Kerry P.S.J. Murphy

Autonomous activity of CaMKII is only transiently increased following the induction of long-term potentiation in the rat hippocampus

A major role has been postulated for a maintained increase in the autonomous activity of CaMKII in the expression of long‐term potentiation (LTP). However, attempts to inhibit the expression of LTP with CaMKII inhibitors have yielded inconsistent results. Here we compare the changes in CaMKII autonomous activity and phosphorylation at Thr286 of αCaMKII in rat hippocampal slices using chemical or tetanic stimulation to produce either LTP or short‐term potentiation (STP). Tetanus‐induced LTP in area CA1 requires CaMKII activation and Thr286 phosphorylation of αCaMKII, but we did not observe an increase in autonomous activity. Next we induced LTP by 10 min exposure to 25 mm tetraethyl‐ammonium (TEA) or 5 min exposure to 41 mm potassium (K) after pretreatment with calyculin A. Exposure to K alone produced STP. These protocols allowed us to monitor temporal changes in autonomous activity during and after exposure to the potentiating chemical stimulus. In chemically induced LTP, autonomous activity was maximally increased within 30 s whereas this increase was significantly delayed in STP. However, in both LTP and STP the two‐fold increase in autonomous activity measured immediately after stimulation was short‐lived, returning to baseline within 2–5 min after re‐exposure to normal ACSF. In LTP, but not in STP, the phosphorylation of αCaMKII at Thr286 persisted for at least 60 min after stimulation. These results confirm that LTP is associated with a maintained increase in autophosphorylation at Thr286 but indicate that a persistent increase in the autonomous activity οf CaMKII is not required for the expression of LTP.

Bi-directional changes in synaptic plasticity induced at corticostriatal synapses in vitro.

Abstract. Long-term changes in the synaptic efficacy of corticostriatal synapses are believed to be important for regulating the excitatory input to the basal ganglia, and hence for motor learning and certain forms of cognition. Previous reports have suggested that long-term depression (LTD) is the predominant form of plasticity at corticostriatal synapses. However, we report here that tetanic stimulation of the white matter can readily induce long-term potentiation (LTP) at corticostriatal synapses in a sagittal slice preparation. Furthermore, we find that corticostriatal LTP is obtained in the absence of pharmacological manipulation, and is dependent on NMDA receptor activation. In contrast, LTD is rarely observed following tetanic stimulation of the white matter, but in fact requires direct stimulation within the striatum. This striatally induced depression is blocked by both D1 and D2 dopamine receptor antagonists and by NMDA receptor blockade. Pairing of striatal stimulation with tetanic stimulation of the white matter does not prevent the induction, but significantly enhances the magnitude of LTP at corticostriatal synapses. We suggest that the corticostriatal depression reported here most likely involves the recruitment of local striatal circuits and dopaminergic inputs, and thus might explain the predominance of LTD previously reported. Our observation that it is indeed possible to induce LTP at corticostriatal synapses under physiological conditions in vitro has implications for the normal function and control of the basal ganglia in motor learning and cognition.

Chemically induced long‐term potentiation increases the number of perforated and complex postsynaptic densities but does not alter dendritic spine volume in CA1 of adult mouse hippocampal slices

Examination of the morphological correlates of long‐term potentiation (LTP) in the hippocampus requires the analysis of both the presynaptic and postsynaptic elements. However, ultrastructural measurements of synapses and dendritic spines following LTP induced via tetanic stimulation presents the difficulty that not all synapses examined are necessarily activated. To overcome this limitation, and to ensure that a very large proportion of the synapses and spines examined have been potentiated, we induced LTP in acute hippocampal slices of adult mice by addition of tetraethylammonium (TEA) to a modified CSF containing an elevated concentration of Ca2+ and no Mg+. Quantitative electron microscope morphometric analyses and three‐dimensional (3‐D) reconstructions of both dendritic spines and postsynaptic densities (PSDs) in CA1 stratum radiatum were made on serial ultrathin sections. One hour after chemical LTP induction the proportion of macular (unperforated) synapses decreased (50%) whilst the number of synapses with simple perforated and complex PSDs (nonmacular) increased significantly (17%), without significant changes in volume and surface area of the PSD. In addition, the surface area of mushroom spines increased significantly (13%) whilst there were no volume differences in either mushroom or thin spines, or in surface area of thin spines. CA1 stratum radiatum contained multiple‐synapse en passant axons as well as multiple‐synapse spines, which were unaffected by chemical LTP. Our results suggest that chemical LTP induces active dendritic spine remodelling and correlates with a change in the weight and strength of synaptic transmission as shown by the increase in the proportion of nonmacular synapses.

