Duchenne's muscular dystrophy (DMD) is a debilitating disease that affects approximately 1 in 3500 boys, with many DMD patients dying before the age of 20 due to cardio-respiratory complications. DMD is the result of defects in the gene that encodes dystrophin, an integral muscle membrane protein. Although the genetic defect has been identified, the relation between the absence of expressed dystrophin and the mechanisms leading to its onset are still unclear. One possibility is that disrupted calcium (Ca²⁺) handling by the sarcoplasmic reticulum (SR) leads to an increased cytosolic Ca²⁺ concentration that activates proteolytic and apoptotic pathways that initiate muscle fiber death. However, little is known about the role of disrupted SR function in the onset of DMD. The purpose of this study was to test the hypothesis that altered calcium cycling by the SR could contribute to elevated cytosolic Ca²⁺ levels in the early stages of DMD, and thereby account for the onset of disease pathogenesis. Rates of SR Ca²⁺ uptake and release were determined in quadriceps muscles obtained from maturing dystrophic and control mice prior to the overt signs of the disease at ages ~9 and 21 days. In addition, the content of several key Ca²⁺ handling proteins, including two isoforms of the sarco(endo)plasmic reticulum ATPase pump (SERCA 1 & 2), ryanodine receptor type 1 (RyR1), parvalbumin, and calsequestrin were determined by Western analysis. Two dystrophic mouse models were used, the mdx mouse which lacks dystrophin, and the mdx:utrophin-deficient (mdx:utrn^-/-) mouse which also lacks utrophin, a protein homolog of dystrophin. The rate of SR Ca²⁺ uptake in quadriceps muscles of mdx/utrn^-/- mice aged 21 days was 73.1% and 61.3% higher than age-matched control and mdx muscles, respectively (p < 0.05). There was no difference in SR Ca²⁺ release rates between the genotypes at either age. There were significant increases in the content of each of the calcium handling proteins with age (p < 0.05), but no significant differences were detected between genotypes at either age. These data demonstrate the Ca²⁺ release rates of dystrophic SR are not compromised, but suggest the increased uptake rates of mdx:utrn^-/- SR may be an adaptation to increased cytosolic calcium levels, and/or be due to changes in intrinsic SERCA function and/or regulation. The role of increased SR Ca²⁺ uptakes rates in onset of DMD pathogenesis can not be directly determined from the present data; therefore it is suggested that future studies directly assess cytosolic Ca²⁺ concentration and examine the role of SERCA regulatory proteins in intact fibers obtained from mdx:utrn^-/- muscles at age 21 days. / Master of Science

The sonic muscle of toadfish is the fastest vertebrate muscle ever measured, and the rate of transport of Ca2+ and dissociation of cross-bridge are also fatest. Parvalbumins are Ca2+-binding proteins present in vertebrate muscle, and they can aid muscle relaxation. Several isoforms of parvalbumin had been identified and presented in variable proportion in different kinds of muscles (e.g. red muscle, white muscle and pink muscle). Both male and female grunting toadfish (Allenbatrachus grunniens) have intrinsic sonic muscles attached on swim bladders. The morphology of male and female sonic muscle was compared, and no significant differences in both length, width, thickness and weight were found. SDS-PAGE and western blotting were used to determine the total parvalbumin expression and identify the parvalbumins from sonic muscle and body white muscle. There were no significant differences in total parvalbumin expression in sonic muscle and body white muscle. The result indicates that there is no positive correlation between high content of parvalbumins and speed of muscle relaxation. In native-PAGE, two and four parvalbumin isoforms were identified from sonic muscle and body white muscle, respectively. The estimated size of Parv1, Parv2 and Parv3 size in grunting toadfish¡¦s sonic muscle were 10kDa, 10.5kDa and 10.5kDa, respectively, and the isoelectric points of Parv1, Parv2 and Parv3 in grunting toadfish were 4.77, 4.58 and 4.42, respectively. In the sonic muscle, the major parvalbumin isoform was parvalbumin isoform 1 (Parv1), which comprised more than 94% of total parvalbumin, and parvalbumin isoform 2 (Pav2) comprised vi only 5% of total parvalbumin content. In body white muscle, on the other hand, the major isoform was parvalbumin isoform 2 (Parv2) which comprised 58% of toal parvalbumin. Both Parv1 (with Parv1a) and Parv3 comprised about 20%. Parvalbumin isoforms were be discussed. The result supports that Parv1 has a highest effect on the relaxation of the grunting-toadfish¡¦s sonic muscle.

