Several recent reviews have described the architecture of the adult cerebellar cortex (e.g., Apps and Garwicz, 2005; Apps and Hawkes, 2009; Ruigrok, 2011). In brief, a range of expression markers expressed in subsets of Purkinje cells have revealed an orthogonal matrix of transverse zones and parasagittal stripes (Figure 1). First, the cerebellar cortex is divided by transverse boundaries into transverse zones. These are most easily recognized in the vermis but appear to have their counterparts in the hemispheres as well. Each transverse zone is further subdivided into long narrow stripes that run parasagittally from rostral to caudal. The most-studied example is the expression pattern of zebrin II/aldolase C, which identifies a stereotyped array of zebrin II+ and zebrin II- stripes (e.g., Brochu et al., 1990; Hawkes and Gravel, 1991; Ahn et al., 1994; Hawkes and Herrup, 1995; Figures 1A, B). The combination of multiple such patterns adds up to a cerebellar cortex with several hundred distinct topographical units (e.g., Hawkes, 1997; Hawkes et al., 1997, 1999; Armstrong et al., 2000).

The Purkinje cell expression domains are reproducible between individuals to a remarkable level—individual stripes comprised of no more than 100 or so Purkinje cells are faithfully reproduced (e.g., the P4b+/P5a+ stripes in the hemispheres: Hawkes and Leclerc, 1987; Figures 1A, B). Indeed, although the size of a particular zone or stripe may be modified to suit the animal’s mode of life a common ground plan is conserved across all mammals studied to date (~30 species—Sillitoe et al., 2005; Marzban and Hawkes, 2011) and is also found in birds (Pakan et al., 2007; Iwanuik et al., 2009; Marzban et al., 2010).

Stripes of Purkinje cells are targets of specific afferent subsets during development and restrict their terminal fields in the adult, with the result that specific afferent subsets terminate in stripes. Studies over the past 25 years or so have shown that afferent terminal fields are precisely aligned with Purkinje cell stripes. These studies have combined immunocytochemistry for stripe antigens with anterograde tracing to generate detailed topographical maps, in particular relating afferent terminal fields to zebrin II+/− stripes (e.g., climbing fibers—Gravel et al., 1987; Voogd et al., 2003; Sugihara and Shinoda, 2004; Voogd and Ruigrok, 2004; Sugihara and Quy, 2007 etc.; mossy fibers—Gravel and Hawkes, 1990; Akintunde and Eisenman, 1994; Ji and Hawkes, 1994; Armstrong et al., 2009; etc.). In some cases, molecular differences have also been demonstrated between afferent subsets. For example, mossy fibers that express somatostatin terminate on Purkinje cell stripes that express the small heat shock protein (HSP25; Armstrong et al., 2009), and climbing fibers immunoreactive for corticotropin-releasing factor (CRF) terminate selectively on zebrin II+ Purkinje cells (Sawada et al., 2008; see Section Corticotropin-releasing Factor).

Although striped patterns of Purkinje cell gene expression are aligned with stripes of afferent innervation, the formation and maintenance of stripes is not contingent upon afferent input: chemical or surgical afferent lesions do not alter the pattern (zebrin—Leclerc et al., 1988; Zagrebelsky et al., 1996, 1997; sphingosine kinase 1a—Terada et al., 2004; HSP25—Armstrong et al., 2001; L7/pcp2—Oberdick et al., 1993; etc.), and subtype phenotypes are expressed in slice and dissociated cerebellar cultures and after grafting the cerebellar anlage to an ectopic location (e.g., Wassef et al., 1990; Seil et al., 1995).