Abnormal cortical synaptic plasticity in a mouse model of Huntington's disease.

Huntingtons disease is a fatal neurodegenerative disorder characterised by a progressive motor, psychiatric and cognitive decline and associated with a marked loss of neurons in the cortex and striatum of affected individuals. The disease is inherited in an autosomal dominant fashion and is caused by a trinucleotide (CAG) repeat expansion in the gene encoding the protein huntingtin. Predictive genetic testing has revealed early cognitive deficits in asymptomatic gene carriers such as altered working memory, executive function and recognition memory. The perirhinal cortex is believed to process aspects of recognition memory. Evidence from primate studies suggests that decrements in neuronal firing within this cortical region encode recognition memory and that the underlying mechanism is an activity-dependent long-term depression (LTD) of excitatory neurotransmission, the converse of long-term potentiation (LTP). We have used the R6/1 mouse model of HD to assess synaptic plasticity in the perirhinal cortex. This mouse model provides an ideal tool for investigating early and progressive changes in synaptic function in HD. We report here that LTD at perirhinal synapses is markedly reduced in R6/1 mice. We also provide evidence to suggest that a reduction in dopamine D2 receptor signalling may be implicated.

Bi-directional plasticity and age-dependent long-term depression at mouse CA3-CA1 hippocampal synapses

Low-frequency stimulation (LFS) is used to induce long-term depression (LTD) and depotentiation at rodent CA3-CA1 hippocampal synapses. The relationship between the efficacy of LFS induction and postnatal age remains to be clearly defined in rat and had not been studied in mouse. The data presented here show that in acute mouse hippocampal slices LFS-induced LTD and depotentiation at CA3-CA1 synapses are: synapse specific; NMDA receptor-dependent; and metabotropic glutamate (mGlu) receptor type I/II independent. Furthermore LFS-induced LTD is highly age-dependent whilst long-term potentiation (LTP) and depotentiation are not. In slices from very young mice (P6-9) LFS induced a robust and stable LTD (-31.1 +/- 5.9%, n = 8, P < 0.01) of CA1 field excitatory post-synaptic potentials (fEPSPs), measured 55-60 min after conditioning. LFS also induced LTD in slices from mice aged P10-13 and P14-17 (-16.0 +/- 3.0%, n = 35, P < 0.001 and -17.9 +/- 5.5%, n = 12, P < 0.01, respectively). However, LTD was not expressed in slices from animals aged P18-21 ( -7.0 +/- 4.1%, n = 16, P > 0.05) or older.

The role of dopamine in huntington's disease

Alterations in dopamine (DA) neurotransmission in Parkinsons disease are well known and widely studied. Much less is known about DA changes that accompany and underlie some of the symptoms of Huntingtons disease (HD), a dominant inherited neurodegenerative disorder characterized by chorea, cognitive deficits, and psychiatric disturbances. The cause is an expansion in CAG (glutamine) repeats in the HTT gene. The principal histopathology of HD is the loss of medium-sized spiny neurons (MSNs) and, to a lesser degree, neuronal loss in cerebral cortex, thalamus, hippocampus, and hypothalamus. Neurochemical, electrophysiological, and behavioral studies in HD patients and genetic mouse models suggest biphasic changes in DA neurotransmission. In the early stages, DA neurotransmission is increased leading to hyperkinetic movements that can be alleviated by depleting DA stores. In contrast, in the late stages, DA deficits produce hypokinesia that can be treated by increasing DA function. Alterations in DA neurotransmission affect glutamate receptor modulation and could contribute to excitotoxicity. The mechanisms of DA dysfunction, in particular the increased DA tone in the early stages of the disease, are presently unknown but may include initial upregulation of DA neuron activity caused by the genetic mutation, reduced inhibition resulting from striatal MSN loss, increased excitation from cortical inputs, and DA autoreceptor dysfunction. Targeting both DA and glutamate receptor dysfunction could be the best strategy to treat HD symptoms.

