parallel fiber stimulation

B

Paired 5 mV 20 ms

Unpaired Post

C

5

EPSP Amplitude Percent Change

Pre

0

LTD

-5 -10 -15 -20 -25 -30

Paired (n=20) Unpaired (n=12)

-35 0

3

6

9

12

15

18

21

Time (min) eyeblink conditioning (Attwell, Ivarsson, Millar, & Yeo, 2002; Boele, Koekkoek, & De Zeeuw, 2010; Christian & Thompson, 2003; Freeman, Halverson, & Poremba, 2005; Heiney, Wohl, Chettih, Ruffolo, & Medina, 2014; Lavond, Kim, & Thompson, 1993; López-Ramos, Houdek, Cendelín, Vožeh, & Delgado-García, 2018; Mojtahedian, Kogan, Kanzawa, Thompson, & Lavond, 2007; Pakaprot, Kim, & Thompson, 2009; Thurling et al., 2015; Wikgren & Korhonen, 2001) but see Perciavalle et al. (2013). As a result, changes in the DCN resulting from eyeblink conditioning have long been hypothesized to occur and there is some evidence from electron microscopy for eyeblink conditioning-specific increases in the number of excitatory synapses in the DCN (Kleim et al., 2002). Previous studies have shown changes in the

cells from unpaired animals showed pairing-specific depression, 50% of cells from animals given eyeblink conditioning showed no depression and, in several cases, actually showed potentiation (Schreurs et al., 1997). These data established there were localized learning-specific changes in membrane and synaptic excitability of Purkinje cells in lobule HVI of the rabbit and, importantly, the long-term changes within the Purkinje cells that affect this enhanced excitability may have occluded subsequent attempts to induce long-term depression. Leaning-specific changes in membrane excitability of deep cerebellar nuclei Lesion and recording studies have long implicated the DCN in 7

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

strength of eyeblink conditioning – in this case, the smaller the AHP, the stronger the eyeblink conditioning.

membrane properties of neurons in rat DCN as a function of development, but due to technical difficulties in obtaining viable DCN slices from adult animals, it has remained unclear whether there are learningrelated alterations in the membrane properties of DCN neurons in adult rats. After some modifications to our slicing procedures (Wang & Schreurs, 2014), we were able to record from DCN pyramidal neurons in cerebellar slices from young rats (P25–26) that had a relatively mature sensory nervous system and were able to acquire eyeblink conditioning compared to rats that received unpaired stimulus presentations or were placed in the training chamber without stimulus presentations (Wang et al., 2018). We used young rats because of the extensive perineuronal net that forms around the DCN and makes patch-clamp recording extremely difficult as mammals develop (Carulli et al., 2007; Hirono et al., 2018; Mueller et al., 2016). Because the ontogeny of eyeblink conditioning overlaps with the development of the perineuronal net, we were limited to only modest average levels of eyeblink conditioning in these rats (Andrews, Freeman, Carter, & Stanton, 1995; Freeman, Nicholson, Muckler, Rabinak, & DiPietro, 2003; Goldsberry & Freeman, 2017). Nevertheless, whole-cell recordings of DCN neurons revealed that delay eyeblink conditioning induced significant changes in membrane properties of evoked DCN action potentials including a reduction in AHP amplitude and a shortened latency. Similar findings were also obtained in hyperpolarization-induced rebound spikes of these DCN neurons. Importantly, as shown in Fig. 7, we found that the size of the AHP was significantly correlated with the

2. Conclusions Taken together, our slice recording experiments show: (1) there are significant, persistent, replicable changes in intrinsic membrane excitability and synaptic plasticity of Purkinje cell dendrites that result from eyeblink conditioning, and (2) that these changes may be attributable, in part, to persistent changes in potassium channels. We have now also shown that there are similar changes in the intrinsic membrane excitability of principal neurons in the deep cerebellar nuclei as a result of eyeblink conditioning. As in other preparations, there is a growing consensus that both intrinsic and synaptic plasticity are required for eyeblink conditioning (Alkon, 1984; Bekisz et al., 2010; Daoudal & Debanne, 2003; Debanne, Inglebert, & Russier, 2019; Disterhoft, Coulter, & Alkon, 1986; Jang & Kim, 2019; Johnston et al., 2003; Lorenzetti, Mozzachiodi, Baxter, & Byrne, 2006; Mozzachiodi & Byrne, 2010; Oh & Disterhoft, 2015; Ross et al., 2019; Sehgal, Song, Ehlers, & Moyer, 2013; Shim, Lee, & Kim, 2018; Song, Ehlers, & Moyer, 2015; Titley, Brunel, & Hansel, 2017; Yang & Santamaria, 2016; Zhang & Linden, 2003). It now remains to document synaptic plasticity in the deep cerebellar nuclei as a result of eyeblink conditioning – a phenomenon that has long been studied in naïve rodents (Hirono et al., 2018; Longley & Yeo, 2014; Pedroarena & Schwarz, 2003; Pugh &

