Long-term depression (LTD) induced by low frequency stimulation (LFS; 900x1Hz) at medial perforant path (MPP) synapses in the rat dentate gyrus (DG) has been described as both developmentally regulated and N-methyl D-aspartate receptor (NMDAr) independent, yet sufficient evidence suggest that the processes is not entirely independent of NMDAr activity. In the present study, in vitro DG-LTD LFS was induced in hippocampal slices prepared from rats at postnatal day (PND) 14, 21 and 28 to investigate how the sensitivity of DG-LTD~LFS to the NMDAr antagonist amino-5-phosphonovaleric acid (AP5; 50µM) changes throughout the juvenile developmental period (jDP; PNDs 12-29) that occurs immediately after the period of peak neurogenesis. We further examined the acute effects of the partial NMDAr antagonist ethanol (EtOH) on DG-LTD LFS and NMDAr excitatory post synaptic currents (NMDAr-EPSCs) in dentate granule cells (DGCs) using 50 and 100mM concentrations (50mM ~0.2%BAC) of EtOH. The magnitude of LTD induced at all three time points was not statistically different between age groups, but the probability of successfully inducing LTD did decrease with age. We found that AP5 was insufficient to inhibit DG-LTD LFS at PND14, but significantly inhibited DG-LTD LFS at PND21 and PND28. We also found that 50mM EtOH, but not 100mM EtOH, significantly attenuated the mag-nitude of DG-LTD LFS induced at each time point. Acute effects of 50mM EtOH had relatively little effect on NMDAr-EPSCs at PND14, and showed a slight potentiation of the response at PND21. 50mM EtOH at PND28, and 100mM EtOH at all three developmental time points showed inhibition of the NMDAr-EPSC. These findings provide insight on how developmental changes to the DG network and dentate gran-ule cells (DGCs) influences mechanisms and processes involved in the induction and expression of synaptic plasticity in the DG. / Graduate

Primary visual cortex is a classic model to study experience-dependent brain plasticity. In early life, if one eye is deprived of normal vision, there can be a dramatic change in the ocular dominance of the striate cortex such that the large majority of neurons lose responsiveness to the deprived eye and, consequently, the ocular dominance distribution shifts in favor of the open eye. Interestingly, the visual experience dependent plasticity following monocular deprivation (MD) occurs during a transient developmental period, which is called the critical period. MD hardly induces ocular dominance plasticity beyond critical period. The mechanisms underlying ocular dominance plasticity during the critical period are not fully understood. It has been proposed that long-term depression (LTD) may underlie the loss of cortical neuronal responsiveness to the deprived eye. However, discordant results have been reported in terms of the role of LTD and LTP in visual plasticity due to the lack of specific blockers. Here we report the prevention of the normally-occurring ocular dominance (OD) shift to the open eye following MD by using a specific long-term depression (LTD) blocking peptide derived from the GluR2 subunit of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR). We were able to prevent the shift of OD to the open eye with systemic or local administration of the GluR2 peptide. Both electrophysiological and anatomical approaches were taken to demonstrate the peptide effect. Moreover, enhancing LTD with D-serine, a NMDA receptor co-agonist, brought back the ocular dominance plasticity in adult mice subject to four-day MD and, therefore, reopened the critical period. Our data indicate that LTD plays an essential role in visual plasticity during the critical period and the developmental regulation of LTD may account for the closure of critical period in adult. In an additional study, we have found anisomycin, a protein synthesis inhibitor, produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in mouse primary visual cortex and that this anisomycin-mediated fEPSP depression occludes NMDA receptor dependent LTD. In contrast, another two protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission and or on LTD. We propose that anisomycin-LTD might be mediated by p38 MAP kinase since anisomycin is also a potent activator of the P38/JNK MAPK pathway. In agreement with notion, the decline of the fEPSP caused by anisomycin can be rescued by the application of the P38 inhibitor SB203580, but not by the JNK inhibitor SP600125. The occlusion of LFS-LTD by anisomycin-induced fEPSP decline suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of the membrane permeant peptide discussed above, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-LTD, prior to anisomycin treatment significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, this study indicates that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating P38 MAPK-mediated endocytosis of AMPA receptors in neonatal mouse visual cortex.