Activation of cyclic AMP-dependent protein kinase is required for long-term enhancement at corticostriatal synapses in rats

The induction of long-term potentiation (LTP) at corticostriatal synapses is dependent on the activation of postsynaptic NMDA receptors, but the mechanisms involved in the maintenance of LTP are not known. We report here that forskolin, an activator of adenylyl cyclase, induces a lasting enhancement of the corticostriatal synaptic response. This enhancement is associated with a lasting decrease in paired-pulse ratio, and is blocked by inhibitors of adenylyl cyclase and cyclic AMP-dependent protein kinase (PKA), but not by a PKA inhibitor injected into the postsynaptic cell. Tetanically-induced LTP is also associated with a decrease in paired-pulse ratio and partially occludes the subsequent action of forskolin. Our results suggest that activation of presynaptic PKA can enhance neurotransmission at corticostriatal synapses; a mechanism required for the expression of LTP at these synapses.

Impaired long-term potentiation in the prefrontal cortex of Huntington's disease mouse models: rescue by D1 dopamine receptor activation.

Background: The introduction of gene testing for Huntington’s disease (HD) has enabled the neuropsychiatric and cognitive profiling of human gene carriers prior to the onset of overt motor and cognitive symptoms. Such studies reveal an early decline in working memory and executive function, altered EEG and a loss of striatal dopamine receptors. Working memory is processed in the prefrontal cortex and modulated by extrinsic dopaminergic inputs. Objective: We sought to study excitatory synaptic function and plasticity in the medial prefrontal cortex of mouse models of HD. Methods: We have used 2 mouse models of HD, carrying 89 and 116 CAG repeats (corresponding to a preclinical and symptomatic state, respectively) and performed electrophysiological field recording in coronal slices of the medial prefrontal cortex. Results: We report that short-term synaptic plasticity and long-term potentiation (LTP) are impaired and that the severity of impairment is correlated with the size of the CAG repeat. Remarkably, the deficits in LTP and short-term plasticity are reversed in the presence of a D1 dopamine receptor agonist (SKF38393). Conclusion: In a previous study, we demonstrated that a deficit in long-term depression (LTD) in the perirhinal cortex could also be reversed by a dopamine agonist. These and our current data indicate that inadequate dopaminergic modulation of cortical synaptic function is an early event in HD and may provide a route for the alleviation of cognitive dysfunction.

Progressive CAG expansion in the brain of a novel R6/1-89Q mouse model of Huntington's disease with delayed phenotypic onset.

Transgenic models representing Huntingtons disease (HD) have proved useful for understanding the cascade of molecular events leading to the disease. We report an initial characterisation of a novel transgenic mouse model derived from a spontaneous truncation event within the R6/1 transgene. The transgene is widely expressed, carries 89 CAG repeats and the animals exhibit a significantly milder neurological phenotype with delayed onset compared to R6/1. Moreover, we report evidence of progressive somatic CAG expansions in the brain starting at an early age before an overt phenotype has developed. This novel line shares a common genetic ancestry with R6/1, differing only in CAG repeat number, and therefore, provides an additional tool with which to examine early molecular and neurophysiological changes in HD.

Dysfunctional Dopaminergic Neurones in Mouse Models of Huntington's Disease: A Role for SK3 Channels

Background: Huntingtons disease (HD) is a late-onset fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the gene coding for the protein huntingtin and is characterised by progressive motor, psychiatric and cognitive decline. We previously demonstrated that normal synaptic function in HD could be restored by application of dopamine receptor agonists, suggesting that changes in the release or bioavailability of dopamine may be a contributing factor to the disease process. Objective: In the present study, we examined the properties of midbrain dopaminergic neurones and dopamine release in presymptomatic and symptomatic transgenic HD mice. Methods and Results: Using intracellular sharp recordings and immunohistochemistry, we found that neuronal excitability was increased due to a loss of slow afterhyperpolarisation and that these changes were related to an apparent functional loss and abnormal distribution of SK3 channels (KCa2.3 encoded by the KCNN3 gene), a class of small-conductance calcium-activated potassium channels. Electrochemical detection of dopamine showed that this observation was associated with an enhanced dopamine release in presymptomatic transgenic mice and a drastic reduction in symptomatic animals. These changes occurred in the context of a progressive expansion in the CAG repeat number and nuclear localisation of mutant protein within the substantia nigra pars compacta. Conclusions: Dopaminergic neuronal dysfunction is a key early event in HD disease progression. The initial increase in dopamine release appears to be related to a loss of SK3 channel function, a protein containing a polyglutamine tract. Implications for polyglutamine-mediated sequestration of SK3 channels, dopamine-associated DNA damage and CAG expansion are discussed in the context of HD.