Functional Classification of Neurons in the Mouse Lateral Cerebellar Nuclei
The deep cerebellar nuclei (DCN) are at the center of the cerebellum not only anatomically but also functionally. Classical anatomical studies have described different types of DCN neurons according to their expression of various marker proteins, but only recently have we begun to characterize these different cell types according to their electrophysiological properties. These efforts have benefited greatly from the availability of transgenic mouse lines that express green fluorescent protein under the control of the glutamic acid decarboxylase (GAD67) and glycine transporter (GlyT2) promoters, which are markers for GABAergic and glycinergic neurons, respectively. These studies have identified several types of neurons within the lateral cerebellar nuclei, each of which exhibits distinct active membrane properties. In addition to their differential use of neurotransmitters (glutamate, GABA, or glycine), these cell types also receive and provide synaptic information from different sources and to different targets.
KeywordsCerebellum Cerebellar nuclei GAD67 GlyT2 GFP Transgenic mouse Electrophysiology Morphology
Our understanding of cerebellar function has historically been based on the premise that the cerebellar cortex is the site of information processing, or at least memory storage and retrieval . This corticocentric viewpoint assumes that cerebellar output is mainly carried by the projection neurons of the deep cerebellar nuclei (DCN). However, evidence clearly suggests that the DCN are not merely a relay station, but rather constitute a site of signal integration if not signal generation. Indeed, a role in adaptive information processing and perhaps pattern generation may be deduced from the presence of specific forms of synaptic plasticity within the DCN [2, 3, 4]. Understanding the information-processing capabilities of a neuronal circuit generally requires the elucidation of the distinct cell types that comprise it. Several excellent studies have made an effort to examine the diversity of DCN neurons using morphological staining [5, 6], either alone or in combination with electrophysiological recordings . These studies have revealed considerable diversity of DCN neurons but have found only modest correlations between electrophysiological features and neuronal morphology, indicating that these neurons cannot be classified solely based on morphometric criteria . To overcome this challenge, our laboratory took advantage of transgenic mouse lines that express fluorescent proteins under the promoters for glutamic acid decarboxylase (GAD67 ) and a glycine transporter (GlyT2 ), which are often used as markers for GABAergic and glycinergic neurons, respectively [11, 12]. A careful examination of cerebellar structures in GAD67-EGFP mice confirmed that all GABAergic (GABA-containing) neurons in the DCN express EGFP. Since we did not directly identify the transmitters released by these EGFP-expressing cells, and in keeping with the terminology applied in the original publications, we have addressed the various neuronal subtypes based on the presence of these markers (e.g., GAD67-positive, GAD+) rather than the most probable neurotransmitter (e.g., GABA). Neurons that were not identified by either of the two markers are likely to be glutamatergic (for a more detailed discussion see ).
This review will compile work from our three original studies of the neuronal types found in the lateral cerebellar nucleus of the mouse as well as complementary information from other previous and concurrent studies, without attempting to comprehensively review the available literature on other features of the cerebellar nuclei. We will therefore focus primarily on four types of neurons identified in the mouse lateral DCN based on GAD67 and GlyT2 promoter activity, with the data from each neuron type indicated by color code: black, large GAD67-negative (putative non-GABAergic) DCN cells (GADnL); orange, small GAD67-negative DCN cells (GADnS); green, GAD67-positive (GAD+, GABAergic) cells; and blue, non-spontaneously active GlyT2+ cells (Gly-I). The GAD+ neurons include both local and inferior olive-projecting GABAergic neurons. Our own data from the GABAergic cells are strongly biased towards the larger (soma size >12 um) cells, among which we have observed no evidence for axons projecting outside the nucleus. The GABAergic neurons that project to the inferior olive (IO) have been described as very small [18, 19], and for this reason, we believe that they constitute a separate cell type, which we refer to as GAD+IO. To our knowledge, there are no studies that have specifically examined the electrophysiology of these small IO-projecting neurons. To acknowledge the undoubtedly great functional importance of this cell population, we have included the GAD+IO cells in our schematic representations while noting that a more detailed examination remains a goal for future studies.