Parvalbumin (PV) and cholecystokinin (CCK) proteins are found in the basolateral amygdala nuclei, particularly in gamma-aminobutyric acid (GABA) interneurons. PV+ neurons were localized to the basolateral amygdala and they expressed the GABA neuron marker glutamic acid decarboxylase (GAD). Here, we used Cre recombinase mouse lines to induce expression of mutant muscarinic inhibitory (hM4D) and excitatory (hM3D) receptors on PV+ or CCK+ neurons. Activation of the mutant receptors with clozapine-n-oxide (CNO) was used to measure how amygdala neural changes affect the acoustic startle reflex (ASR). Excitation of amygdala PV+ neurons potentiated the ASR. Activation of basolateral amygdalar CCK+ neurons potentiated the ASR and caused seizures, possibly by activating glutamate CCK+ neurons. The CCK+ subset of GAD neurons were targeted with a new triple transgenic mouse line (Dlx5-flpe/CCK-Cre/FrePe) to show that most CCK+ neurons were GAD negative. These findings are compared with optogenetic approaches to target specific neuronal populations.

Parvalbumin (PV) and cholecystokinin (CCK) proteins are found in the basolateral amygdala nuclei, particularly in gamma-aminobutyric acid (GABA) interneurons. PV+ neurons were localized to the basolateral amygdala and they expressed the GABA neuron marker glutamic acid decarboxylase (GAD). Here, we used Cre recombinase mouse lines to induce expression of mutant muscarinic inhibitory (hM4D) and excitatory (hM3D) receptors on PV+ or CCK+ neurons. Activation of the mutant receptors with clozapine-n-oxide (CNO) was used to measure how amygdala neural changes affect the acoustic startle reflex (ASR). Excitation of amygdala PV+ neurons potentiated the ASR. Activation of basolateral amygdalar CCK+ neurons potentiated the ASR and caused seizures, possibly by activating glutamate CCK+ neurons. The CCK+ subset of GAD neurons were targeted with a new triple transgenic mouse line (Dlx5-flpe/CCK-Cre/FrePe) to show that most CCK+ neurons were GAD negative. These findings are compared with optogenetic approaches to target specific neuronal populations.

