The spatial pattern of discrete beta-amyloid deposits in Alzheimer's disease reflects synaptic disconnection

The spatial pattern of discrete beta-amyloid (A beta) deposits was studied in the superficial laminae of cortical fields of different types and in the hippocampus in 6 cases of Alzheimer's disease (AD). In 41/42 tissues examined, discrete A beta deposits were aggregated into clusters and in 34/41 tissues (25/34 of the cortical tissues), there was evidence for a regular periodicity of the A beta deposit clusters parallel to the tissue boundary. The dimensions of the clusters varied from 400 to > 12,800 microns in different tissues. Although the A beta deposit clusters were larger than predicted, the regular periodicity suggests that they develop in relation to groups of cells associated with specific projections. This would be consistent with the hypothesis that the distribution of discrete A beta deposits in AD could reflect progressive synaptic disconnection along interconnected neuronal pathways. This implies that amyloid deposition could be a response to, rather than a cause of, synaptic disconnection in AD.

Simultaneous estimation of global background synaptic inhibition and excitation from membrane potential fluctuations in layer III neurons of the rat entorhinal cortex in vitro

It is becoming clear that the detection and integration of synaptic input and its conversion into an output signal in cortical neurons are strongly influenced by background synaptic activity or "noise." The majority of this noise results from the spontaneous release of synaptic transmitters, interacting with ligand-gated ion channels in the postsynaptic neuron [Berretta N, Jones RSG (1996); A comparison of spontaneous synaptic EPSCs in layer V and layer II neurones in the rat entorhinal cortex in vitro. J Neurophysiol 76:1089-1110; Jones RSG, Woodhall GL (2005) Background synaptic activity in rat entorhinal cortical neurons: differential control of transmitter release by presynaptic receptors. J Physiol 562:107-120; LoTurco JJ, Mody I, Kriegstein AR (1990) Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex. Neurosci Lett 114:265-271; Otis TS, Staley KJ, Mody I (1991) Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release. Brain Res 545:142-150; Ropert N, Miles R, Korn H (1990) Characteristics of miniature inhibitory postsynaptic currents in CA1 pyramidal neurones of rat hippocampus. J Physiol 428:707-722; Salin PA, Prince DA (1996) Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75:1573-1588; Staley KJ (1999) Quantal GABA release: noise or not? Nat Neurosci 2:494-495; Woodhall GL, Bailey SJ, Thompson SE, Evans DIP, Stacey AE, Jones RSG (2005) Fundamental differences in spontaneous synaptic inhibition between deep and superficial layers of the rat entorhinal cortex. Hippocampus 15:232-245]. The function of synaptic noise has been the subject of debate for some years, but there is increasing evidence that it modifies or controls neuronal excitability and, thus, the integrative properties of cortical neurons. In the present study we have investigated a novel approach [Rudolph M, Piwkowska Z, Badoual M, Bal T, Destexhe A (2004) A method to estimate synaptic conductances from membrane potential fluctuations. J Neurophysiol 91:2884-2896] to simultaneously quantify synaptic inhibitory and excitatory synaptic noise, together with postsynaptic excitability, in rat entorhinal cortical neurons in vitro. The results suggest that this is a viable and useful approach to the study of the function of synaptic noise in cortical networks. © 2007 IBRO.

An investigation into the synaptic conditions and celluar mechanisms underlying cerebellar synaptic plasticity

