The emotion hypothesis of startle eyeblink modification posits that potentiated eyeblinks are observed in response to fear/disgust (aversive) pictures and eyeblink inhibition occurs in response to pleasant (appetitive) pictures due to the degree to which the stimuli match with the aversive startle probe. Stimuli high in arousal elicit exaggerated responses. Four studies sought to investigate the effect of angering pictures on the startle eyeblink response. Three potential hypotheses were posed: 1) given anger's high levels of arousal and negativity, eyeblinks will be potentiated like those to fear/disgust pictures; 2) given anger's arousing and appetitive qualities, eyeblinks will be inhibited like those to pleasant pictures; 3) anger's arousal, negativity, and approach qualities will balance each other out causing eyeblinks resembling those in response to neutral pictures. Study 1 supported the third hypothesis in that eyeblinks to angering and neutral pictures did not differ, despite angering pictures being rated higher on arousal and anger and lower in valence. These results replicated in Study 2 with a different set of angering pictures. Also, Study 2 demonstrated that dysphoric participants exhibited potentiated eyeblinks during angering pictures much like eyeblinks during fear/disgust stimuli, whereas non-dysphoric participants did not. Ratings of pictures on arousal, valence, and anger did not differ between groups. Constructive patriotism related to inhibited eyeblinks during angering pictures. Study 3 found that dysphoric participants rated angering pictures higher in fear than did non-dysophoric participants, suggesting that the potentiated eyeblinks observed in Study 2 were a result of greater perceived fear. Study 4 again showed that eyeblinks during angering and neutral pictures did not differ, and that constructive patriotism related to inhibited eyeblinks. Taken together, results are consistent with the third hypothesis and suggest that angering stimuli elicit eyeblinks much like those to neutral stimuli due to the competing influences of arousal, valence, and motivation on the startle eyeblink reflex.

Research concerning the development of learning and memory suggests that there are multiple memory systems. These systems differ in complexity, underlying neural substrates, and consequently, their developmental emergence. Pavlovian conditioning, and specifically eyeblink conditioning (EBC), allows researchers to investigate both simple and complex forms of learning and memory early in development. Delay EBC, which is considered a relatively simple form of learning, involves the association of a conditioned stimulus (CS) with an unconditioned stimulus (US). Research from our laboratory suggests that the emergence of delay EBC is dependent on the development of sensory input to the pontine nucleus. Trace EBC, a more complex form of learning, involves the association of a CS with a US over a stimulus-free trace interval. Due to its relatively late emergence, the developmental time course of trace EBC has been traditionally regarded as independent of sensory system development. Rather, it is the involvement of late-developing structures such as the hippocampus which is considered the principle limiting factor in the emergence of trace EBC. The current collection of studies investigates the developmental emergence of delay and trace conditioning. We found that both delay and trace conditioning are facilitated by using an early-developing somatosensory CS. This suggests that the sensory system development plays a role in even late-developing trace EBC. Moreover, hippocampal CA1 neuronal activity shows increased responsiveness in even very young animals when trained with an early-developing somatosensory CS compared to those trained with a tone CS. Combined, these data suggest that both hippocampal and sensory system development may play key roles in the developmental emergence of learning.

Cannabinoid receptors (CBR) are the most abundant G-protein coupled receptors in the mammalian brain with the highest densities within the cerebellum (Herkenham et al., 1990). Cannabinoid manipulations have been reported to cause deficits in cerebellar-dependent learning (Kishimoto and Kano, 2006; Skosnik et al., 2007; Steinmetz and Freeman, 2010; 2013). Cannabinoid receptors-1 (CB1R) have been hypothesized to be important in the establishment of long-term depression within the cerebellar cortex (Levenes et al., 1998; Safo and Regehr, 2005). However, all investigations in vivo have used global manipulations and have not attempted to localize or characterize these receptors during cerebellar-dependent learning. Chapter 2 systematically examined localization within the cerebellar cortex of cannabinoid effects on eyeblink conditioning, a type of cerebellum-dependent learning. Local infusions into a specific portion of the cerebellar cortex, the eyeblink conditioning microzone, resulted in deficits in learning similar to systemic injections. Additionally, infusions of cannabinoids into the eyeblink conditioning microzone, and no other parts of the cerebellar cortex or deep nuclei, were responsible for the deficits. Finally, tetrode recordings were made in Purkinje cells while receiving either CBR agonist or vehicle injections prior to training. Fewer Purkinje cells exhibited learning-related decreases in activity when the rat was administered a CBR agonist as compared to when it was injected with the vehicle. The CBR administered Purkinje cells also showed earlier onsets and smaller amplitudes in their learning-related activity. Purkinje cells that show a learning-related increase in activity were not affected by cannabinoid administration. The impairment in Purkinje cell plasticity was not observed after the rats reached asymptotic levels of learning. These results indicate that CBR agonist administration disrupts the induction of plasticity within the cerebellar cortex and this may account for the behavioral deficit in eyeblink conditioning. Chapter 3 examined whether infusions of the CBR agonist into the cerebellar cortex impaired forebrain-dependent learning as well as forebrain-independent associative learning. Similar to subcutaneous injections, forebrain-dependent trace eyeblink conditioning was unimpaired, whereas forebrain independent delay eyeblink conditioning was impaired. These findings provide evidence that plasticity mechanisms that are modulated by cannabinoids do not play a significant role in trace eyeblink conditioning. Finally, in Chapter 4 the role of CBRs and endocannabinoids during memory consolidation were examined. CBR and endocannabinoid manipulations prior to training resulted in impaired eyeblink conditioning. However, a CBR agonist or a drug increasing endocannabinoid levels resulted in enhanced consolidation when administered 1 hour post-training. In contrast, a CBR antagonist or an endocannabinoid decreasing drug resulted in impairments 1 hour post-training. Thus, CBRs and endocannabinoids appear to be important in learning and consolidation of cerebellar-dependent learning.