B

A

PD

Sit

AHP amplitude (mV) 60

R=0.688

50 40 30 20

R=-0.06

10

R=-0.05 -16

-14

-12

-10

-8

-6

-4

-20 p<0.05 -30

p<0.01

Conditioned response (%)

PD UP Sit

70

-10

D

80

C

-18

UP

0

0 -2

0

AHP amplitude (mV) Fig. 7. Conditioning-specific changes in principal neurons of rat deep cerebellar nuclei. Panel A shows an individual fluorescently-labeled neuron in the deep cerebellar nuclei (DCN, red scale bar 20 μm). The top inset shows a group of labeled DCN neurons at lower magnification (inset scale bar 200 μm) and the bottom inset shows a biocytin-labeled principal neuron with multiple processes (inset scale bar 50 μm). Panel B depicts individual and mean action potential after-hyperpolarization amplitudes, used to assess membrane excitability, were significantly smaller for rats receiving tone-shock pairings (PD) than those given unpaired presentations of tone and shock (UP) or those that sat in the chamber without stimulus presentations (Sit). Panel C shows a significant positive correlation between AHP amplitude and the percent conditioned responses in rats given tone-shock pairings but not in unpaired or sit control rats. Panel D shows a waterfall plot of conditioned eyeblinks for an individual rat given tone-shock pairings. Figure adapted from Wang et al. (2018). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 8

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

Raman, 2009; Wu & Raman, 2017) and hinted at in histological studies (Kleim et al., 2002).