Purkinje cell stripe boundaries are also restriction boundaries for interneurons. Most prominent among these are the granule cells. First, the analysis of murine chimeras has identified a reproducible set of lineage boundaries within the granular layer that align with the transverse boundaries seen in the Purkinje cells (Hawkes et al., 1999). Multiple expression boundaries are also found at these locations in the adult and in the external granular layer during development (reviewed in Armstrong and Hawkes, 2000; Consalez and Hawkes, 2013). This strongly suggests that different granule cell lineages exploit the underlying Purkinje cell zonal architecture as the external granular layer spreads to cover the embryonic cerebellar anlage. Secondly, in the adult granular layer a complex array of patches and stripes can be revealed (e.g., nitric oxide (NO) synthase or its surrogate, reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase: Hawkes and Turner, 1994; Schilling et al., 1994; Ozol and Hawkes, 1997; Hawkes et al., 1998). These also align with the Purkinje cell architecture. It is difficult to credit that these represent cell autonomous properties of the granule cells, given the challenges such a model would present for the targeting of granule cell migration and settling, so it is more likely that the expression patterns are secondary to the local environment (e.g., Purkinje cells or mossy fibers).

First of all a reproducible pattern of stripes is revealed in the molecular layer of the cerebellar cortex by using immunocytochemistry for the synaptic vesicle protein synaptophysin (Hawkes et al., 1985), with stripes of higher expression alternating with those of lower expression, both in the granular layer associated with mossy fiber synaptic glomeruli and in the molecular layer, associated primarily with pf-PC synapses (Hawkes et al., 1985; Leclerc et al., 1989). The interpretation of the observation is less obvious—does stronger staining reflect more antigen per vesicle, antibody access, more vesicles per synapse, a higher Purkinje cell spine density …? Appropriate comparisons with the expression patterns of other synaptic markers that might resolve this question have not been reported. A patchy/striped arrangement of mossy fiber terminals in the granular layer is reported with other presynaptic markers, but in these cases the expression in the molecular layer appears to be uniform (e.g., dysbindin—Sillitoe et al., 2003; neuronal nitric oxide synthase (nNOS); see Section Nitric Oxide). A uniform distribution in the molecular layer may be misleading in that differential expression of granule cell markers is easier to discern in the granular layer, where the somata are segregated into stripes and clusters, than in the molecular layer where the long parallel fiber trajectories extensively overlap and smooth out different expression levels from different granule cell populations.

Structural synaptic proteins are also differentially expressed. For example, a prominent striped expression pattern is revealed by immunocytochemical staining for the postsynaptic membrane glycoprotein neuroplastin (Marzban et al., 2003; note—in the hippocampus, neuroplastin has been linked to the inhibition of LTP; Empson et al., 2006). High levels of neuroplastin expression are preferentially associated with the zebrin II- Purkinje cell stripes.

Glutamate released into the synaptic cleft at both the climbing fiber and the pf-PC synapse binds to three types of glutamate receptor in the postsynaptic membrane—metabotropic (mGluR), GluRδ2 and AMPAR. mGluRs are G protein-coupled receptors with 7 transmembrane segments that do not form ion channels but rather signal via intracellular chemical messenger systems. Eight genes coding for different subtypes of mGluRs have been identified, seven of which are expressed in the cerebellum. In particular, Purkinje cell mGluR1 is localized in the peri- and extra-synaptic membranes. It is functionally coupled to PLC through which it modulates the IP₃ (1,4,5)/Ca2+ signaling pathway and plays a key role in the induction of pf-PC LTD (reviewed in Knöpfel and Grandes, 2002).

There are at least 4 mGluR1 splice variants with differing subcellular and cellular distributions (mGluR1a-d: e.g., Conn and Pin, 1997). In the case of mGluR1b in the cerebellum, expression is striped in the molecular layer and co-located with zebrin II- stripe markers (Mateos et al., 2001). mGluR1a is also located in the Purkinje cell dendritic spine (e.g., Mateos et al., 2000) but whether or not there is an mGluR subtype restricted to the zebrin II+ Purkinje cell dendritic spines is not known.

GluRδ2 is also highly expressed in cerebellar Purkinje cells and is localized specifically to pf-PC synapses (Araki et al., 1993; reviewed in Hirano, 2006). GluRδ2 neither binds glutamate nor conducts current but rather regulates mGluR1-mediated synaptic transmission via PKCγ (e.g., Kato et al., 2012). Loss-of-function mutations in GluRδ2 result in multiple defects including impairment of LTD (Kashiwabuchi et al., 1995). There is no evidence that its expression is stripe-restricted (gain-of-function mutation of the GluRδ2 gene in the lurcher mouse (Grid^Lc/+) results in striped Purkinje cell degeneration (Zuo et al., 1997; reviewed in Armstrong et al., 2011) but this likely reflects differential sensitivity to the insult rather than selective GluRδ2 expression).