Primary visual cortex is a classic model to study experience-dependent brain plasticity. In early life, if one eye is deprived of normal vision, there can be a dramatic change in the ocular dominance of the striate cortex such that the large majority of neurons lose responsiveness to the deprived eye and, consequently, the ocular dominance distribution shifts in favor of the open eye. Interestingly, the visual experience dependent plasticity following monocular deprivation (MD) occurs during a transient developmental period, which is called the critical period. MD hardly induces ocular dominance plasticity beyond critical period. The mechanisms underlying ocular dominance plasticity during the critical period are not fully understood. It has been proposed that long-term depression (LTD) may underlie the loss of cortical neuronal responsiveness to the deprived eye. However, discordant results have been reported in terms of the role of LTD and LTP in visual plasticity due to the lack of specific blockers. Here we report the prevention of the normally-occurring ocular dominance (OD) shift to the open eye following MD by using a specific long-term depression (LTD) blocking peptide derived from the GluR2 subunit of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR). We were able to prevent the shift of OD to the open eye with systemic or local administration of the GluR2 peptide. Both electrophysiological and anatomical approaches were taken to demonstrate the peptide effect. Moreover, enhancing LTD with D-serine, a NMDA receptor co-agonist, brought back the ocular dominance plasticity in adult mice subject to four-day MD and, therefore, reopened the critical period. Our data indicate that LTD plays an essential role in visual plasticity during the critical period and the developmental regulation of LTD may account for the closure of critical period in adult. In an additional study, we have found anisomycin, a protein synthesis inhibitor, produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in mouse primary visual cortex and that this anisomycin-mediated fEPSP depression occludes NMDA receptor dependent LTD. In contrast, another two protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission and or on LTD. We propose that anisomycin-LTD might be mediated by p38 MAP kinase since anisomycin is also a potent activator of the P38/JNK MAPK pathway. In agreement with notion, the decline of the fEPSP caused by anisomycin can be rescued by the application of the P38 inhibitor SB203580, but not by the JNK inhibitor SP600125. The occlusion of LFS-LTD by anisomycin-induced fEPSP decline suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of the membrane permeant peptide discussed above, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-LTD, prior to anisomycin treatment significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, this study indicates that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating P38 MAPK-mediated endocytosis of AMPA receptors in neonatal mouse visual cortex.

Primary visual cortex is a classic model to study experience-dependent brain plasticity. In early life, if one eye is deprived of normal vision, there can be a dramatic change in the ocular dominance of the striate cortex such that the large majority of neurons lose responsiveness to the deprived eye and, consequently, the ocular dominance distribution shifts in favor of the open eye. Interestingly, the visual experience dependent plasticity following monocular deprivation (MD) occurs during a transient developmental period, which is called the critical period. MD hardly induces ocular dominance plasticity beyond critical period. The mechanisms underlying ocular dominance plasticity during the critical period are not fully understood. It has been proposed that long-term depression (LTD) may underlie the loss of cortical neuronal responsiveness to the deprived eye. However, discordant results have been reported in terms of the role of LTD and LTP in visual plasticity due to the lack of specific blockers. Here we report the prevention of the normally-occurring ocular dominance (OD) shift to the open eye following MD by using a specific long-term depression (LTD) blocking peptide derived from the GluR2 subunit of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor (AMPAR). We were able to prevent the shift of OD to the open eye with systemic or local administration of the GluR2 peptide. Both electrophysiological and anatomical approaches were taken to demonstrate the peptide effect. Moreover, enhancing LTD with D-serine, a NMDA receptor co-agonist, brought back the ocular dominance plasticity in adult mice subject to four-day MD and, therefore, reopened the critical period. Our data indicate that LTD plays an essential role in visual plasticity during the critical period and the developmental regulation of LTD may account for the closure of critical period in adult. In an additional study, we have found anisomycin, a protein synthesis inhibitor, produces a time-dependent decline in the magnitude of the field EPSP (fEPSP) in mouse primary visual cortex and that this anisomycin-mediated fEPSP depression occludes NMDA receptor dependent LTD. In contrast, another two protein synthesis inhibitors, emetine and cycloheximide, have no effect either on baseline synaptic transmission and or on LTD. We propose that anisomycin-LTD might be mediated by p38 MAP kinase since anisomycin is also a potent activator of the P38/JNK MAPK pathway. In agreement with notion, the decline of the fEPSP caused by anisomycin can be rescued by the application of the P38 inhibitor SB203580, but not by the JNK inhibitor SP600125. The occlusion of LFS-LTD by anisomycin-induced fEPSP decline suggests that common mechanisms may be shared between the two forms of synaptic depression. Consistent with this view, bath application of the membrane permeant peptide discussed above, which specifically blocks regulated AMPA receptor endocytosis, thereby preventing the expression of LFS-LTD, prior to anisomycin treatment significantly reduced the anisomycin-induced decline of the fEPSP. In conclusion, this study indicates that anisomycin produces long-lasting depression of AMPA receptor-mediated synaptic transmission by activating P38 MAPK-mediated endocytosis of AMPA receptors in neonatal mouse visual cortex. / Medicine, Faculty of / Graduate