Trends in Neurosciences, 29, 587–599. Ekerot, C. F., & Kano, M. (1985). Long-term depression of parallel fibre synapses following stimulation of climbing fibres. Brain Research, 342, 357–360. Farley, J., & Alkon, D. L. (1985). Cellular mechanisms of learning, memory, and information storage. Annual Review of Psychology, 36, 419–494. Finch, E. A., Tanaka, K., & Augustine, G. J. (2012). Calcium as a trigger for cerebellar long-term synaptic depression. Cerebellum, 11, 706–717. Freeman, J. H., Jr., Halverson, H. E., & Poremba, A. (2005). Differential effects of cerebellar inactivation on eyeblink conditioned excitation and inhibition. Journal of Neuroscience, 25, 889–895. Freeman, J. H., Jr., Nicholson, D. A., Muckler, A. S., Rabinak, C. A., & DiPietro, N. T. (2003). Ontogeny of eyeblink conditioned response timing in rats. Behavioral Neuroscience, 117, 283–291. Freeman, J. H., Jr., Scharenberg, A. M., Olds, J. L., & Schreurs, B. G. (1998). Classical conditioning increases membrane-bound protein kinase C in rabbit cerebellum. Neuroreport, 9, 2669–2673. Freeman, J. H., Jr., Shi, T., & Schreurs, B. G. (1998). Pairing-specific long-term depression prevented by blockade of PKC or intracellular Ca2+. Neuroreport, 9, 2237–2241. Gilbert, P. F. C., & Thach, W. T. (1977). Purkinje cell activity during motor learning. Brain Research, 128, 309–328. Goldsberry, M. E., & Freeman, J. H. (2017). Sensory system development influences the ontogeny of trace eyeblink conditioning. Developmental Psychobiology, 59, 70–76. Gonzalez-Joekes, J. (2014). Transsynaptic Delineation of the Rabbit Eyeblink Premotor Pathway: Identifying Anterior Interpositus Neurons and Their Synaptic Changes as a Function of Learning. Unpublished Doctoral DissertationMorgantown, WV: West Virginia University124. Gormezano, I., Kehoe, E. J., & Marshall, B. S. (1983). Twenty years of classical conditioning research with the rabbit. In J. M. Sprague (Ed.). Progress in Psychobiology and Physiological Psychology (pp. 197–275). New York: Academic Press. Gould, T. J., Sears, L. L., & Steinmetz, J. E. (1993). Possible CS and US pathways for rabbit classical eyelid conditioning: Electrophysiological evidence for projections from the pontine nuclei and inferior olive to cerebellar cortex and nuclei. Behavioral and Neural Biology, 60, 172–185. Hansel, C., Linden, D. J., & D'Angelo, E. (2001). Beyond parallel fiber LTD: The diversity of synaptic and non-synaptic plasticity in the cerebellum. Nature Neuroscience, 4, 467–475. Hartell, N. A. (1996). Strong activation of parallel fibers produces localized calcium transients and a form of LTD that spreads to distant synapses. Neuron, 16, 601–610. Hattori, S., Yoon, T., Disterhoft, J. F., & Weiss, C. (2014). Functional reorganization of a prefrontal cortical network mediating consolidation of trace eyeblink conditioning. The Journal of Neuroscience, 34, 1432–1445. Heiney, S. A., Wohl, M. P., Chettih, S. N., Ruffolo, L. I., & Medina, J. F. (2014). Cerebellardependent expression of motor learning during eyeblink conditioning in head-fixed mice. Journal of Neuroscience, 34, 14845–14853. Hesslow, G., Jirenhed, D. A., Rasmusson, A., & Johansson, F. (2013). Classical conditioning of motor responses: What is the learning mechanism? Neural Networks, 47, 81–87. Hilgard, E. R., & Campbell, A. A. (1936). The course of acquisition and retention of conditioned eyelid responses in man. Journal of Experimental Psychology, 19, 227–247. Hilgard, E. R., & Marquis, D. G. (1935). Acquisition, extinction, and retention of conditioned responses to light in dogs. Journal of Comparative Psychology, 19, 29–58. Hirano, T. (2018). Regulation and interaction of multiple types of synaptic plasticity in a Purkinje neuron and their contribution to motor learning. Cerebellum, 17, 756–765. Hirono, M., Watanabe, S., Karube, F., Fujiyama, F., Kawahara, S., Nagao, S., ... Misonou, H. (2018). Perineuronal nets in the deep cerebellar nuclei regulate GABAergic transmission and delay eyeblink conditioning. The Journal of Neuroscience, 38, 6130–6144. Hoxha, E., Tempia, F., Lippiello, P., & Miniaci, M. C. (2016). Modulation, plasticity and pathophysiology of the parallel fiber-Purkinje cell synapse. Frontiers in Synaptic Neuroscience, 8, 35. Huang, C. M., & Liu, G. (1985). Electrophysiological mapping of the auditory areas in the cerebellum of the cat. Brain Research, 335, 121–129. Hughes, B., & Schlosberg, H. (1938). Conditioning in the white rat. IV. The conditioned lid reflex. Journal of Experimental Psychology, 23, 641–650. Ito, M. (1998). Cerebellar learning in the vestibulo-ocular reflex. Trends in Cognitive Sciences, 2, 313–321. Ito, M. (2001). Cerebellar long-term depression: Characterization, signal transduction, and functional roles. Physiological Reviews, 81, 1143–1195. Ito, M., & Kano, M. (1982). Long-lasting depression of parallel fiber-Purkinje cell transmission induced by conjunctive stimulation of parallel fibers and climbing fibers in the cerebellar cortex. Neuroscience Letters, 33, 253–258. Ito, M., Sakurai, M., & Tongroach, P. (1982). Climbing fibre induced depression of both mossy fibre responsiveness and glutamate sensitivity of cerebellar Purkinje cells. Journal of Physiology (London), 324, 113–134. Ito, M., Yamaguchi, K., Nagao, S., & Yamzaki, T. (2014). Long-term depression as a model of cerebellar plasticity. Progress in Brain Research, 210, 1–30. Jang, D. C., & Kim, S. J. (2019). Plasticity leading to cerebellum-dependent learning: Two different regions, two different types. Pflügers Archiv - European Journal of Physiology, 471(7), 927–934. Johansson, F., Jirenhed, D. A., Rasmussen, A., Zucca, R., & Hesslow, G. (2018). Absence of parallel fibre to purkinje cell LTD during eyeblink conditioning. Scientific Reports, 8, 14777. Johnston, D., Christie, B. R., Frick, A., Gray, R., Hoffman, D. A., Schexnayder, L. K., ... Yuan, L.-L. (2003). Active dendrites, potassium channels and synaptic plasticity. Philosophical Transactions of the Royal Society London B, 358, 667–674.

