Gary Zenitsky

The cerebellum and eye-blink conditioning: learning versus network performance hypotheses.

Classical conditioning of the eye-blink reflex in the rabbit is a form of motor learning that is uniquely dependent on the cerebellum. The cerebellar learning hypothesis proposes that plasticity subserving eye-blink conditioning occurs in the cerebellum. The major evidence for this hypothesis originated from studies based on a telecommunications network metaphor of eye-blink circuits. These experiments inactivated parts of cerebellum-related networks during the acquisition and expression of classically conditioned eye blinks in order to determine sites at which the plasticity occurred. However, recent evidence revealed that these manipulations could be explained by a network performance hypothesis which attributes learning deficits to a non-specific tonic dysfunction of eye-blink networks. Since eye-blink conditioning is mediated by a spontaneously active, recurrent neuronal network with strong tonic interactions, differentiating between the cerebellar learning hypothesis and the network performance hypothesis represents a major experimental challenge. A possible solution to this problem is offered by several promising new approaches that minimize the effects of experimental interventions on spontaneous neuronal activity. Results from these studies indicate that plastic changes underlying eye-blink conditioning are distributed across several cerebellar and extra-cerebellar regions. Specific input interactions that induce these plastic changes as well as their cellular mechanisms remain unresolved.

Video recording system for the measurement of eyelid movements during classical conditioning of the eyeblink response in the rabbit.

Classical conditioning of the eyeblink response in the rabbit is a popular model for studying the neural substrates of associative learning. Most of the common eyeblink recording techniques require invasive procedures. To perform experiments in intact animals, a non-invasive, high-speed video recording system was developed to measure eyeblink responses of rabbits during classical conditioning experiments. Besides being non-invasive, this method does not require excessive restraint of the animal. The PC-based system combines a Pulnix video camera with National Instruments video capture and timing hardware to control the experiment and acquire images of the peri-ocular region. The software developed for controlling these experiments also detects the eyeblink by measuring the movement of the upper and lower eyelids, the area of the exposed surface of the eye, and head movements in the sagittal plane. The time resolution of this relatively inexpensive system is 8.33 ms, and at a working distance of 0.8 m, it can detect movements as small as 0.11 mm.

Manganese exposure induces neuroinflammation by impairing mitochondrial dynamics in astrocytes

HighlightsMn induces mitochondrial dysfunction in astrocytes.Mn exposure induces inflammatory response in astrocytes and exacerbates &agr;Synagg‐induced inflammatory response in astrocytes.Mito‐apocynin attenuates Mn‐induced inflammatory response in astrocytes by reducing mitochondrial damage.Intranasal Mn exposure induces neuroinflammation and behavioral deficits in mouse model.Thus, Mn‐induced mitochondrial defects contribute to astroglial neuroinflammation. ABSTRACT Chronic manganese (Mn) exposure induces neurotoxicity, which is characterized by Parkinsonian symptoms resulting from impairment in the extrapyramidal motor system of the basal ganglia. Mitochondrial dysfunction and oxidative stress are considered key pathophysiological features of Mn neurotoxicity. Recent evidence suggests astrocytes as a major target of Mn neurotoxicity since Mn accumulates predominantly in astrocytes. However, the primary mechanisms underlying Mn‐induced astroglial dysfunction and its role in metal neurotoxicity are not completely understood. In this study, we examined the interrelationship between mitochondrial dysfunction and astrocytic inflammation in Mn neurotoxicity. We first evaluated whether Mn exposure alters mitochondrial bioenergetics in cultured astrocytes. Metabolic activity assessed by MTS assay revealed an IC50 of 92.68 &mgr;M Mn at 24 h in primary mouse astrocytes (PMAs) and 50.46 &mgr;M in the human astrocytic U373 cell line. Mn treatment reduced mitochondrial mass, indicative of impaired mitochondrial function and biogenesis, which was substantiated by the significant reduction in mRNA of mitofusin‐2, a protein that serves as a ubiquitination target for mitophagy. Furthermore, Mn increased mitochondrial circularity indicating augmented mitochondrial fission. Seahorse analysis of bioenergetics status in Mn‐treated astrocytes revealed that Mn significantly impaired the basal mitochondrial oxygen consumption rate as well as the ATP‐linked respiration rate. The effect of Mn on mitochondrial energy deficits was further supported by a reduction in ATP production. Mn‐exposed primary astrocytes also exhibited a severely quiescent energy phenotype, which was substantiated by the inability of oligomycin to increase the extracellular acidification rate. Since astrocytes regulate immune functions in the CNS, we also evaluated whether Mn modulates astrocytic inflammation. Mn exposure in astrocytes not only stimulated the release of proinflammatory cytokines, but also exacerbated the inflammatory response induced by aggregated &agr;‐synuclein. The novel mitochondria‐targeted antioxidant, mito‐apocynin, significantly attenuated Mn‐induced inflammatory gene expression, further supporting the role of mitochondria dysfunction and oxidative stress in mediating astrogliosis. Lastly, intranasal delivery of Mn in vivo elevated GFAP and depressed TH levels in the olfactory bulbs, clearly supporting the involvement of astrocytes in Mn‐induced dopaminergic neurotoxicity. Collectively, our study demonstrates that Mn drives proinflammatory events in astrocytes by impairing mitochondrial bioenergetics.

