Cerebellar cortex contributions to the expression and timing of conditioned eyelid responses.

We used micro-infusions during eyelid conditioning in rabbits to investigate the relative contributions of cerebellar cortex and the underlying deep nuclei (DCN) to the expression of cerebellar learning. These tests were conducted using two forms of cerebellum-dependent eyelid conditioning for which the relative roles of cerebellar cortex and DCN are controversial: delay conditioning, which is largely unaffected by forebrain lesions, and trace conditioning, which involves interactions between forebrain and cerebellum. For rabbits trained with delay conditioning, silencing cerebellar cortex by micro-infusions of the local anesthetic lidocaine unmasked stereotyped short-latency responses. This was also the case after extinction as observed previously with reversible blockade of cerebellar cortex output. Conversely, increasing cerebellar cortex activity by micro-infusions of the GABA(A) antagonist picrotoxin reversibly abolished conditioned responses. Effective cannula placements were clustered around the primary fissure and deeper in lobules hemispheric lobule IV (HIV) and hemispheric lobule V (HV) of anterior lobe. In well-trained trace conditioned rabbits, silencing this same area of cerebellar cortex or reversibly blocking cerebellar cortex output also unmasked short-latency responses. Because Purkinje cells are the sole output of cerebellar cortex, these results provide evidence that the expression of well-timed conditioned responses requires a well-timed decrease in the activity of Purkinje cells in anterior lobe. The parallels between results from delay and trace conditioning suggest similar contributions of plasticity in cerebellar cortex and DCN in both instances.

Cerebellar cortex involvement in classical eyelid conditioning

A model for cerebellar involvement in motor learning was tested using classical eyelid conditioning in the rabbit. Briefly, we assume that modifications of the strength of granule cell synapses at Purkinje cells in the cerebellar cortex and mossy fiber (MF) synapses at cerebellar interpositus nuclei are responsible for the acquisition, adaptively-timed expression, and extinction of conditioned eyelid responses (CRs). A corollary of these assumptions is that the cerebellar cortex is necessary for acquisition and extinction. This model also suggests a mechanism whereby the cerebellar cortex can discriminate different times during a conditioned stimulus (CS) and thus mediate the learned timing of CRs. Therefore, experiments were done to determine the role of the cerebellar cortex in the timing, extinction, and acquisition of CRs. Lesions of the cerebellar cortex that included the anterior lobe disrupted the learned timing of CRs such that they occurred at extremely short latencies. Stimulation of MFs in the middle cerebellar peduncle as the CS could support differently timed CRs in the same animal. These data indicate that synaptic plasticity in the cerebellar cortex mediates the learned timing of CRs. These short-latency CRs which resulted from anterior lobe damage did not extinguish, while CRs in animals receiving lesions which did not include the anterior lobe extinguished normally. Preliminary data suggests that lesions of the anterior lobe which produce short-latency responses prevent the acquisition of CRs to a novel CS. These findings indicate that the anterior lobe of cerebellar cortex is necessary for eyelid conditioning. The involvement of the anterior lobe in eyelid conditioning has not been previously reported, however, the anterior lobe has generally been spared in lesion studies examining cerebellar cortex involvement in eyelid conditioning due to its relatively inaccessible location. The observation that the anterior lobe of the cerebellar cortex is not always required for the basic expression of CRs, but is necessary for response timing, extinction, and acquisition, is consistent with the hypothesis that eyelid conditioning can involve plasticity in both the cerebellar cortex and interpositus nucleus and that plasticity in the nucleus is controlled by Purkinje cell activity. ^

Characterization of the interactions between two sites of plasticity engaged during eyelid conditioning

Here, we investigate the involvement of two sites of plasticity in the learning and expression of a simple associative motor behavior—the classically conditioned eyelid response. While previous studies clearly demonstrate that lesions of the anterior interpositus nucleus of the cerebellum abolish learned responses and prevent subsequent learning, studies investigating the effects of lesions of the cerebellar cortex on learning and retention have produced discrepant results. We complement ablative lesion studies of the cortex with the use of reversible, pharmacological blockade of cerebellar cortical transmission to investigate the role of the cerebellar cortex in eyelid conditioning. We demonstrate that both pharmacological blockade as well as focused ablative lesions of the cortex abolish timed responses and unmask responses with a fixed, short latency that are not displayed by the intact animal. Pharmacological blockade of cerebellar cortex output at various stages of acquisition and extinction reveals appropriate, learning dependent changes in the amplitude and probability of short latency responses during training. Acquisition of both short latency as well as timed responses is prevented by ablative lesions of the anterior lobe of the cerebellar cortex. These convergent results from technically distinct methods of removing the influence of the cerebellar cortex from conditioned behavior are consistent with the proposal that (1) eyelid conditioning engages two cerebellar sites of plasticity-one in the cortex and one in the anterior interpositus nucleus, (2) plasticity in the cerebellar cortex is necessary for proper response timing, (3) plasticity in the nucleus mediates the short latency responses unmasked by lesions of the cerebellar cortex, and (4) cerebellar cortical output is necessary for the induction of plasticity in the nucleus. ^