Acknowledgments This work was supported by the NINDS intramural research program, NIH Grant NS094009, a bridge grant from West Virginia University Health Sciences Research Office, and Rockefeller Neuroscience Institute funds. The contents of this article are solely the responsibility of the author and do not necessarily represent the official views of the NIH. The author is grateful to all the wonderful undergraduate and graduate students and, most importantly, the postdoctoral fellows without whom this research would not have been possible. References Albus, J. S. (1971). A theory of cerebellar function. Mathematical Biosciences, 10, 25–61. Alkon, D. L. (1984). Calcium-mediated reduction of ionic currents: A biophysical memory trace. Science, 226, 1037–1045. Andrews, S. J., Freeman, J. H., Jr., Carter, C. S., & Stanton, M. E. (1995). Ontogeny of eyeblink conditioning in the rat: Auditory frequency and discrimination learning effects. Developmental Psychobiology, 28, 307–320. Attwell, P. J. E., Ivarsson, M., Millar, L., & Yeo, C. H. (2002). Cerebellar mechanisms in eyeblink conditioning. Annals of the New York Academy of Sciences, 978, 79–92. Bekisz, M., Garkun, Y., Wabno, J., Hess, G., Wrobel, A., & Kossut, M. (2010). Increased excitability of cortical neurons induced by associative learning: An ex vivo study. European Journal of Neuroscience, 32, 1715–1725. Berger, T. W., Alger, B., & Thompson, R. F. (1976). Neuronal substrate of classical conditioning in the hippocampus. Science, 192, 483–485. Berthier, N. E., & Moore, J. W. (1986). Cerebellar Purkinje cell activity related to the classically conditioned nictitating membrane response. Experimental Brain Research, 63, 341–350. Blosa, M., Bursch, C., Weigel, S., Holzer, M., Jager, C., Janke, C., ... Morawski, M. (2016). Reorganization of synaptic connections and perineuronal nets in the deep cerebellar nuclei of Purkinje cell degeneration mutant mice. Neural Plasticity, 2016, 1–17. Boele, H.-J., Koekkoek, S. K. E., & De Zeeuw, C. I. (2010). Cerebellar and extracerebellar involvement in mouse eyeblink conditioning; the ACDC model. Frontiers in Cellular Neuroscience, 3, 19. Cajal, S. R. (1894). The Croonian lecture. - La fine structure des centres nerveux. Proceedings of the Royal Society of London, 55, 444–468. Carey, M. R., & Lisberger, S. G. (2002). Embarrassed, but not depressed: Eye opening lessons for cerebellar learning. Neuron, 35, 223–226. Carulli, D., Rhodes, K. E., & Fawcett, J. W. (2007). Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum. Journal of Comparative Neurology, 501, 83–94. Christian, K. M., & Thompson, R. F. (2003). Neural substrates of eyeblink conditioning: Acquisition and retention. Learning and Memory, 11, 427–455. Clark, R. E., & Zola, S. (1998). Trace eyeblink classical conditioning in the monkey: A nonsurgical method and behavioral analysis. Behavioral Neuroscience, 112, 1062–1068. Cleary, L. J., Lee, W. L., & Byrne, J. H. (1998). Cellular correlates of long-term sensitization in Aplysia. Journal of Neuroscience, 18, 5988–5998. Coleman, S. R. (1985). The problem of volition and the conditioned reflex. Part I: Conceptual background, 1900–1940. Behaviorism, 13, 99–124. Coleman, S. R., & Webster, S. (1988). The problem of volition and the conditioned reflex. Part II: Voluntary-responding subjects, 1951–1980. Behaviorism, 16, 17–49. Cormier, R. J., Greenwood, A. C., & Connor, J. A. (2000). Bidirectional synaptic plasticity correlated with the magnitude of dendritic calcium transients above a threshold. Journal of Neurophysiology, 85, 399–406. Coulter, D. A., Lo, T., Kubota, M., Disterhoft, J. F., Moore, J. W., & Alkon, D. L. (1989). Classical conditioning reduces amplitude and duration of calcium-dependent afterhyperpolarization in rabbit hippocampal pyramidal cells. Journal of Neurophysiology, 61, 971–981. Crepel, F., & Jaillard, D. (1991). Pairing of pre- and postsynaptic activities in cerebellar Purkinje cells induces long-term changes in synaptic efficacy in vitro. Journal of Physiology (London), 432, 123–141. D’Angelo, E., Mapelli, L., Casellato, C., Garrido, J. A., Luque, N., Monaco, J., & Ros, E. (2015). Distributed circuit plasticity: new clues for the cerebellar mechanisms of learning. Cerebellum, 15, 139–151. Daoudal, G., & Debanne, D. (2003). Long-term plasticity of intrinsic excitability: Learning rules and mechanisms. Learning and Memory, 10, 456–465. Debanne, D., Inglebert, Y., & Russier, M. (2019). Plasticity of intrinsic neuronal excitability. Current Opinion in Neurobiology, 54, 73–82. Delgado-Garcia, J. M., & Gruart, A. (2002). The role of interpositus nucleus in eyelid conditioned responses. The Cerebellum, 1, 289–308. Delgado-Garcia, J. M., & Gruart, A. (2006). Building new motor responses: Eyelid conditioning revisited. Trends in Neurosciences, 29, 330–338. Disterhoft, J. F., Coulter, D. A., & Alkon, D. L. (1986). Conditioning-specific membrane changes of rabbit hippocampal neurons measured in vitro. Proceedings of the National Academy of Sciences, 83, 2733–2737. Disterhoft, J. F., & Oh, M. M. (2006). Learning, aging and intrinsic neuronal plasticity.