The Four DCN Neuron Subtypes Exhibit Distinct Active Membrane Properties
Rather unexpectedly, the presumably GABAergic GAD+ cell population exhibits the greatest AP width (Fig. 2c, e), in contrast to the cerebral cortex, where many GABAergic interneurons are defined by their short-lasting APs. AP width in GAD− cells can be clustered into two groups with values above or below 0.6 ms, suggesting the presence of more than one neuron population. In fact, plotting the AP width against cell capacitance suggested the existence of two cellular populations, one characterized by large size (C m > 100 pF) and shorter APs and another being small in somatic size (C m < 100 pF) with broader APs. These cell groups were respectively classified as GADnL and GADnS populations, divided by a (somewhat arbitrary) boundary value of 100 pF.
One attractive hypothesis is that these different AP widths are a consequence of different expression levels of delayed-rectifier type potassium channels, particularly, Kv3 subtypes [25, 26]. Our own studies [27, 28] and subsequent investigations by Joho and coworkers [29, 30] do not indicate a strong correlation between neuronal subtypes and Kv3 channel expression. However, it seems that Kv3 channel expression levels are somewhat lower in neurons containing GABA (local and/or IO-projecting) than in the large glutamatergic projection neurons, which would be consistent with GABAergic cells showing broader APs than glutamatergic ones.
AP width is not the only clearly distinguishing feature between GADnL and GADnS cells; GADnS cells also exhibit a stronger late component in the afterhyperpolarization (Fig. 2c, double arrowhead) and somewhat longer rebound depolarization and associated spiking after release from a hyperpolarizing step (Fig. 2d; [7, 31, 32]). With the exception of Gly-I cells, which have not yet been thoroughly characterized in this aspect, all cell types consistently show a pronounced sag in their response to hyperpolarizing current steps, indicative of hyperpolarization-activated cation current (Ih) and rebound bursting that may involve the activity of T-type calcium currents (Fig. 2d and e) [31, 33, 34]. As recently reviewed by Turner and coworkers , differences in rebound burst behavior may reflect distinct expression patterns of T-type calcium channel-forming CaV3 isoforms. However, this study focused on spontaneously active large-diameter neurons, and their data pool is unlikely to contain a significant fraction of the sparse, Gly-I neurons that are also characterized by lack of spontaneous activity. Our preliminary examinations of Gly-I cells suggest that these intrinsically silent neurons are also less prone to rebound bursting than the other, pacemaker-like neurons of the DCN (M. Uusisaari, unpublished observations).
Finally, these four cell types differ in spike frequency versus injected current density and their spike frequency accommodation. Most notably, GADnL neurons can be driven to the highest firing frequencies with a nearly linear current–frequency (I-F) relationship while also exhibiting the smallest amount of spike frequency adaption during sustained firing (Fig. 2e—iv, v). Local GAD+ and GADnS cells respond to depolarization with saturating values of firing frequency, and show a significant amount of frequency adaptation. Again, this is in contrast to the situation in the cerebral cortex, where GABAergic interneurons typically fire at high and sustained frequencies. The Gly-I cells exhibit the strongest accommodation, to the extent that they tend to stop firing after a few initial spikes. It would thus seem that the DCN neurons cover a range of signal transduction modes, from tonic firing and rate-coding (GADnL) to burst-firing and onset or time-interval coding (GAD+, Gly-I).
