Date of Award

5-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Neuroscience

Research Advisor

Detlef H. Heck, Ph.D.

Committee

William E. Armstrong, Ph.D. Matthew Ennis, Ph.D. Hitoshi Kita, Ph.D. Guy Mittleman, Ph.D.

Abstract

Rodents consume water by performing stereotypical, rhythmic licking movements which are believed to be driven by central pattern generating circuits located in the brainstem. Temporal aspects of rhythmic licking behavior have been shown to be represented in the olivo-cerebellar system in the form of population complex spike activity. These findings suggest that the olivo-cerebellar system is involved in the generating circuitry responsible for licking rhythm in rodents. However, the representation of licking in the simple spike activity of Purkinje cells and the consequences of loss of cerebellar function on licking behavior has not been quantified. I investigated the influence of the cerebellum on the maintenance of rhythm in murine fluid-licking.

In one set of experiments, I characterized Purkinje cell activity in healthy mice during fluid licking. Use of a head-restrained preparation allowed recordings of well-isolated single units during repeated experimental sessions. Thus, a large number of neurons were tested for their relationship with behavior and detailed spatial maps of behavior related neuronal activity were generated as exemplified here with recordings from lick-related Purkinje cells in the cerebellum. The data show a multifaceted representation of licking behavior in the simple spike activity of a large population of Purkinje cells distributed across Crus I, Crus II, and lobus simplex of mouse cerebellar cortex. Lick related Purkinje cell simple spike activity was changed in a manner that was either rhythmic, in phase with the lick rhythm, or nonrhythmic with a decrease or increase in firing in relation to licks but not phasically. For rhythmically responsive units, signal modulation was marked by the introduction of a phasic variation in the frequency of spikes. A subpopulation of lick related Purkinje cells exhibited different activity patterns during short and long interlick intervals (ILIs).

I examined the role of the cerebellum in fluid-licking by using several models of cerebellar ataxia with distinct causes. First, I observed fluid-licking in animals over several days to determine how the microstructure of the behavior may also be altered. The first model involved animals that underwent cerebellectomies. Surgical removal of the cerebellum resulted in significant slowing of the lick rhythm but did not affect the mouse’s ability to perform the gross licking movement. Thus, the cerebellum is involved in the modulation but not in the generation of the licking rhythm. Next, I observed changes to behavior in animals with a genetic cause to their ataxia, the Cerebellin1 (CBLN1) knockout and heterozygous mice (Morgan et al., 1988). The CBLN1 gene is a member of a family of proteins that have been found primarily in the Purkinje cell/parallel fiber synapse and is thought to stabilize the connection. Although removal of the gene does not alter the numbers of neurons or their spatial relations, the mutation results in moderate to severe ataxia. While these animals also varied significantly from their wild type counterparts in lick rate and microstructure, the changes were not all similar to the cerebellectomized model of ataxia. For example, cerebellectomized mice licked significantly slower with an average ILI of 135 ± 8 ms (mean ± S.D.) compared to 117 ± 7 ms whereas in cbln1 KO had a faster lick rate (110 ± 4 ) than wild type counterparts (121 ± 6 ), with all of these values significant with p < 0.05. These observations show that the removal of the normal functions of the cerebellum can alter fluid-licking resulting in bidirectional rate changes. An alternative possibility is that there may be compensatory process. Lastly, I used a chemically-induced model of cerebellar ataxia by injecting the GABA agonist muscimol in the medial and lateral deep cerebellar nuclei. This transient cerebellar ataxia resulted in a similar slowing of the licking rhythm as in the cerebellectomized mice with the eventual recovery of the fluid-licking behavior to normal as the effect of muscimol wore off.

My work to characterize the role of the cerebellum in the maintenance of fluid- licking rhythm and behavior microstructure has resulted in the development of experimental procedures for the recording of neuronal activity in awake and behaving mice. It is an important and necessary step towards neurophysiological investigation of normal and pathological mouse brain function. I have presented the first characterization of simple spike activity, the main cerebellar cortical output signal, during fluid-licking. Furthermore, my results show that the cerebellum is also involved in the control of fluid intake or homeostasis as the intervals between drinking events were abnormally long in mice with cerebellar ataxia. Electrophysiological recordings of individual Purkinje cells from the cerebellar cortex demonstrated variations in spike activity capable of influencing the rhythmicity of fluid licking. While licking still occurred with relative regularity in ataxic animals, the lick rates slowed significantly for mice with surgically induced ataxia and pharmalogically induced ataxia. For animals with a genetic origin to ataxia, lick rates increased. Regularity of licking remained evident despite the change in interlick interval duration. Any alteration of lick timing could ultimately affect the coordination of licking with other orofacial movements. Future investigations may benefit from this work by investigating if therapeutic interventions for cerebellar ataxias show a recovery of typical behavior or adapt the neurophysiological recordings to other behaviors in awake mice.

DOI

10.21007/etd.cghs.2010.0038

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