The direction of synaptic plasticity at the connection between parallel fibres (PFs) and Purkinje cells can be modified by PF stimulation alone. Strong activation (Hartell, 1996) or high frequency stimulation (Schreurs and Alkon, 1993) of PFs induced a long-term depression (LTD) of PF-mediated excitatory postsynaptic currents. Brief raised frequency molecular layer stimulation produced a cAMP-dependent long-temi potentiation (LTP) of field potential (FP) responses (Salin et al., 1998). Thin slices of cerebellar vermis were prepared from 14-21 day old male Wistar rats decapitated under Halothane anaesthesia. FP's were recorded from the Purkinje cell layer in response to alternate 0.2Hz activation of stimulating electrodes placed in the molecular layer. In the presence of picrotoxin, FPs displayed two tetrodotoxin-sensitive, negative-going components termed N1 and N2. EPs were graded responses with paired pulse facilitation and were selectively blocked by 101AM 6-cyano-7-nitroquinoxaline-2,3-dicne (CNQX) an antagonist at iy,-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type ionotropic glutamate receptors (AMPAR) suggesting that they were primarily PE-mediated. The effects of raised stimulus intensity (RS) and/or increased frequency (IF) activation of the molecular layer on FP responses were examined. In sagittai and transverse slices combined RS and IF molecular layer activation induced a LTD of the N2 component of FP responses. RSIF stimulation produced fewer incidences of LTD in sagittal slices when an inhibitor of nitric oxide synthase (NOS), guanylate cyclase (GC), protein kinase G (PKG) or the GABAB receptor antagonist CGP62349 was included into the perfusion medium. Application of a nitric oxide (NO) donor, a cyclic guanosine monophosphate (cGMP) analogue or a phosphodiesterase (PDE) type V inhibitor to prevent cGMP breakdown paired with IF stimulation produced an acute depression, Raised frequency (RF) molecular layer stimulation produced a slowly emerging LTD of N2 in sagittal slices that was largely blocked in the presence of NOS, cGMP or PKG inhibitors. In transverse slices RE stimulation produced a LTP of the N2 component that was prevented by an inhibitor of protein kinase A or NOS. Inhibition of cGMP-signalling frequently revealed an underlying potentiation suggesting that cGMP activity might mask the effects of cAMP. In sagittal slices RE stimulation resulted in a potentiation of FPs when the cAMP-specific PDE type IV inhibitor rolipram was incorporated into the perfusion medium. In summary, raised levels of PE stimulation can alter the synaptic efficacy at PF-Purkinje cell synapses. The results provide support for a role of NO/cGMP/PKG signalling in the induction of LTD in the cerebellar cortex and suggest that activation of GABAa receptors might also be important. The level of cyclic nucleotide-specific PDE activities may be crucial in determining the level of cGMP and CAMP activity and hence the direction of synaptic plasticity.

On the mechanisms of cerebellar synaptic plasticity

Changes in the strength of signalling between neurones are thought to provide a cellular substrate for learning and memory. In the cerebellar cortex, raising the frequency and the strength of parallel fibre (PF) stimulation leads to a long-term depression (LTD) of the strength of signalling at the synapse between PFs and Purkinje cells (PCs), which spreads to distant synapses to the same cell via a nitric oxide (NO) dependent mechanism. At the same synapse, but under conditions of reduced post-synaptic calcium activity, raised frequency stimulation (RFS) of PFs triggers a long-term potentiation of synaptic transmission. The aims of the work described in this thesis were to investigate the conditions necessary for LTD and LTP at this synapse following RFS and to identify the origins and second messenger cascades involved in the induction and spread of LTP and LTD. In thin, parasagittal cerebellar slices whole cell patch clamp recordings were made from PCs and the effects of RFS of one of two, independent PF inputs to the same PC were examined under a range of experimental conditions. Under conditions designed to reduce post-synaptic calcium activity, RFS to a single PF input led to LTP and a decreases in paired pulse facilitation (PPF) in both pathways. This heterosynaptic potentiation was prevented by inhibition of protein kinase A (PKA) or by inhibition of NO synthase with either 7-nitroindazole (7-NI) or NG Nitro-L-argenine methyl ester. Inhibition of guanylate cyclase (GC) or protein kinase G (PKG) had no effect. A similar potentiation was observed upon application of the adenylyl cyclase (AC) activator forskolin or the NO donor spermine NONOate. Both of these treatments also resulted in an increase in the frequency of mEPSCs, which provides further evidence for a presynaptic origin of LTP. Forskolin induced potentiation and the increase in mEPSC frequency were blocked by 7-NI. The styryl dye FM1-43, a fluorescent reporter of endo- and exocytosis, was also used to further examine the possible pre-synaptic origins of LTP. RFS or forskolin application enhanced FM1-43 de-staining and NOS inhibitors blocked this effect. Application of NONOate also enhanced FM1-43 de-staining. When post-synaptic calcium activity was less strictly buffered, RFS to a single PF input led to a transient potentiation that was succeeded by LTD in both pathways. This LTD, which resembled previously described forms, was prevented by inhibition of the NO/cGMP/PKG cascade. Modification of the AC/cAMP/PKA cascade had no effect. In summary, the direction of synaptic plasticity at the PF-PC synapse in response to RFS depends largely on the level of post-synaptic calcium activity. LTP and LTD were non-input specific and both forms of plasticity were dependent on NOS activity. Induction of LTP was mediated by a presynaptic mechanism and depended on NO and cAMP production. LTD on the other hand was a post-synaptic process and required activity of the NO/cGMP/PKG signalling cascade.