Inactivation of cerebellar output axons impairs acquisition of conditioned eyeblinks

Acquisition of classically conditioned eyeblink responses (CRs) in the rabbit critically depends on intermediate cerebellum-related neural circuits. A highly efficient method for determining possible sites of plasticity within eyeblink circuits is the reversible inactivation of circuit components during learning. Inactivation of either the HVI region of the cerebellar cortex or the cerebellar interposed nuclei (IN) during learning is known to prevent CR acquisition. In contrast, inactivating cerebellar efferent axons in the brachium conjunctivum (BC) with small injections of tetrodotoxin (TTX) has been reported to have no effect on CR acquisition. This suggested that the intermediate cerebellum is essential for learning CRs and that activity mediated by the BC is not required for this process. Since we previously found that BC inactivation blocks CR extinction we re-examined its role in CR acquisition. To ensure complete and long-lasting inactivation of the BC, we injected before each training session doses of TTX that were larger than those in the previous acquisition study. Contrary to the previous negative findings, we found that this temporary block of axons in the brachium conjunctivum prevented normal acquisition of CRs. Injecting TTX directly in the adjacent lateral lemniscus, which could possibly influence CR acquisition, had no effect on learning. In addition, a functional test of TTX diffusion around the BC indicated that the inactivation did not affect other known parts of eyeblink circuits, such as the cerebellar interposed nuclei, the middle cerebellar peduncle or the contralateral red nucleus. We conclude that this form of associative learning in the rabbit eyeblink system requires extra-cerebellar learning and/or cerebellar learning that depends on the operation of cerebellar feedback loops.

Inactivation of the brachium conjunctivum prevents extinction of classically conditioned eyeblinks

It is well established that the intermediate cerebellum is involved in the acquisition of classically conditioned eyeblink responses (CRs). Recent studies that inactivated the interposed nuclei (IN) demonstrated that blocking the intermediate cerebellum also interrupts CR extinction. Is this extinction deficit related to interrupting the information flow to efferent targets of the IN? To address this question, we inactivated axons of IN neurons in the brachium conjunctivum (BC). This treatment blocked the output of the intermediate cerebellum without directly affecting neurons in the deep cerebellar nuclei. Rabbits were trained in a delay classical conditioning paradigm, using a tone as the conditioned stimulus (CS) and a corneal air puff as the unconditioned stimulus (US). Then, the BC was microinjected with a sodium channel blocker, tetrodotoxin, during 4 extinction sessions in which rabbits were presented only with the CS. Tests performed after the 4-day injection period revealed that CRs did not extinguish in BC inactivation sessions but extinguished at a normal rate in the absence of the drug. CRs were then re-acquired. These data show that the normal flow of information along axons of cerebellar nuclear cells is required for CR extinction.

A trigeminal conditioned stimulus yields fast acquisition of cerebellum-dependent conditioned eyeblinks.