Synaptic plasticity is the process whereby connections between neurons can be altered in an experience dependent manner. For example, drugs of abuse alter plasticity in the ventral tegmental area (VTA) of the midbrain. A large amount of research has been applied to uncovering the mechanism whereby synapses on the reward signaling dopamine cells is altered, however, less is known regarding the VTA inhibitory GABA neurons. Our objective was to examine the ability of GABA neurons to exhibit plasticity, and determine how drugs of abuse could influence it. Here we report a novel type of plasticity of excitatory neurotransmission onto VTA GABA cells. This plasticity is dependent on the metabotropic glutamate receptor 5, to signal for diacylglycerolipase alpha to make the endocannabinoid 2-arachadonoyl glycerol to signal via cannabinoid receptor 1 (CB1). Marijuana and cocaine are drugs of abuse that have been shown to alter the endocannabinoid system. Tetrahydrocannabinol is the active ingredient in marijuana, and is a known agonist of CB1, and cocaine is able to attenuate endocannabinoid signals. We tested the effects of these drugs on VTA GABA plasticity and found that it can be blocked by chronic injections of tetrahydrocannabinol, as well as acute and chronic injections of cocaine. If VTA GABA neurons are depressing excitatory inputs, that could lead to less inhibition onto VTA dopamine cells, and therefore, more reward signaling in the brain. This new type of plasticity could be an additional mechanism whereby cocaine and marijuana exert their rewarding and addictive effects. Another brain structure known to exhibit use-dependent plasticity is the hippocampus, which is involved in learning and memory. The stratum oriens is a layer of inhibitory interneurons in the hippocampus that is involved in feedback inhibition onto the principle excitatory cells in the stratum pyramidale. Our goal was to determine whether oriens interneurons were capable of producing an endocannabinoid signal, and if so, whether they could influence plasticity. We identified 2 major subtypes of oriens interneurons, oriens lacunosum-moleculare cells, and parvalbumin-positive basket cells, which are capable of receiving and producing an endocannabinoid signal. Furthermore, we demonstrated that one such endocannabinoid, anandamide, is responsible for signaling for synaptic plasticity. This plasticity is also dependent on CB1, and is unique in that there are few examples of CB1 signaling for potentiation rather than depression. Collectively, these experiments demonstrate two mechanisms of endocannabinoid mediated synaptic plasticity, which could influence reward signaling, addiction and memory.

Learning and memory in the brain are thought to be dependent on synaptic plasticity. In response to sensory input, synapses can be strengthened or weakened, known as long-term potentiation or long-term depression (LTD), respectively. Transient receptor potential vanilloid 1 (TRPV1) has been shown to mediate a novel form of presynaptic LTD in hippocampal interneurons. TRPV1 is currently being heavily studied in the PNS and being targeted by pharmaceuticals for its anti-nociceptive and anti-inflammatory properties. However, much less is known regarding TRPV1 function in the CNS, including the signal mechanism mediating hippocampal LTD despite its obvious importance. Here we performed whole-cell voltage clamp electrophysiology experiments from CA1 hippocampal interneurons to identify this signaling mechanism. Because calcineurin (CaN) is reported to be linked to multiple forms of synaptic plasticity, we hypothesized that TRPV1 activates presynaptic CaN, which is required for this presynaptic LTD. In order to distinguish between presynaptic and postsynaptic CaN activity we added the specific CaN inhibitors cyclosporin A (CsA) or FK-506 to the bath to block CaN activity ubiquitously in the slice, both presynaptically and postsynaptically, and to the internal solution to block CaN only in the postsynaptic neuron. CsA or FK-506 present in the internal solution, blocking only postsynaptic CaN, showed no effect on TRPV1-dependant LTD. Bath application of CsA or FK-506, inhibiting CaN in the presynaptic neuron as well, blocked LTD elicited by both a high frequency stimulation protocol (P < 0.05) and by direct TRPV1 activation with specific agonists resiniferotoxin and capsaicin (P < 0.05). This demonstrates that CsA and FK506 block both high frequency stimulation induced LTD and also TRPV1 specific depression. We are thus able to show that calcineurin is required for this form of presynaptic TRPV1 mediated LTD in the hippocampus. This finding is the first to demonstrate a TRPV1-induced signaling mechanism in CA1 hippocampus.

The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.