Morphometric Parameters of the DCN Neuron Subtypes
Despite the cell-to-cell variability of dendritic structures, the above-described subtypes could not be readily identified by their morphology alone, even with information about GAD67 expression. Indeed, quantitative analysis of several measures of dendritic morphology and, surprisingly, cell body size revealed clearly overlapping distributions . Importantly, GAD+ cells could not be reliably differentiated from GAD− cells on the basis of cell body size, and the Gly-I cells described in  also significantly overlap with GAD− cells in terms of cell body size. Since many of the electrophysiological recordings found in literature are reported to originate from “large DCN neurons,” these data are likely biased towards glutamatergic cells, but presumably also include GABAergic or glycinergic cells. Since the electrophysiological differences between GAD+ and GAD− cells are subtle, it is not surprising that most reports did not find clearly distinguishable cell types. In this context, it should be noted that the Gly-I cells might have been omitted in previous slice studies as “unhealthy cells,” as they are not necessarily spontaneously active. This is unfortunate, as our targeted staining of these silent, GlyT2+ cells revealed that they project directly to the cerebellar cortex, unlike any other cell type that we have encountered in the DCN so far , making further elucidation of their behavior mandatory. The actual synaptic targets within the cerebellar cortex remain unknown for these putatively glycinergic—and thus, inhibitory—neurons, but are likely to be cerebellar Golgi cells as these are the only neurons in most areas of the cerebellar granule cell layer that express glycine receptors .
Spontaneous Synaptic Activity of DCN Neuron Subtypes
Purkinje neuron (PN) axons from the cerebellar cortex provide the main efferent input to the DCN, and this is their main target. Based on counts of Purkinje and DCN neurons (estimates for mouse are ~200,000 PNs  and ~30,000 neurons in all three DCN ), signal transmission from the cortex to the nuclei shows overall convergence, but the true extent of this convergence at the cellular level is not known. Based on the count of Purkinje axon terminals on large DCN cell bodies, and taking into account various estimates of how many terminals a single axon will form on a given neuronal body and the spread of PN axons within the nuclei, estimates of the number of PNs contacting a single large DCN neuron range between several tens and several hundreds [39, 40, 41]. Despite anatomical evidence [5, 42] suggesting that the number of PN synapses on DCN cells is variable across different types of DCN cells, it is generally assumed that all DCN neurons are equally controlled by PN activity.
Unfortunately, we have only limited data on the properties of the synaptic inputs to Gly-I cells. Preliminary results obtained in our laboratory suggest that these cells resemble GADnS cells more than GAD+ cells with respect to IPSC kinetics and frequency (M. Uusisaari, unpublished observations), which suggests that Gly-I cell bodies are being targeted by Purkinje neuron axons. As Gly-I cells seem to be endowed with the capability to control cerebellar cortical activation via their putative inhibitory synaptic contacts in the granule cell layer, elucidation of their afferent inputs is a pressing open task.
Synaptic Inputs to DCN Neuron Subtypes
Clearly, we presently lack even a basic description of all of the various cell types in the DCN and are far from compiling a circuit diagram that reflects an understanding of their functional properties. Fundamental issues that need to be addressed in future work include the examination of the neurons that project from the DCN to the IO, determining the extent to which our observations in the lateral nucleus can be generalized to the other two nuclei, and the completion of a careful histological examination of the spatial distribution of cell types within the different nuclei . Following on the identification of these different cell types, we also need to examine their afferent and intrinsic connections. Notably, previous studies aimed at characterizing the properties of MF and CF synapses were specifically biased towards large neurons, whereas there is clear anatomical evidence for afferent connections targeting different cell types . Even less is known about the functional role of the connectivity within the DCN, which is likely to consist of local GABAergic, glutamatergic, and glycinergic synaptic communication [5, 51, 52]. It seems likely that the DCN is organized in a modular fashion, since afferent and efferent connections segregate according to their extra-nuclear sources . There is a possibility that synaptic crosstalk may occur between these modules in a manner analogous to the parallel fibers that provide information flow across the zones of the cerebellar cortex.
Finally, future research efforts should explore the possible function of inhibitory nucleo-cortical synaptic connections formed by Gly-I cell axons [54, 55, 56]. Our growing toolbox of mouse strains in which neuronal subtypes are conveniently identifiable via cell type-specific expression of fluorescent protein reporters, in conjunction with techniques for neural circuit labeling via transsynaptic gene delivery, should enable us to effectively address these issues in the years to come .
Conflict of Interest
The authors declare that they have no conflict of interest with respect to this article.
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