9

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

Nolan, M. F., Malleret, G., Lee, K. H., Gibbs, E., Dudman, J. T., Santoro, B., ... Morozov, A. (2003). The hyperpolarization-activated HCN1 channel is important for motor learning and neuronal integration by cerebellar Purkinje cells. Cell, 115, 551–564. Oh, M. M., & Disterhoft, J. F. (2015). Increased excitability of both principal neurons and interneurons during associative learning. Neuroscientist, 21, 372–384. Olds, J. L., Anderson, M. L., McPhie, D. L., Staten, L. D., & Alkon, D. L. (1989). Imaging of memory-specific changes in the distribution of protein kinase C in the hippocampus. Science, 245, 866–869. Pakaprot, N., Kim, S., & Thompson, R. F. (2009). The role of the cerebellar Interpositus nucleus in short and long term memory for trace eyeblink conditioning. Behavioral Neuroscience, 123, 54–61. Pedroarena, C. M., & Schwarz, C. (2003). Efficacy and short-term plasticity at GABAergic synapses between Purkinje and cerebellar nuclei neurons. Journal of Neurophysiology, 89, 704–715. Perciavalle, V., Apps, R., Bracha, V., Delgado-Garcia, J. M., Gibson, A. R., Leggio, M., ... Sanchez-Campusano, R. (2013). Consensus paper: Current views on the role of cerebellar interpositus nucleus in movement control and emotion. Cerebellum, 12, 738–757. Pugh, J. R., & Raman, I. M. (2009). Nothing can be coincidence; synaptic inhibition and plasticity in the cerebellar nuclei. Trends in Neurosciences, 32, 170–177. Raymond, J. L., Lisberger, S. G., & Mauk, M. D. (1996). The cerebellum: A neuronal learning machine? Science, 272, 1126–1131. Ross, M. T., Flores, D., Bertram, R., Johnson, F., Wu, W., & Hyson, R. L. (2019). Experience-dependent intrinsic plasticity during auditory learning. The Journal of Neuroscience, 39, 1206–1221. Sakurai, M. (1987). Synaptic modification of parallel fibre-Purkinje cell transmission in in vitro guinea-pig cerebellar slices. Journal of Physiology (London), 394, 463–480. Sakurai, M. (1990). Calcium is an intracellular mediator of the climbing fiber in induction of cerebellar long-term depression. Proceedings of the National Academy of Sciences, USA, 87, 3383–3385. Scharenberg, A. M., Olds, J. L., Schreurs, B. G., Craig, A. M., & Alkon, D. L. (1991). Protein kinase C redistribution within CA3 stratum oriens during acquisition of nictitating membrane conditioning in the rabbit. Proceedings of the National Academy of Sciences, USA, 88, 6637–6641. Schneiderman, N. (1966). Interstimulus interval function of the nictitating membrane response of the rabbit under delay versus trace conditioning. Journal of Comparative and Physiological Psychology, 62, 397–402. Schneiderman, N., Fuentes, I., & Gormezano, I. (1962). Acquisition and extinction of the classically conditioned eyelid response in the albino rabbit. Science, 136, 650–652. Schreurs, B. G., & Alkon, D. L. (1993). Rabbit cerebellar slice analysis of long-term depression and its role in classical conditioning. Brain Research, 631, 235–240. Schreurs, B. G., Gusev, P. A., Tomsic, D., Alkon, D. L., & Shi, T. (1998). Intracellular correlates of acquisition and long-term memory of classical conditioning in Purkinje cell dendrites in slices of rabbit cerebellar lobule