Astrocyte-neuron signalling by synaptic stimulation in the ventrobasal thalamus

In the Ventrobasal (VB) thalamus, astrocytes are known to elicit NMDA-receptor mediated slow inward currents (SICs) spontaneously in neurons. Fluorescence imaging of astrocytes and patch clamp recordings from the thalamocortical (TC) neurons in the VB of 6-23 day old Wistar rats were performed. TC neurons exhibit spontaneous SICs at low frequencies (~0.0015Hz) that were inhibited by NMDA-receptor antagonists D-AP5 (50µM), and were insensitive to TTX (1µM) suggesting a non-neuronal origin. The effect of corticothalamic (CT) and sensory (Sen) afferent stimulation on astrocyte signalling was assessed by varying stimulus parameters. Moderate synaptic stimulation elicited astrocytic Ca2+ increases, but did not affect the incidence of spontaneous SICs. Prolonged synaptic stimulation induced a 265% increase in SIC frequency. This increase lasted over one hour after the cessation of synaptic stimulation, so revealing a Long Term Enhancement (LTE) of astrocyte-neuron signalling. LTE induction required group I mGluR activation. LTE SICs targeted NMDA-receptors located at extrasynaptic sites. LTE showed a developmental profile: from weeks 1-3, the SIC frequency was increased by an average 50%, 240% and 750% respectively. Prolonged exposure to glutamate (200µM) increased spontaneous SIC frequency by 1800%. This “chemical” form of LTE was prevented by the broad-spectrum excitatory amino acid transporter (EAAT) inhibitor TBOA (300µM) suggesting that glutamate uptake was a critical factor. My results therefore show complex glutamatergic signalling interactions between astrocytes and neurons. Furthermore, two previously unrecognised mechanisms of enhancing SIC frequency are described. The synaptically induced LTE represents a form of non-synaptic plasticity and a glial “memory” of previous synaptic activity whilst enhancement after prolonged glutamate exposure may represent a pathological glial signalling mechanism.

Modulation of network oscillatory activity and GABAergic synaptic transmission by CB1 cannabinoid receptors in the rat medial entorhinal cortex

Cannabinoids modulate inhibitory GABAergic neurotransmission in many brain regions. Within the temporal lobe, cannabinoid receptors are highly expressed, and are located presynaptically at inhibitory terminals. Here, we have explored the role of type-1 cannabinoid receptors (CB1Rs) at the level of inhibitory synaptic currents and field-recorded network oscillations. We report that arachidonylcyclopropylamide, an agonist at CB1R, inhibits GABAergic synaptic transmission onto both superficial and deep medial entorhinal (mEC) neurones, but this has little effect on network oscillations in beta/gamma frequency bands. By contrast, the CB1R antagonist/inverse agonist LY320135 (500?nM), increased GABAergic synaptic activity and beta/gamma oscillatory activity in superficial mEC, was suppressed, whilst that in deep mEC was enhanced. These data indicate that cannabinoid-mediated effects on inhibitory synaptic activity may be constitutively active in vitro, and that modulation of CB1R activation using inverse agonists unmasks complex effects of CBR function on network activity.