Schizophrenia is a relatively poorly understood, debilitating psychiatric disorder affecting around 0.5% of the population worldwide. The main characteristics of the disease are hallucinations, delusions and cognitive impairment such as difficulty in learning. It has been recently suggested that Disrupted-in-Schizophrenia-1 (DISC1) might be one of the main genetic risk factors for this disease. Mouse Disc1 has been implicated in brain development, mainly in neurite outgrowth, integration of newborn neurons, neuronal precursor proliferation/differentiation and neuronal migration. Disc1 function in the cortical excitatory cells was studied in fair detail but there is little data on Disc1 role in cortical interneuron development. In this study I have investigated development of the cortical interneurons in 21 days old mice with ENU-induced point mutations in the mouse Disc1 sequence - L100P and Q31L; previously characterized as ‘schizophrenic-like’ and ‘depressive-like’ respectively. Bin analysis was performed on five brain regions: frontal and central primary somatosensory (fSSp and SSp respectively) cortices, ventral auditory (vAud) cortex, visual (Vis) cortex and medial prefrontal cortex (MPFC); for four major interneuronal markers: parvalbumin (PV), somatostatin (STT), calretitnin (CLR) and glutamate decarboxylase 67 (GAD67). A significant decrease in PV (protein and mRNA) expression was observed in a subclass of the cortical interneurons in the fSSp, SSp, vAud and Vis cortices of L100P homozyogous (L100P) and heterozygous (L100P +/-) mouse brains when compared to their wild-type (WT) littermates. No such difference in the PV positive cells was found in the MPFC in the L100P mouse brain. Other interneuronal markers expression was not different in the L100P and L100P +/- brain from that in the WT littermate controls. Furthermore, there was no significant difference in any of the interneuronal markers expression in the Q31L mouse brain cortex. A minor change in the relative distribution of the interneurons (GAD67 positive cells) was found in the L100P but not Q31L brain. With no difference in the number of the interneurons and the nature of PV expression regulation, the cell non-autonomous effect of L100P Disc1 on this subpopulation of intereneurons was investigated. Overexpression of the mouse Disc1-100P in utero in the radial glia cells born at E14.5 (future layer II/III and IV excitatory cells) resulted in a significant decrease in the PV positive cells in all of the electroporated regions (fSSp, SSp, vAud and Vis cortices) when compared to mouse WT Disc1 overexpression. Furthermore, a decrease in the PV cells on the contralateral side was observed in the SSp and Vis cortices. This study demonstrates that mouse Disc1 is involved in the generation of parvalbumin expressing interneurons within the cortex in a cell non-autonomous way. The L100P point mutation in Disc1 led to downregulation of parvalbumin, which in turn would result in abnormal inhibitory properties of this interneuron subtype.

BACKGROUND: Schizophrenia is a debilitating disorder that has a profound impact on quality of life due to the presence of both cognitive deficits and psychotic symptoms. Despite having significant global economic and social costs and a worldwide prevalence of 1%, schizophrenia is still not well understood. Research has been making strides in uncovering the pathophysiology and the etiology that drive this disease, ranging from genetic abnormalities, disrupted circuitry, changes in microarchitecture, to impaired synaptic connectivity. Evidence suggests that disrupted-in-schizophrenia-1 (DISC1) driven genetic disturbances in fast-spiking parvalbumin (PV) neurons and their surrounding perineuronal nets (PNNs) likely contribute to schizophrenia etiology as they are part of the microcircuits required for working memory, a cognitive function that has been consistently impaired in schizophrenic patients. OBJECTIVE: To identify the neuroanatomical changes in PV neurons and surrounding PNNs in the superficial and deep layers of the prelimbic and infralimbic prefrontal cortex of a rat DISC-1 knockout model. METHODS: 19 DISC1-KO male rats and 15 wildtype rats were treated with saline or MK-801. They were sacrificed between P268-269 and brains were extracted and separated at the corpus callosum. After fixing and preserving, the brains were sliced then stained to visualize parvalbumin and perineuronal nets with immunohistochemistry. Slices were imaged and analyzed for PV, PNN, and PV+PNN counts in the superficial and deep regions of the prelimbic and infralimbic cortices. Averages counts within each group were taken and analyzed via 2-way ANOVAs for each brain region and dependent variable. RESULTS: DISC1-KO rats displayed the following trending changes: decreased PV cells in deep layers of infralimbic and decreased PNNs throughout the prelimbic cortex. MK-801 appears to increase the number of unsheathed PV cells in the superficial layers of prelimbic and infralimbic cortex. It decreased the number of PNNs in the prelimbic of wildtype animals but not in the DISC1-KO cohort. MK-801 moderately increased PV counts in DISC1-KO. CONCLUSIONS: This DISC1-KO model is a promising model of schizophrenia as we see the same directionality of decreases in PV and PNN as post mortem human studies. Furthermore, MK-801 is seen to have an increasing trend effect on PV cells, which should be considered when interpreting findings in future studies that look at these markers.