HVI. Journal of Neuroscience, 18, 5498–5507. Schreurs, B. G., Oh, M. M., & Alkon, D. L. (1996). Pairing-specific long-term depression of Purkinje cell excitatory postsynaptic potentials results from a classical conditioning procedure in the rabbit cerebellar slice. Journal of Neurophysiology, 75, 1051–1060. Schreurs, B. G., Sanchez-Andres, J. V., & Alkon, D. L. (1991). Learning-specific differences in Purkinje-cell dendrites of lobule HVI (lobulus simplex): Intracellular recording in a rabbit cerebellar slice. Brain Research, 548, 18–22. Schreurs, B. G., Sanchez-Andres, J. V., & Alkon, D. L. (1992). GABA-induced responses in purkinje cell dendrites of the rabbit cerebellar slice. Brain Research, 597, 99–107. Schreurs, B. G., Tomsic, D., Gusev, P. A., & Alkon, D. L. (1997). Dendritic excitability microzones and occluded long-term depression after classical conditioning of the rabbit's nictitating membrane response. Journal of Neurophysiology, 77, 86–92. Schwartzkroin, P. A. (1975). Characteristics of CA1 neurons recorded intracellularly in the hippocampalin vitro slice preparation. Brain Research, 85, 423–436. Sehgal, M., Song, C., Ehlers, V. L., & Moyer, J. R. (2013). Learning to learn - Intrinsic plasticity as a metaplasticity mechanism for memory formation. Neurobiology of Learning and Memory, 105, 186–189. Shim, H. G., Jang, D. C., Lee, J., Chung, G., Lee, S., Kim, Y. G., ... Kim, S. J. (2017). Longterm depression of intrinsic excitability accompanied by synaptic depression in cerebellar Purkinje cells. The Journal of Neuroscience, 37, 5659–5669. Shim, H. G., Lee, Y. S., & Kim, S. J. (2018). The emerging concept of intrinsic plasticity: Activity-dependent modulation of intrinsic excitability in cerebellar purkinje cells and motor learning. Experimental Neurobiology, 27, 139–154. Shimohama, S., Saitoh, T., & Gage, F. H. (1990). Differential expression of protein kinase C isozymes in rat cerebellum. Journal of Chemical Neuroanatomy, 3, 367–375. Siegel, J. J., & Mauk, M. D. (2013). Persistent activity in prefrontal cortex during trace eyelid conditioning: Dissociating responses that reflect cerebellar output from those that do not. Journal of Neuroscience, 33, 15272–15284. Sjostrom, P. J., & Nelson, S. B. (2002). Spike timing, calcium signals and synaptic plasticity. Current Opinion in Neurobiology, 12, 305–314. Solomon, P. R., & Moore, J. W. (1975). Latent inhibition and stimulus generalization of the classically conditioned nictitating membrane response in rabbits (Oryctolagus cuniculus) following dorsal hippocampal ablation. Journal of Comparative and Physiological Psychology, 89, 1192–1203. Solomon, P. R., Vander Schaaf, E. R., Thompson, R. F., & Weisz, D. J. (1986). Hippocampus and trace conditioning of the rabbit's classically conditioned nictitating membrane response. Behavioral Neuroscience, 100, 729–744. Song, C., Ehlers, V. L., & Moyer, J. R., Jr. (2015). Trace fear conditioning differentially modulates intrinsic excitability of medial prefrontal cortex-basolateral complex of amygdala projection neurons in infralimbic and prelimbic cortices. Journal of Neuroscience, 35, 13511–13524. Sotelo, C. (2008). Viewing the cerebellum through the eyes of Ramon Y Cajal. Cerebellum,