The time that glutamate resides in the synaptic cleft, and hence is available for receptor binding, is governed by EAAT. In particular, EAAT4 has been implicated at the pf-PC synapse. It is therefore striking that the expression of EAAT4 is different from stripe to stripe with high levels associated with the zebrin II+ stripes (Dehnes et al., 1998). As a result, regional differences in glutamate transporter expression affect the degree of mGluR1 receptor stimulation, with the result that pf-PC LTD is dampened in Purkinje cells expressing high levels of EAAT4 (= zebrin II+; Wadiche and Jahr, 2005).

One consequence of mGluR1 activation is Ca²⁺ influx via VDCC in the postsynaptic dendritic membrane. There is no evidence that VDCCs are expressed differentially by Purkinje cell subsets (As for the lurcher mouse, it has been shown that a mutation of the VDCCα1a channel in the tottering mouse (Cacna1a^tg) results in the selective Purkinje cell death of the zebrin II- Purkinje cell subset, but again the evidence suggests that this is due to differential sensitivity to an abnormal Ca²⁺ influx rather than restricted expression of the α1a channel; Fletcher et al., 1996).

The downstream response to dendritic Ca²⁺ influx is modulated by calpacitins, notably the 2 Purkinje cell proteins PEP-19 and neurogranin/RC3. Sutcliffe and colleague have proposed that calpacitins regulate calmodulin availability in dendritic spines and thus regulates their ability to amplify the mobilization of Ca²⁺ in response to metabotropic glutamate receptor stimulation, releasing calmodulin rapidly in response to large influxes of Ca²⁺ and slowly in response to small increases. This action is inhibited by PKC-mediated phosphorylation (reviewed in Gerendasy and Sutcliffe, 1997; Díez-Guerra, 2010). α-Calmodulin kinase KII is also shown to be required for LTD at the pf-PC synapse (Hansel et al., 2006). Neurogranin knockout mice show deficits in the induction of hippocampal LTP (e.g., Pak et al., 2000) but no cerebellar phenotype is reported. On the other hand, the much more abundant PEP19 is directly implicated: in the PEP19 null mouse both motor learning and pf-PC LTD are impaired (Wei et al., 2011). During cerebellar development, both PEP19 (Wassef et al., 1992) and neurogranin (Larouche et al., 2006) expression is restricted to Purkinje cell subsets. However, in the adult PEP19 expression is uniformly expressed by all Purkinje cells (Mugnaini et al., 1987) whereas neurogranin has disappeared (Larouche et al., 2006), so any significance for patterned LTD at the adult pf-PC synapse is doubtful.

In addition to releasing glutamate, parallel fibers also release NO. NO acts through inhibition of protein phosphatases in the Purkinje cell dendritic spine and thus enhance AMPAR phosphorylation. LTD is abolished in transgenic mice lacking nNOS (Lev-Ram et al., 1997). There is clear evidence of different stripes of nNOS in the granular layer of the cerebellar cortex (e.g., Yan et al., 1993; Hawkes and Turner, 1994; Schilling et al., 1994; Baader and Schilling, 1996). Similar striping is harder to discern in the molecular layer, perhaps obscured by the overlapping parallel fiber populations. The nNOS pathway is activated via N-methyl-D-aspartate (NMDA)-type glutamate receptors located at the pf-PC synapse (and/or located in the presynaptic terminals of inhibitory interneurons; Shin and Linden, 2005). Functional NMDA receptors are also expressed at climbing fiber-PC synapses, and channel blocking inhibits LTD (Piochon et al., 2010).

It is noteworthy that NMDA receptor expression, as revealed by an NRC2 subunit knock-in mouse (NR2C^nlacZ), reveals stripes of granule cells similar to those revealed by PLCβ4 expression (= zebrin II-; Karavanova et al., 2007). There is no evidence that Purkinje cell NMDA receptors are expressed in stripes.