The brain is composed of multiple systems that interact during cognition and behavior. Concurrently performing two or more tasks that compete for processing from a common system resource typically results in decremented performance, referred to as interference, for one or more of the tasks as compared to single-task performance. This interference is a hallmark of the dual-task experimental design. The purpose of this experiment is to investigate the involvement of cerebellar cortex in trace eyeblink conditioning by utilizing the traditional dual-task design. Timed-interval finger tapping, a motor function mediated by the cerebellar cortex, and several different cognitive tasks representing dissociable distributed brain memory systems were co-administered with trace eyeblink conditioning. If cerebellar cortex is involved in trace eyeblink conditioning, then timed-interval finger tapping should significantly interfere with acquisition rate for conditioned responding. Performance variables from trace eyeblink conditioning were analyzed to investigate interference effects of timed-interval finger tapping and the various cognitive tasks. Results from a series of mixed model repeated measures ANOVAs indicate that the acquisition rate and magnitude of trace conditioning was not significantly reduced in the timed-interval tapping group compared to the control group, although participants did demonstrate evidence of inferior learning. This finding suggests that cerebellar cortex is not critically involved during acquisition of trace conditioning, however it is to be interpreted with caution as methodological and theoretical confounds may preclude a straightforward conclusion. / Psychology

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The neural mechanisms that support learning and memory are still poorly understood. Much work has focused on changes in neurotransmitter receptor expression, while changes in voltage-gated ion channel expression have been largely unexplored, despite the fact that voltage-gated ion channels govern neuronal excitability. Here we used eyeblink conditioning (EBC) in rats, a model of learning and memory with a well-understood neural circuit, to examine regulation of voltage-gated ion channels as a consequence of learning. EBC is a form of classical conditioning that involves pairings of a behaviorally neutral conditioned stimulus (CS) and an eyeblink eliciting unconditioned stimulus (US) over many trials to produce an eyeblink conditioned response (CR) to the CS in anticipation of the US. The acquisition and generation of the eyeblink CR is governed by plasticity at various sites in the cerebellum, both in the cerebellar cortex and the interpositus nucleus (IPN). Purkinje cells (PCs) are the primary neuron in the cerebellar cortex and these cells represent the sole output of the cerebellar cortex. PCs tonically inhibit the neurons of the IPN; the IPN is the start of the eyeblink pathway. In order for a CR to be generated, the inhibition of the IPN by PCs must be lifted. Basket cells (BCs) are small inhibitory interneurons that form synapses near the PC soma. These neurons are strategically located to strongly regulate PC output through inhibitory input near the axon hillock. BC axon terminals have the highest expression of Kv1.2, an alpha subunit of the Kv1 (Shaker) family of voltage-gated potassium channels, in the cerebellum. In addition, significant Kv1.2 expression is found on PC dendrites. Blocking Kv1.2 leads to increased GABAergic input to PCs and facilitates EBC. In the current work, we addressed the question of whether EBC itself regulates surface expression of Kv1.2 in cerebellar cortex. Rats received three days of either EBC, explicitly unpaired stimulus presentations, or no stimuli, and cerebellar tissue was harvested and analyzed via biotinylation/western blot (WB) and multiphoton microscopy (MP) techniques. In the first experiment, the Unpaired group showed significantly reduced surface Kv1.2 expression at BC axon terminals as measured by MP, but no changes observed with the WB measure, which measures expression at both BC axon terminals and PC dendrites. The second experiment used the same procedures but examined cerebellar tissue following a shorter training procedure. We hypothesized that the Paired and Unpaired groups would show similar Kv1.2 surface expression earlier in training. The Unpaired group showed increased surface Kv1.2 compared to the other two groups in the WB measures, but no differences were observed in the MP measure. Paired group rats that did not exhibit CRs showed the same pattern as the Unpaired group. Overall, we observed training and location specific changes in surface Kv1.2 expression, suggesting that learning does appear to regulate voltage-gated ion channel expression in the mammalian brain. Increased surface Kv1.2 early in training before CR expression emerges may set the stage for other mechanisms to govern the expression of the learned response. Prolonged stimulus input that is unmodulated by expression of a learned response, such as in the Unpaired group in the first experiment, leads to long-term changes in surface Kv1.2 expression exclusively at BC axon terminals.