We conducted experiments to understand the role of Ventral Hippocampus (vHPC) projections to the Nucleus Accumbens (NAc) in exploratory locomotion, and to determine if the reduced vHPC parvalbumin neuron activity can result in behaviors associated with schizophrenia. Through the use of optogenetics, we activated vHPC neurons and vHPC terminals in the NAc. Both manipulations significantly increased locomotor activity in the open field. Selective inhibition of vHPC terminals in the NAc during a test for novel environment exploration significantly reduced preference for novel environments over familiar environments. DREADD-mediated inhibition of activation of vHPC parvalbumin neuron activity did not significantly alter amphetamine-induced locomotion. Overall, these experiments provide support for the role of the vHPC-NAc pathway in mediating exploratory behavior in novel environments, but it remains inconclusive whether dysregulated vHPC activity due to the loss of parvalbumin neurons leads to behaviors associated with schizophrenia.

We conducted experiments to understand the role of Ventral Hippocampus (vHPC) projections to the Nucleus Accumbens (NAc) in exploratory locomotion, and to determine if the reduced vHPC parvalbumin neuron activity can result in behaviors associated with schizophrenia. Through the use of optogenetics, we activated vHPC neurons and vHPC terminals in the NAc. Both manipulations significantly increased locomotor activity in the open field. Selective inhibition of vHPC terminals in the NAc during a test for novel environment exploration significantly reduced preference for novel environments over familiar environments. DREADD-mediated inhibition of activation of vHPC parvalbumin neuron activity did not significantly alter amphetamine-induced locomotion. Overall, these experiments provide support for the role of the vHPC-NAc pathway in mediating exploratory behavior in novel environments, but it remains inconclusive whether dysregulated vHPC activity due to the loss of parvalbumin neurons leads to behaviors associated with schizophrenia.

Earlier human and animal studies have indicated abnormal striatal GABAergic interneurons relating to autism spectrum disorder’s (ASD) core features such as stereotypic repetitive behaviors, impaired language and motor skills, and social interactions. Purkinje cells (PCs) in the cerebellum are of great interest in ASD; earlier research has reported a loss of PCs, irregularities within deep cerebellar nuclei, a lower level of GAD67 (glutamic acid decarboxylase) mRNA expressed on PCs, and reduced parvalbumin (PV)-positive interneurons in cortex and hippocampus. In this study, in-situ hybridization was used to quantify the levels of PV mRNA in PCs in post-mortem human autism and control cerebellum sections. Two-tailed t-test analysis of the data showed a significant decrease (p<0.05) in PV mRNA level on PCs in autism compared to control sections. In addition, when comparing two groups (seizure and no seizure) in autism sections, no statistical significance was observed. Post-mortem interval (PMI) and age was compared between the PV mRNA levels in autism and control. Only weak negative correlation was found among age and PV mRNA levels in both groups. This report of decreased PV mRNA level in autism cases further supported previous research findings related to PCs and also confirmed interference with the inhibitory function of PCs to deep cerebellar nuclei and the cortex resulting in behavioral and motor impairments in ASD.

Individuals with Autism Spectrum Disorder (ASD) experience a variety of symptoms that vary dramatically across individuals and can range from severe impairments to minor issues with social interactions and communication. The underlying cause of ASD is still unknown, and the level of influence that genetic and environmental factors have on the severity and occurrence of ASD is still a topic of great debate. Since the theories concerning cause or causes of ASD are multifactorial, the treatment options available are extremely limited and are based on behavioral testing. Alternatively, genetic testing might be considered in a diagnosis protocol. This study is designed to investigate ASD by assessing the variability of three genes associated with neuronal inhibition. Based on previous studies this experiment hypothesized that GAD1, GAD2, and PAVLB expression is decreased in Broca’s area in individuals with ASD when compared to controls, with the premise that this alteration could contribute to the symptoms involving language and communication. In situ hybridization was used to quantify the expression of the GAD1, GAD2, and PVALB genes in Broca’s area in postmortem human tissue. The variability of these three genes was quantified by measuring the amount of radioactively tagged mRNA in fifty cell bodies in each brain sample. This study used twenty-two brains of individuals with ASD and twenty-one control brains, including age matched males and females. The variables of age and sex are analyzed and discussed as well as the emulsion and film analyses. A decrease in parvalbumin expression was found between the ASD and control groups in Broca’s area. These finding were discussed in the context of symptoms and neuropathological features of ASD.