Glutamate release and binding to mGluR1 is also the first step in signaling via the climbing fiber pathway. In parallel to glutamate release, climbing fibers also secrete CRF (Barmack and Young, 1990), which plays a permissive role in LTD that is probably mediated through PKC (Miyata et al., 1999). However, not all climbing fibers express CRF. Whole mount immunocytochemistry shows that CRF is restricted to (or is expressed at higher levels in) a striped subset of climbing fiber terminals that terminate in zebrin II+ Purkinje cell stripes (mouse—Sawada et al., 2008). However, the significance of this may not be straightforward as previous studies reported uniform CRF expression (e.g., cat—Cummings, 1989) or expression differences between lobules but not in the form of stripes (e.g., developing mouse—Overbeck and King, 1999). There is no evidence that G-protein coupled CRF receptor (CRFR1) expression is similarly striped (e.g., Allen Brain Atlas).

As well as releasing CRF, climbing fiber synapses also store and release IGF-1 (Torres-Aleman et al., 1994). It is not known if IGF-1 or its receptor tyrosine kinase (IGF1R) is expressed in stripes in the adult cerebellum (in general in the brain IGF1R is broadly expressed—it is the ligands that show regional restriction: e.g., reviewed in D’Ercole et al., 1996). During early postnatal development, IGF-1 is also expressed in a zebrin II- Purkinje cell subset, where it acts to block apoptosis (Croci et al., 2011), but it is unclear whether selective expression is retained in the adult.

mGluR1 signals via the Gq subclass of G-proteins to PLCβ (reviewed in Knöpfel and Grandes, 2002). There are four PLCβ isoforms, encoded by distinct genes (PLCβ1-4; Bahk et al., 1994). Strikingly, PLCβ3 and PLCβ4 are expressed by distinct, non-overlapping subsets of Purkinje cells. PLCβ3 is confined to the zebrin II+ Purkinje cell subset (Figure 1C) and PLCβ4 expression is coextensive with the zebrin II- Purkinje cell subset (Figures 1D, E; Sarna et al., 2006; Marzban et al., 2007) (Unexpectedly, a small subset of zebrin II+ Purkinje cell stripes in the nodular zone of the mouse cerebellum (~ lobules IX and X)—those that express HSP25—is reproducibly immunonegative for both PLCβ3 and PLCβ4 (Sarna et al., 2006)—the implication of this is unclear).

Parallel to the PLCβ pathway, there is also a signaling pathway via phospholipase A (PLA)—in particular, the PLA2 isoform: e.g., Linden, 1995; Le et al., 2010), which acts to break down phospholipids into arachidonic acid, a potent activator of PK Cγ (e.g., Shearman et al., 1989). At least 20 PLA2 isoforms have been identified, three of which have been reported in Purkinje cells (cPLA2α, sPLA2IIA, and iPLA2; Shirai and Ito, 2004). There is no evidence that any of these is restricted to a particular Purkinje cell subset.

Two signaling pathways leading to LTD lie downstream of PLC. The first involves IP₃ release from intracellular stores to bind to its receptor on the endoplasmic reticulum (IP₃R; e.g., Furuichi et al., 1989; Maeda et al., 1989). The cerebellar distribution of IP₃R has recently been reported in a transgenic mouse in which the IP₃R promoter was fused to a β-galactosidase reporter and a nuclear localization signal (IP₃R1nls-lacZ; Furutama et al., 1996, 2010; Figure 1F). Transgene expression in the heterozygote reveals a striking array of Purkinje cell stripes that can be traced continuously through embryogenesis through to adulthood. In general, IP₃R1nls-lacZ transgene expression is restricted to the zebrin II+ Purkinje cell subset. The extent to which this distribution reflects any feature of the true restriction of the receptor—perhaps developmentally—or is a transgene artifact (due to the transgene insertion, promoter truncation, enhancer trapping, etc.), is questionable. Immunocytochemistry with antibodies against IP₃R do not show Purkinje cell stripes (e.g., Mignery et al., 1989).