Jorntell, H. (2016). Cerebellar synaptic plasticity and the credit assignment problem. Cerebellum, 15, 104–111. Kehoe, E. J., & Macrae, M. (2002). Fundamental behavioral methods and findings in classical conditioning. In J. W. Moore (Ed.). A neuroscientist's guide to classical conditioning (pp. 171–231). New York: Springer. Kelly, T. M., Zuo, C. C., & Bloedel, J. R. (1990). Classical conditioning of the eyelid reflex in the decerebrate-decerebellate rabbit. Behavioural Brain Research, 38, 7–18. Kleim, J. A., Freeman, J. H., Jr., Bruneau, R., Nolan, B. C., Cooper, N. R., Zook, A., & Walters, D. (2002). Synapse formation is associated with memory storage in the cerebellum. Proceedings of the National Academy of Sciences, USA, 99, 13228–13231. Koekkoek, S. K. E., Hulscher, H. C., Dortland, B. R., Hensbroek, R. A., Elgersma, Y., Ruigrok, T. J. H., & De Zeeuw, C. I. (2003). Cerebellar LTD and learning-dependent timing of conditioned eyelid responses. Science, 301, 1736–1739. Kohda, K., Inoue, T., & Mikoshiba, K. (1995). Ca2+ release from Ca2+ stores, particularly from ryanodine-sensitive Ca2+ stores, is required for the induction of LTD in cultured cerebellar Purkinje cells. Journal of Neurophysiology, 74, 2184–2188. Konnerth, A., Dreessen, J., & Augustine, G. J. (1992). Brief dendritic calcium signals initiate long-lasting synaptic depression in cerebellar Purkinje cells. Proceedings of the National Academy of Sciences, USA, 89, 7051–7055. Koppensteiner, P., Galvin, C., & Ninan, I. (2019). Lack of experience-dependent intrinsic plasticity in the adolescent infralimbic medial prefrontal cortex. Synapse, 73, e22090. Lavond, D. G., Kim, J. J., & Thompson, R. F. (1993). Mammalian brain substrates of aversive classical conditioning. Annual Review of Psychology, 44, 317–342. Linden, D. J., & Connor, J. A. (1993). Cellular mechanisms of long-term depression in the cerebellum. Current Opinion in Neurobiology, 3, 401–406. Lisberger, S. G. (1998). Cerebellar LTD: A molecular mechanism of behavioral learning? Cell, 92, 701–704. Lisman, J., Cooper, K., Sehgal, M., & Silva, A. J. (2018). Memory formation depends on both synapse-specific modifications of synaptic strength and cell-specific increases in excitability. Nature Neuroscience, 21, 309–314. Llinas, R., & Sugimori, M. (1980a). Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. Journal of Physiology (London), 305, 197–213. Llinas, R., & Sugimori, M. (1980b). Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. Journal of Physiology (London), 305, 171–195. Llinas, R. R. (2014). Intrinsic electrical properties of mammalian neurons and CNS function: A historical perspective. Frontiers in Cellular Neuroscience, 8. https://doi. org/10.3389/fncel.2014.00320. Longley, M., & Yeo, C. H. (2014). Distribution of neural plasticity in cerebellum-dependent motor learning. Progress in Brain Research, 210, 79–101. López-Ramos, J. C., Houdek, Z., Cendelín, J., Vožeh, F., & Delgado-García, J. M. (2018). Timing correlations between cerebellar interpositus neuronal firing and classically conditioned eyelid responses in wild-type and Lurcher mice. Scientific Reports, 8, 10697. Lorenzetti, F. D., Mozzachiodi, R., Baxter, D. A., & Byrne, J. H. (2006). Classical and operant conditioning differentially modify the intrinsic properties of an identified neuron. Nature Neuroscience, 9, 17–19. Marr, D. (1969). A theory of cerebellar cortex. Journal of Physiology (London), 202, 437–470. Mauk, M. D., Garcia, K. S., Medina, J. F., & Steele, P. M. (1998). Does cerebellar LTD mediate motor learning? Toward a resolution without a smoking gun. Neuron, 20, 359–362. McCormick, D. A., Clark, G. A., Lavond, D. G., & Thompson, R. F. (1982). Initial localization of the memory trace for a basic form of learning. Proceedings of the National Academy of Sciences, USA, 79, 2731–2735. McCormick, D. A., & Thompson, R. F. (1984a). Cerebellum: Essential involvement in the classically conditioned eyelid response. Science, 223, 296–299. McCormick, D. A., & Thompson, R. F. (1984b). Neuronal responses of the rabbit cerebellum during acquisition and performance of a classically conditioned nictitating membrane-eyelid response. Journal of Neuroscience, 4, 2811–2822. McEchron, M. D., Bouwmeester, H., Tseng, W., Weiss, C., & Disterhoft, J. F. (1998). Hippocampectomy disrupts auditory trace fear conditioning and contextual fear conditioning in the rat. Hippocampus, 8, 638–646. McEchron, M. D., Tseng, W., & Disterhoft, J. F. (2003). Single neurons in CA1 hippocampus encode trace interval duration during trace heart rate (fear) conditioning in rabbit. Journal of Neuroscience, 23, 1535–1547. McGlinchey-Berroth, R., Carillo, M. C., Gabrieli, J. D. E., Brawn, C. M., & Disterhoft, J. F. (1997). Impaired trace eyeblink conditioning in bilateral, medial-temporal lobe amnesia. Behavioral Neuroscience, 111, 873–882. Meadows, J. P., Guzman-Karlsson, M. C., Phillips, S., Brown, J. A., Strange, S. K., Sweatt, J. D., & Hablitz, J. J. (2016). Dynamic DNA methylation regulates neuronal intrinsic membrane excitability. Science Signaling, 9, 1–15. Misonou, H., Mohaptra, D. P., Park, E. W., Leung, V., Zhen, D., Misonou, K., ... Trimmer, J. S. (2004). Regulation of ion channel localization and phosphorylation by neuronal activity. Nature Neuroscience, 7, 711–718. Mojtahedian, S., Kogan, D. R., Kanzawa, S. A., Thompson, R. F., & Lavond, D. G. (2007). Dissociation of conditioned eye and limb responses in the cerebellar interpositus. Physiology and Behavior, 91, 9–14. Moye, T. B., & Rudy, J. W. (1987). Ontogenesis of trace conditioning in young rats: Dissociation of associative and memory processes. Developmental Psychobiology, 20, 405–414. Mozzachiodi, R., & Byrne, J. H. (2010). More than synaptic plasticity: Role of nonsynaptic plasticity in learning and memory. Trends in Neurosciences, 33, 17–26. Mueller, A. L., Davis, A., Sovich, S., Carlson, S. S., & Robinson, F. R. (2016). Distribution of N-acetylgalactosamine-positive perineuronal nets in the macaque brain: Anatomy and implications. Neural Plasticity, 2016, 19.

