Multiple behavior-specific cancellation signals contribute to suppressing predictable sensory reafference in a cerebellum-like structure.

Movement induces sensory stimulation of an animal's own sensory receptors, termed reafference. With a few exceptions, notably vestibular and proprioception, this reafference is unwanted sensory noise and must be selectively filtered in order to detect relevant external sensory signals. In the cerebellum-like electrosensory nucleus of elasmobranch fish, an adaptive filter preserves novel signals by generating cancellation signals that suppress predictable reafference. A parallel fiber network supplies the principal Purkinje-like neurons (called ascending efferent neurons, AENs) with behavior-associated internal reference signals, including motor corollary discharge and sensory feedback, from which predictive cancellation signals are formed. How distinct behavior-specific cancellation signals interact within AENs when multiple behaviors co-occur and produce complex, changing patterns of reafference is unknown. Here, we show that when multiple streams of internal reference signals are available, cancellation signals form that are specific to parallel fiber inputs temporally correlated with, and therefore predictive of, sensory reafference. A single AEN has the capacity to form more than one cancellation signal, and AENs form multiple cancellation signals simultaneously and modify them independently during co-occurring behaviors. Cancellation signals update incrementally during continuous behaviors, as well as episodic bouts of behavior that last minutes to hours. Finally, individual AENs, independently of their neighbors, form unique AEN-specific cancellation signals that depend on the particular sensory reafferent input it receives. Together, these results demonstrate the capacity of the adaptive filter to utilize multiple cancellation signals to suppress dynamic patterns of reafference arising from complex co-occurring and intermittently performed behaviors.

A cerebellum-like circuit in the lateral line system of fish cancels mechanosensory input associated with its own movements.

An animal's own movement exerts a profound impact on sensory input to its nervous system. Peripheral sensory receptors do not distinguish externally generated stimuli from stimuli generated by an animal's own behavior (reafference) - although the animal often must. One way that nervous systems can solve this problem is to provide movement-related signals (copies of motor commands and sensory feedback) to sensory systems, which can then be used to generate predictions that oppose or cancel out sensory responses to reafference. Here, we studied the use of movement-related signals to generate sensory predictions in the lateral line medial octavolateralis nucleus (MON) of the little skate. In the MON, mechanoreceptive afferents synapse on output neurons that also receive movement-related signals from central sources, via a granule cell parallel fiber system. This parallel fiber system organization is characteristic of a set of so-called cerebellum-like structures. Cerebellum-like structures have been shown to support predictive cancellation of reafference in the electrosensory systems of fish and the auditory system of mice. Here, we provide evidence that the parallel fiber system in the MON can generate predictions that are negative images of (and therefore cancel) sensory input associated with respiratory and fin movements. The MON, found in most aquatic vertebrates, is probably one of the most primitive cerebellum-like structures and a starting point for cerebellar evolution. The results of this study contribute to a growing body of work that uses an evolutionary perspective on the vertebrate cerebellum to understand its functional diversity in animal behavior.

Morphological development of the dorsal hindbrain in an elasmobranch fish (Leucoraja erinacea).

The developmental anatomy of the dorsal hindbrain in an elasmobranch fish, Leucoraja erinacea, is described. We focus on the cerebellum, which is a synapomorphy for gnathostomes. Cerebellar development in L. erinacea, a representative of the most basal gnathostome lineage, may be a proxy for the ancestral state of cerebellar development. We also focus on sensory processing regions termed 'cerebellum-like' structures due to common anatomical and physiological features with the cerebellum. These structures may be considered generatively homologous if they share common developmental features. To test this hypothesis, the morphological development of the cerebellum and cerebellum-like structures must first be described. Of particular importance is the development of common features, such as the molecular layer, which is the defining characteristic of these structures. The molecular layers of the cerebellum and cerebellum-like structures are supplied with parallel fiber axons from distinct granule cell populations. These are the lateral granule mass, the dorsal granular ridge, the medial granule mass, and the granular eminences of the cerebellum. Cerebellar and cerebellar-like development in L. erinacea is similar to development in other elasmobranchs. The temporal order in which these granule cell populations develop suggests an evolutionary history of duplication or expansion of an existing developmental event.

Evidence for generative homology of cerebellum and cerebellum-like structures in an elasmobranch fish based on Pax6, Cbln1 and Grid2 expression.

The majority of neurons in the mammalian brain reside within the cerebellum (Cb). Yet, the evolutionary origins of the Cb are not well understood. There are several sensory nuclei present across vertebrate phylogeny collectively termed cerebellum-like structures (CbLS) due to a shared anatomy and physiology with the Cb. Despite the similarities, the CbLS are clearly not phylogenetically homologous with the Cb. Common structure and function may arise due to a shared genetic and developmental toolkit. To examine this possibility, we used sequence analysis, western blotting, immunohistochemistry and RT-qPCR to test for the expression of three genes that are critical for mammalian cerebellar development in the Cb and CbLS of an elasmobranch fish, Leucoraja erinacea. In the mammalian Cb, Pax6 is necessary for parallel fiber development, while Cbln1 and Grid2 code for proteins necessary for parallel fiber-principal cell synaptogenesis. Pax6 and Cbln1 are expressed by granule cells in the Cb and CbLS of the adult skate and stage 31 embryo. Grid2 is expressed by principal cells in the Cb and CbLS of the adult and stage 31 embryo. RT-qPCR showed this expression is spatially and temporally restricted to the Cb and CbLS. If Pax6, Cbln1 and Grid2 perform the same functions in the skate Cb and CbLS as they do in the mammalian Cb, then these structures may develop using a shared genetic toolkit and be considered generatively homologous. It is possible that the evolutionary genesis of the Cb was the result of duplication or expansion of the cerebellum-like developmental toolkit.