An additional second-messenger signaling pathway between glutamate release and the induction of LTD goes via the generation of DAG by PLC, which in turn activates PKC (Crépel and Krupa, 1988; for a general review of PKC, see Newton, 1995). There are seven PKC subtypes—three (α, β and γ) activated in a Ca²⁺/DAG-dependent manner and 4 (δ, Є, η and θ) Ca²⁺-independent (reviewed in Tanaka and Nishizuka, 1994). Activation of Ca²⁺-dependent PKC is necessary for induction of LTD at the pf-PC synapse (e.g., Ito, 1989, 2002; Daniel et al., 1998). LTD induction at the pf-PC synapse is blocked by the intracellular application of PKC inhibitors in Purkinje cells (Linden and Connor, 1991). De Zeeuw et al. (1998) constructed a transgenic mouse in which a Purkinje cell-specific promoter (pcp2-L7) was used to target the expression of a broad-spectrum PKC inhibitor (the pseudosubstrate PKC[19–31]) and thereby showed that PKC activation in the Purkinje cell is a prerequisite for the induction of LTD. None of the Ca²⁺/DAG-dependent PKC isoforms is expressed selectively by a Purkinje cell subset (e.g., Barmack et al., 2000).

The role(s), if any, of the four Ca²⁺-independent PKCs in LTD induction is unclear. However, they deserve attention here because while most PKC isoform distributions are uniform across the molecular layer the one exception is PKCδ, whose expression in the nodular zone of the rat reveals a reproducible striped expression pattern with higher levels in the zebrin II+ stripes (Barmack et al., 2000). Furthermore, experimental manipulation of the cerebellar afferent inputs by labyrinthectomy demonstrated an activity-dependent targeting of the PKCδ isoform to the pf-PC synapse (Barmack et al., 2001). PKCδ has been implicated in hippocampal LTP (e.g., Kim et al., 2013) but no specific role in cerebellar LTD is known.

LTD at the pf-PC synapse is also modulated by an unusual form of γ-aminobutyric acid (GABA) receptor signaling. In the adult cerebellum GABA_BRs are predominantly located perisynaptically at the dendritic spines of Purkinje cells (e.g., Turgeon and Albin, 1993; Kaupmann et al., 1997; Bischoff et al., 1999; Kulik et al., 2002; Fritschy et al., 2004; Luján and Shigemoto, 2006). Both GABA_A (reviewed in Fritschy and Panzanelli, 2006) and GABA_B receptor classes are expressed in the cerebellum but only GABA_BRs have been implicated in pf-PC LTD. GABA_BRs are G-protein-coupled receptors formed as heteromers of 2 subunits (GABA_BR1/2; Marshall et al., 1999; Möhler and Fritschy, 1999). Postsynaptic GABA_BR signaling enhances LTD (Tabata et al., 2004; Kamikubo et al., 2007), possibly as a Ca²⁺-dependent cofactor of mGluR1 signaling. The role of GABA_BRs in the modulation of LTD is unconventional in that it does not require GABA. Rather, extracellular Ca²⁺ binds to the GABA_BR and constitutively increases the glutamate sensitivity of mGluR1 (Tabata et al., 2004). The role of GABA_BR in LTD is relevant for the present discussion because immunocytochemistry for GABA_BR2 shows a strong restriction of receptor immunoreactivity to the zebrin II+ stripes (Albin and Gilman, 1989; Luján and Shigemoto, 2006; Chung et al., 2008; Figure 1E).

The ultimate downstream target of PLC signaling, via both PKC and IP₃R, is the phosphorylation (via both PKC and Src-family protein tyrosine kinases—e.g., Tsuruno et al., 2008) of postsynaptic AMPA receptors (AMPAR; Ito, 1984; Crépel and Krupa, 1988; Hirano, 1991; Linden et al., 1991; Matsuda et al., 2000; Tsuruno et al., 2008; etc.). AMPAR kinetics, agonist affinity and unitary conductances are unchanged by phosphorylation (Linden, 2001) but rather there results a reduction in AMPAR number due to enhanced endocytosis (Matsuda et al., 2000), which is dependent on phosphorylation at ser-880 in the AMPAR GluR2 subunit (Chung et al., 2003; reviewed in Shin and Linden, 2005). There is no evidence of selective expression of either Src kinases or AMPAR by Purkinje cell subsets.