New algorithms for automated phylogenetic reconstruction using artificial intelligence and data mining techniques

Academic and industrial research in the late 90s have brought about an exponential explosion of DNA sequence data. Automated expert systems are being created to help biologists to extract patterns, trends and links from this ever-deepening ocean of information. Two such systems aimed on retrieving and subsequently utilizing phylogenetically relevant information have been developed in this dissertation, the major objective of which was to automate the often difficult and confusing phylogenetic reconstruction process. ^ Popular phylogenetic reconstruction methods, such as distance-based methods, attempt to find an optimal tree topology (that reflects the relationships among related sequences and their evolutionary history) by searching through the topology space. Various compromises between the fast (but incomplete) and exhaustive (but computationally prohibitive) search heuristics have been suggested. An intelligent compromise algorithm that relies on a flexible “beam” search principle from the Artificial Intelligence domain and uses the pre-computed local topology reliability information to adjust the beam search space continuously is described in the second chapter of this dissertation. ^ However, sometimes even a (virtually) complete distance-based method is inferior to the significantly more elaborate (and computationally expensive) maximum likelihood (ML) method. In fact, depending on the nature of the sequence data in question either method might prove to be superior. Therefore, it is difficult (even for an expert) to tell a priori which phylogenetic reconstruction method—distance-based, ML or maybe maximum parsimony (MP)—should be chosen for any particular data set. ^ A number of factors, often hidden, influence the performance of a method. For example, it is generally understood that for a phylogenetically “difficult” data set more sophisticated methods (e.g., ML) tend to be more effective and thus should be chosen. However, it is the interplay of many factors that one needs to consider in order to avoid choosing an inferior method (potentially a costly mistake, both in terms of computational expenses and in terms of reconstruction accuracy.) ^ Chapter III of this dissertation details a phylogenetic reconstruction expert system that selects a superior proper method automatically. It uses a classifier (a Decision Tree-inducing algorithm) to map a new data set to the proper phylogenetic reconstruction method. ^

Learning in the cerebellar nucleus is necessary for acquisition of Pavlovian eyelid conditioning

The cumulative work presented here supports the hypothesis that plasticity in the cerebellar cortex and cerebellar nuclei mediates a simple associative form of motor teaming-Pavlovian eyelid conditioning. It was previously demonstrated that focal ablative lesions of cerebellar anterior lobe or pharmacological block of the cerebellar cortex output disrupted the timing of the conditioned eyeblink response, unmasking a response with a relatively fixed and very short latency to onset. The results of this thesis demonstrate that the short-latency responses are due to associative learning. Unpaired training does not support the acquisition of short-latency responses while the rate of acquisition of short-latency responses during paired training is approximately the same as that of timed conditioned responses. The acquisition of short-latency responses is dependent on an intact cerebellar cortex. Both ablative lesions of the cerebellar cortex and inactivation of cerebellar cortex output with picrotoxin block the acquisition of short-latency responses. However, once the short-latency responses are acquired neither disconnection of cerebellar cortex nor inactivation of the cerebellar nucleus block reacquisition. The results are consistent with the proposal that plasticity in the cerebellar cortex is necessary for learning the timing of conditioned responses, plasticity in the interpositus nucleus mediates the short latency responses, and cerebellar cortical output and mossy fiber input are necessary for the acquisition of short latency responses. ^

Forebrain-cerebellum interactions revealed by trace eyelid conditioning

While it is commonly assumed that brain systems receive and process information from other brain systems, there are few examples of tractable behaviors that allow such interactions to be studied. With the experiments presented in this dissertation we provide evidence that trace eyelid conditioning, a simple form of associative learning, is mediated by cerebellar learning in response to the output of persistent neural activity in the prefrontal cortex (PFC) and thus may be useful in analyses of PFC-cerebellar interactions. In a series of stimulation and reversible inactivation experiments we provide evidence that trace eyelid conditioning is mediated by cerebellar learning in response to a learned forebrain-driven input. Specifically, we provide evidence that this input is driven by the medial PFC and persists through the stimulus free trace interval of trace eyelid conditioning. In the next set of experiments we show that directly presenting the cerebellum with a pattern of input that mimics the classic persistent activity of PFC neurons reconstitutes trace eyelid conditioning, as assessed by a number of stringent tests. Finally, in set of reversible inactivation experiments, we provide evidence that bidirectional learning during trace eyelid conditioning involves the omission of the persistent, PFC-driven input that the cerebellum learns and responds to during trace eyelid conditioning. Given that persistent activity in PFC is often associated with working memory, these experiments suggest that trace eyelid conditioning may be useful in analyses of working memory mechanisms, cerebellar information processing and their interaction. To facilitate future analyses, we conclude with a working hypothesis of forebrain-cerebellum interactions during trace eyelid conditioning that addresses how persistent activity in PFC is induced and how the cerebellum decodes and uses PFC-driven input. ^

Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^