The cerebellum and cerebellum-like structures of cartilaginous fishes.

The cerebellum is well developed in cartilaginous fishes, with the same cell types (barring basket cells) and organizational features found in other vertebrate groups, including mammals. In particular, the lattice-like organization of cerebellar cortex (with a molecular layer of parallel fibers, interneurons, spiny Purkinje cell dendrites, and climbing fibers) is a defining characteristic. In addition to the cerebellum, cartilaginous fishes have cerebellum-like structures in the dorsolateral wall of the hindbrain. These structures are adjacent to and, in part, contiguous with the cerebellum. They are cerebellum-like in that they have a molecular layer of parallel fibers and inhibitory interneurons that has striking organizational similarities to the molecular layer of the cerebellar cortex. However, these structures also have characteristics that differ from the cerebellum. For example, cerebellum-like structures do not have climbing fibers and are clearly sensory. They receive direct afferent input from peripheral sensory receptors and relay their outputs to midbrain sensory areas. As a consequence of this close sensory association and the ability of researchers to characterize signal processing in these structures in a behaviorally relevant context, good progress has been made in determining the fundamental processing algorithm of the cerebellum-like structures. This algorithm enables the molecular layer to act as an adaptive filter that cancels self-generated noise in electrosensory and lateral line systems. Given the fundamental similarities of the molecular layer across these structures and the phylogeny of these structures across basal vertebrates, it is clear that these structures share a common genetic-developmental program. Syngeny is a term that has been used to describe similarity of structure due to a shared genetic-developmental program, whether the structures are phylogenetically homologous or not. Given that the cerebellum and cerebellum-like structures are physically adjacent, we propose that cerebellum-like structures were the evolutionary antecedent of the cerebellum and that the cerebellum arose through a change in the genetic-developmental program, amounting to a duplication of existing structure. Such duplication to form adjacent structures can be considered a special case of syngeny. On this view, the cerebellum is an evolutionary innovation in gnathostomes that is literally superimposed on pre-existing underlying brain structures and pathways. From this perspective, the cerebellum can be considered an example of 'subsumption architecture', a term that describes the addition of modules that add computational power while maintaining existing fundamental functionality. This addition is reflected in the finding that in elasmobranchs with relatively large brains, the size of the telencephalon and cerebellum enlarge disproportionately, while those parts of the brain that contain more direct sensory and motor connections do not. Added 'computational' power in the chondrichthyan brain and the comparative function and evolution of the cerebellum and cerebellum-like structures across the cartilaginous fishes supports the idea of the cerebellum as an example of subsumption architecture.

The importance of N-methyl-D-aspartate (NMDA) receptors in subtraction of electrosensory reafference in the dorsal nucleus of skates.

The dorsal nucleus of the little skate is a cerebellum-like sensory structure that adaptively filters out predictable electrosensory inputs. The filter's plasticity is mediated by anti-Hebbian associative depression at the synapses between parallel fibers and ascending efferent neurons (AENs). Changes in synaptic strength are indicated by the formation of a cancellation signal which is initiated by co-activation of parallel fibers and AENs, and can be reversed by parallel fiber activity in the absence of AEN activation. In other cerebellum-like sensory structures, the formation of the cancellation signal requires activation of postsynaptic NMDA receptors on the principal neurons. We demonstrate here by immunohistochemistry that the somas and the initial portion of both apical and basal dendrites of the AENs are labeled with antibodies raised against the NR1 subunit of NMDA receptors from a South American electric fish. In in vivo physiological experiments, we show that the formation of the cancellation signal induced by coupling an electrosensory stimulus to ventilatory movements or direct parallel fiber stimulation is blocked when either of the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid (APV) or MK801 is injected into the molecular layer above the recorded AEN. Blocking NMDA receptors prevented formation of a cancellation signal in 79% (15/19; APV) and 60% (3/5; MK801) of the AENs. This blockage was reversible in 40% (6/15) of the AENs after APV removal. Thus, in the dorsal nucleus, the activity-dependent, long-lasting but reversible change in synaptic strength of the parallel fiber-AEN synapses appears to be an NMDA receptor-dependent process.

Plasticity in a cerebellar-like structure: suppressing reafference during episodic behaviors.

Detection of relevant sensory signals requires the filtering out of irrelevant noise, including noise created by the animal's own movements (reafference). This is accomplished in the electrosense of little skates (Raja erinacea) by an adaptive filter in the cerebellar-like electrosensory nucleus (dorsal nucleus) in the medulla. We have shown that electrosensory inputs reliably coupled to the regularly recurring movements of breathing over time are eliminated selectively in the principal neurons (ascending efferent neurons, AENs) by a cancellation signal that is a negative of the reafference and is supplied by a parallel fiber system. Similarly, electrosensory inputs repeatedly linked to passive fin movements are eliminated suggesting that the filter also functions in relation to other behaviors besides breathing. To determine whether this adaptive filter can eliminate reafference created by brief and infrequent episodic behaviors like swimming in skates, we initiated a series of coupling tests in which an external electrosensory stimulus was coupled to short bouts of either parallel fiber stimulation or passive fin movements, and then measured the ability of AENs to generate a cancellation signal. Following five brief coupling periods (30-60 s) separated by long rest periods (1-9 min), 38.5% of the AENs developed a cancellation signal when the coupling was to parallel fiber stimulation, and 73% when the coupling was to passive fin movement. We demonstrate that the cancellation signals can be developed incrementally, persist for at least a 3 h rest period without reinforcement, and are extinguished within minutes when the association of sensory stimulus and fin movement or parallel fiber stimulation no longer exists. The results indicate that the adaptive filter has the properties necessary to cancel reafference associated with even brief and infrequent behaviors.