Classical conditioning of the eyeblink response in the rabbit is a form of motor learning whereby the animal learns to respond to an initially irrelevant conditioned stimulus (CS). It is thought that acquired conditioned responses (CRs) are adaptive because they protect the eye in anticipation of potentially harmful events. This protective mechanism is surprisingly inefficient because the acquisition of CRs requires extensive training - a condition that is unlikely to occur in nature. We hypothesized that the rate of conditioning in rabbits could depend on CS modality and that stimulating mystacial vibrissae as the CS could produce CR acquisition faster than the traditional auditory or visual stimulation. We tested this hypothesis by conditioning naïve rabbits in the delay paradigm using a weak airpuff CS (vCS) directed to the ipsilateral mystacial vibrissae. We found that the trigeminal vCS yields significantly faster CR acquisition. We next examined if vCS-evoked CRs are dependent on the intermediate cerebellum in the same fashion as CRs evoked by the traditional auditory CS. We found that vibrissal CRs could be abolished by inactivating the cerebellar interposed nuclei (IN) with muscimol. In addition, injections of picrotoxin in the IN shortened the onset latency of vibrissal CRs. These findings suggest that the tone and vCS-evoked CRs share similar cerebellar dependency.

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinsons, Alzheimers, and Huntingtons Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Blocking Glutamate-Mediated Inferior Olivary Signals Abolishes Expression of Conditioned Eyeblinks But Does Not Prevent Their Acquisition

The inferior olive (IO) is considered a crucial component of the eyeblink conditioning network. The cerebellar learning hypothesis proposes that the IO provides the cerebellum with a teaching signal that is required for the acquisition and maintenance of conditioned eyeblinks. Supporting this concept, previous experiments showed that lesions or inactivation of the IO blocked CR acquisition. However, these studies were not conclusive. The drawback of the methods used by those studies is that they not only blocked task-related signals, but also completely shut down the spontaneous activity within the IO, which affects the rest of the eyeblink circuits in a nonspecific manner. We hypothesized that more selective blocking of task-related IO signals could be achieved by using injections of glutamate antagonists, which reduce, but do not eliminate, the spontaneous activity in the IO. We expected that if glutamate-mediated IO signals are required for learning, then blocking these signals during training sessions should prevent conditioned response (CR) acquisition. To test this prediction, rabbits were trained to acquire conditioned eyeblinks to a mild vibrissal airpuff as the conditioned stimulus while injections of the glutamate antagonist γ-d-glutamylglycine were administered to the IO. Remarkably, even though this treatment suppressed CRs during training sessions, the postacquisition retention test revealed that CR acquisition had not been abolished. The ability to acquire CRs with IO unconditioned stimulus signals that were blocked or severely suppressed suggests that mechanisms responsible for CR acquisition are extremely resilient and probably less dependent on IO-task-related signals than previously thought.

Chronic Traumatic Encephalopathy

Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disorder that can only be diagnosed post-mortem and has been retrospectively linked to a history of neurotrauma involving repeated concussive and subconcussive head injuries. Such a pattern of neurotrauma is commonly found among military veterans and athletes participating in contact sports. A post-mortem diagnosis depends on the detection of abnormal hyperphosphorylated-tau (p-tau) neuropathology that is characterized by an irregular, focal, perivascular distribution largely localized to cortical sulci. Although p-tau is not unique to CTE, this distribution pattern helps distinguish it from other degenerative tauopathies. High molecular weight p-tau proteins tend to accumulate in neurons and glia as pretangles, neurofibrillary tangles, extracellular tangles and neuropil threads. Supportive non-p-tau pathologies include TDP-43 and β-amyloid in neuronal cytoplasmic inclusions and dot-like structures in the tangles. Progressive tauopathies can share a broad range of neuropsychiatric sequelae, which for CTE are identified through next-of-kin interviews or verbal autopsies. A better understanding of how CTE relates to other neuropsychiatric and neurodegenerative disorders will require carefully controlled prospective studies involving both in vitro and in vivo model systems as well as parallel efforts to develop and validate fluid biomarkers and tau-specific neuroimaging technologies. Such an approach will improve the odds of advancing multiple, targeted therapies from preclinical studies to clinical trials. This chapter covers etiology, pathophysiology, experimental model systems, biomarkers and potential therapeutic strategies pertaining to CTE.