10

Neurobiology of Learning and Memory 166 (2019) 107094

B.G. Schreurs

conditioned response acquired by a somatosensory conditioned stimulus in rabbit eyeblink conditioning. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 25, 1161–1168. Woodruff-Pak, D. S., & Steinmetz, J. E. (2000). Past, present, and future of human eyeblink conditioning. In D. S. Woodruff-Pak, & J. E. Steinmetz (Vol. Eds.), Eyeblink classical conditioning. Applications in humans: Vol. 1, (pp. 1–17). Boston: Kluwer Academic Publishers. Woody, C. D. (1970). Conditioned eye blink: Gross potential activity at coronal-pericruciate cortex of the cat. Journal of Neurophysiology, 33, 838–850. Wu, Y., & Raman, I. M. (2017). Facilitation of mossy fibre-driven spiking in the cerebellar nuclei by the synchrony of inhibition. The Journal of Physiology, 595, 5245–5264. Xu, J., & Kang, J. (2005). The mechanisms and functions of activity-dependent long-term potentiation of intrinsic excitability. Reviews in the Neurosciences, 16, 311–323. Yang, Z., & Santamaria, F. (2016). Purkinje cell intrinsic excitability increases after synaptic long term depression. Journal of Neurophysiology, 116, 1208–1217. Yeo, C. H., Hardiman, M. J., & Glickstein, M. (1984). Discrete lesions of the cerebellar cortex abolish the classically conditioned nictitating membrane response of the rabbit. Behavioural Brain Research, 13, 261–266. Yeo, C. H., Hardiman, M. J., & Glickstein, M. (1985a). Classical conditioning of the nictitating membrane response of the rabbit. I. Lesions of the cerebellar nuclei. Experimental Brain Research, 60, 87–98. Yeo, C. H., Hardiman, M. J., & Glickstein, M. (1985b). Classical conditioning of the nictitating membrane response of the rabbit. II. Lesions of the cerebellar cortex. Experimental Brain Research, 60, 99–113. Yeo, C. H., Hardiman, M. J., & Glickstein, M. (1985c). Classical conditioning of the nictitating membrane response of the rabbit. III. Connections of cerebellar lobule HVI. Experimental Brain Research, 60, 114–126. Yuste, R., & Tank, D. W. (1996). Dendritic integrations in mammalian neurons, a century after Cajal. Neuron, 16, 701–716. Zhang, W., & Linden, D. J. (2003). The other side of the engram: Experience-driven changes in neuronal intrinsic excitability. Nature Reviews Neuroscience, 4, 885–900. Zheng, N., & Raman, I. M. (2010). Synaptic inhibition, excitation, and plasticity in neurons of the cerebellar nuclei. Cerebellum, 9, 56–66.

7, 517–522. Spence, K. W., & Spence, J. T. (1966). Sex and anxiety difference in eyelid conditioning. Psychological Bulletin, 65, 137–142. Steinmetz, J. E., & Woodruff-Pak, D. S. (2000). Animal models in eyeblink classical conditioning. In D. S. Woodruff-Pak, & J. E. Steinmetz (Vol. Eds.), Eyeblink classical conditioning. Animal models: Vol. 2, (pp. 1–15). Boston: Kluwer Academic Publishers. Takehara-Nishiuchi, K. (2018). The anatomy and physiology of eyeblink classical conditioning. Current Topics in Behavioral Neurosciences, 37, 297–323. Thompson, R. F. (1986). The neurobiology of learning and memory. Science, 233, 941–947. Thompson, R. F., & Steinmetz, J. E. (2009). The role of the cerebellum in classical conditioning of discrete behavioral responses. Neuroscience, 162, 732–755. Thurling, M., Kahl, F., Maderwald, S., Stefanescu, R. M., Schlamann, M., Boele, H. J., ... Timmann, D. (2015). Cerebellar cortex and cerebellar nuclei are concomitantly activated during eyeblink conditioning: A 7T fMRI study in humans. Journal of Neuroscience, 35, 1228–1239. Titley, H. K., Brunel, N., & Hansel, C. (2017). Toward a neurocentric view of learning. Neuron, 95, 19–32. Tseng, W., Guan, R., Disterhoft, J. F., & Weiss, C. (2004). Trace eyeblink conditioning is hippocampally dependent in mice. Hippocampus, 14, 58–65. Wang, D., & Schreurs, B. G. (2014). Maturation of membrane properties of neurons in the rat deep cerebellar nuclei. Developmental Neurobiology, 74, 1268–1276. Wang, D., Smith-Bell, C. A., Burhans, L. B., O’Dell, D. E., Bell, R. W., & Schreurs, B. G. (2018). Changes in membrane properties of rat deep cerebellar nuclear projection neurons during acquisition of eyeblink conditioning. Proceedings of the National Academy of Sciences, 115, E9419–E9428. Weiss, C., Knuttinen, M.-G., Power, J. M., Patel, R. I., O'Connor, M. S., & Disterhoft, J. F. (1999). Trace eyeblink conditioning in the freely moving rat: Optimizing the conditioning parameters. Behavioral Neuroscience, 113, 1100–1105. Welsh, J. P., & Harvey, J. A. (1989). Cerebellar lesions and the nictitating membrane reflex: Performance deficits of the conditioned and unconditioned response. Journal of Neuroscience, 9, 299–311. Wikgren, J., & Korhonen, T. (2001). Inactivation of the interpositus nucleus blocks the

11