Disruptions in memory are a hallmark feature of several psychiatric diseases. These illnesses are often marred by an inability to recognize that a stimulus or event as been previously experienced, a phenomenon known as recognition memory. Previous study has demonstrated that cognitive disruptions reflect aberrant signaling, including disruptions in synaptic plasticity, in key regions of the brain, such as prefrontal cortex (PFC), hippocampus, and perirhinal cortex (PRh). However, in the case of recognition memory, how these disruptions arise and what specific plasticity mechanisms are involved is less clear. An understanding of the etiological factors underlying disruption and the synaptic processes involved in recognition will greatly advance the treatment and prevention of psychiatric disorders. As a result, the present thesis examined recognition memory in rodents in two experiments. In the first experiment, we blocked the endocytosis of AMPA receptors during the encoding, consolidation, or retrieval phase of object recognition memory using local PRh infusions of the cell membrane permeable Tat-GluA23Y interference peptide. Tat-GluA23Y infusion before the encoding and consolidation phases did not alter memory. In contrast, Tat-GluA23Y infusion prior to the retrieval phase significantly disrupted memory. These results indicate a distinct role for AMPA receptor endocytosis during a specific phase (retrieval) of visual recognition memory. In the second experiment, pregnant dams were treated with PolyI:C (4mg/kg, i.v.) on gestational day (GD) 15, and both the male and female offspring of these rats were tested as young adults in three different recognition memory tests: spontaneous novel object recognition, novel object location recognition, and object-in-place recognition. Male, but not female, rats were impaired in an object-in-place memory test that depends on processing between medial temporal lobe and PFC. However, neither male nor female rats were impaired on tests of simpler discriminations dependent on the medial temporal lobe. These findings support clinical studies demonstrating impaired object location binding in clinical populations and further demonstrate the plausibility of prenatal immune activation as an etiological factor in neurodevelopmental disease. Taken together, these results highlight the importance of a specific form of synaptic plasticity during the recognition of familiar stimuli and demonstrate that early life adversity can disrupt recognition memory processes.

Disruptions in memory are a hallmark feature of several psychiatric diseases. These illnesses are often marred by an inability to recognize that a stimulus or event as been previously experienced, a phenomenon known as recognition memory. Previous study has demonstrated that cognitive disruptions reflect aberrant signaling, including disruptions in synaptic plasticity, in key regions of the brain, such as prefrontal cortex (PFC), hippocampus, and perirhinal cortex (PRh). However, in the case of recognition memory, how these disruptions arise and what specific plasticity mechanisms are involved is less clear. An understanding of the etiological factors underlying disruption and the synaptic processes involved in recognition will greatly advance the treatment and prevention of psychiatric disorders. As a result, the present thesis examined recognition memory in rodents in two experiments. In the first experiment, we blocked the endocytosis of AMPA receptors during the encoding, consolidation, or retrieval phase of object recognition memory using local PRh infusions of the cell membrane permeable Tat-GluA23Y interference peptide. Tat-GluA23Y infusion before the encoding and consolidation phases did not alter memory. In contrast, Tat-GluA23Y infusion prior to the retrieval phase significantly disrupted memory. These results indicate a distinct role for AMPA receptor endocytosis during a specific phase (retrieval) of visual recognition memory. In the second experiment, pregnant dams were treated with PolyI:C (4mg/kg, i.v.) on gestational day (GD) 15, and both the male and female offspring of these rats were tested as young adults in three different recognition memory tests: spontaneous novel object recognition, novel object location recognition, and object-in-place recognition. Male, but not female, rats were impaired in an object-in-place memory test that depends on processing between medial temporal lobe and PFC. However, neither male nor female rats were impaired on tests of simpler discriminations dependent on the medial temporal lobe. These findings support clinical studies demonstrating impaired object location binding in clinical populations and further demonstrate the plausibility of prenatal immune activation as an etiological factor in neurodevelopmental disease. Taken together, these results highlight the importance of a specific form of synaptic plasticity during the recognition of familiar stimuli and demonstrate that early life adversity can disrupt recognition memory processes.

The ubiquitin proteasome system (UPS) depends on three enzymes called E1, E2, and E3 to ubiquitinate proteins and several isopeptidases to de-ubiquitinate them. Ubiquitination serves as a post-translational modification that either tags proteins for degradation by the proteasome or serves to modulate their function. This dynamic system plays a role in synaptic plasticity and dysfunction of the UPS is associated a variety of neurodegenerative diseases. In this study, three inhibitors the UPS, ziram, clasto-lactacystin β-lactone (lactacystin) and G5 were employed to illuminate involvement of the UPS in long-term and short term plasticity in area CA1 of rat hippocampal slices. Ziram, lactacystin and G5 inhibits the E1 ubiquitin-activating enzyme, the proteasome and isopeptidases, respectively. It was found that UPS inhibition enhanced long-term plasticity, by specifically increasing the magnitude of long-term depression (LTD) and altered short term plasticity, measured with paired pulse facilitation (PPF), to varying degrees. These findings establish that the UPS may play a regulatory role in LTD and PPF, and the changes in PPF further indicate that the UPS may be acting presynaptically. Overall, the results suggest ubiquitination and proteasome-mediated proteolysis are important in both long-term and short-term plasticity.