Major depressive disorder (MDD) is a disorder defined by mood disturbance, but the deficits in emotional reactivity that accompany MDD are not yet fully characterized. Researchers have utilized the emotion-modulated startle paradigm to investigate emotional responding among depressed individuals with mixed results. Inconsistent results may be due in part to the heterogeneity of mood disorders, including variation in mood severity. The current study utilized an emotion-modulated startle procedure with 33 individuals currently experiencing a major depressive episode, 25 individuals currently experiencing a minor depressive episode (mD), and 31 healthy controls. Severity of depression, anxiety, and positive and negative mood states were ascertained on the sample. Emotion-modulated startle failed to differentiate between mood disordered individuals and healthy controls. However, results found a significant association between abnormal patterns of emotion responding and positive affect (PA), such that individuals with low PA showed exaggerated responding to unpleasant stimuli. The results suggest that PA may be an important dimension in mood disorders that underlies abnormal emotional responses.

Consolidation of associative memories may depend on communication between the lateral entorhinal cortex (LEC) and hippocampus (HPC) for recently learned memories and the LEC and medial prefrontal cortex (mPFC) for remote memories. To determine whether direct connections between these regions are necessary for the retrieval of a recently or remotely learned memory, rats acquired an associative memory through trace eyeblink conditioning and were tested for memory retention after inactivating the regions of interest with the GABAA agonist, muscimol. Inactivating the LEC-HPC connection did not impair memory retrieval. However, inactivating the LEC-mPFC connection impaired remote, but not recent, memory retrieval. Thus, the LEC and mPFC connection is necessary for the retrieval of a remotely, but not recently learned associative memory. Increased reliance on the entorhinal-prefrontal connection indicates the strengthening of functional connectivity between the two regions, which may be a biological correlate for the proposed reorganization during systems consolidation.

Consolidation of associative memories may depend on communication between the lateral entorhinal cortex (LEC) and hippocampus (HPC) for recently learned memories and the LEC and medial prefrontal cortex (mPFC) for remote memories. To determine whether direct connections between these regions are necessary for the retrieval of a recently or remotely learned memory, rats acquired an associative memory through trace eyeblink conditioning and were tested for memory retention after inactivating the regions of interest with the GABAA agonist, muscimol. Inactivating the LEC-HPC connection did not impair memory retrieval. However, inactivating the LEC-mPFC connection impaired remote, but not recent, memory retrieval. Thus, the LEC and mPFC connection is necessary for the retrieval of a remotely, but not recently learned associative memory. Increased reliance on the entorhinal-prefrontal connection indicates the strengthening of functional connectivity between the two regions, which may be a biological correlate for the proposed reorganization during systems consolidation.

Delay classical eyeblink conditioning is an important model of associative, cerebellar dependent learning. Norepinephrine (NE) plays a significant modulatory role in the acquisition of learning; other neurotransmitter systems are also at play. The goal of this dissertation was to determine whether NE, GABA and glutamate (Glu) release is observed in cerebellar cortex during delay eye blink conditioning, and whether such release was selectively associated with training and not due only to stimulatory sensory input. The data support the hypothesis of noradrenergic and GABAergic system involvement in motor learning with NE as a modulator of early responding and GABA as a mediator of the learned response. In addition to neurotransmitter levels, we found that the local administration into the cerebellum of Rp-cAMP and propranolol impair the consolidation of learning when administered post training on the eyeblink conditioning task indicating that the B-adrenergic receptor and the cAMP downstream signaling cascade are essential for memory consolidation. These results support the hypothesis of NE acting as a neuromodulator in the cerebellum for the acquisition of motor learning. A similar experimental design was applied to aged animals and the neurochemical pattern of release was haracterized by a delay in the response to eyeblink conditioning and smaller amounts of the neurotransmitter evoked by the paired US-CS. It is hypothesized that the impairment in aging could be due to excitotoxicity caused by chronic inflammation. The present study also approached this issue by targeting the pro-inflammatory cytokine TNF-a and we found that suppression of TNF-a in aged animals improved learning.