Neurons Specifically Activated by Fear Learning in Lateral Amygdala Display Increased Synaptic Strength.

The lateral amygdala (LA) plays a critical role in the formation of fear-conditioned associative memories. Previous studies have used c-fos regulated expression to identify a spatially restricted population of neurons within the LA that is specifically activated by fear learning. These neurons are likely to be a part of a memory engram, but, to date, functional evidence for this has been lacking. We show that neurons within a spatially restricted region of the LA had an increase in both the frequency and amplitude of spontaneous postsynaptic currents (sPSC) when compared to neurons recorded from home cage control mice. We then more specifically addressed if this increased synaptic activity was limited to learning-activated neurons. Using a fos-tau-LacZ (FTL) transgenic mouse line, we developed a fluorescence-based method of identifying and recording from neurons activated by fear learning (FTL+ ) in acute brain slices. An increase in frequency and amplitude of sPSCs was observed in FTL+ neurons when compared to nonactivated FTL- neurons in fear-conditioned mice. No learning-induced changes were observed in the action potential (AP) input-output relationships. These findings support the idea that a discrete LA neuron population forms part of a memory engram through changes in synaptic connectivity.

Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes.

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d(6)-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction.

Behavioural analysis of congenic mouse strains confirms stress-responsive Loci on chromosomes 1 and 12.

The way in which animals respond to stressful environments correlates with anxiety-related behaviour. To begin identifying the genetic factors that influence anxiety, we have studied the stress-responsiveness of inbred mouse strains using a modified form of the open field activity test (OFA), termed the elevated (e) OFA. In particular, two strains show high (DBA/2J) or low (C57BL/6J) stress-responsiveness in the eOFA. Genetic studies of an F(2) intercross between these two strains previously identified two regions, on chromosomes (Chr) 1 and 12, linked to anxiety-related behaviour. To confirm that these regions contain loci for stress-responsiveness, we established separate congenic mouse strains for the linked Chr1 and Chr12 regions. Each congenic strain harbours a DBA/2J-derived interval encompassing the linked region on the C57BL/6J genetic background: the congenic intervals are between, but not including approximately 48.6 Mb and approximately 194.8 Mb on Chr1, and approximately 36.2 Mb and the distal end of Chr12. Cohorts of DBA/2J, C57BL/6J and congenic mice were analysed for a series of stress-responsive phenotypes using the eOFA test. Both congenic strains had significantly different stress-responsive phenotypes compared to the low-stress C57BL/6J parental strain, but the DBA/2J-derived Chr12 interval had a greater genetic effect than the DBA/2J-derived Chr1 interval for changing the behavioral phenotype of the parental C57BL/6J mouse strain. These results confirmed the presence of stress-responsive loci on Chr1 and Chr12. New stress-related phenotypes were also identified, which aided in comparing and differentiating DBA/2J, C57BL/6J and congenic mice.

Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes.

Insulin-dependent diabetes mellitus (IDDM) is associated with serum antibodies that precipitate a 64-kDa pancreatic islet cell protein reported to be glutamic acid decarboxylase (GAD; glutamate decarboxylase, EC 4.1.1.15). Previously, antibodies to GAD were found in the rare neurological disorder stiff man syndrome. To demonstrate directly antibodies to GAD, enzymatically active GAD was first purified from fresh human cerebellum. Brain GAD activity was precipitated by noninhibitory antibodies in the sera of 16/26 (62%) subjects defined as having preclinical IDDM (islet cell antibody-positive first-degree relatives of a person with IDDM), 3/13 (23%) with recent-onset IDDM, and 3/3 with the stiff man syndrome. In addition, sera of 5/26 (19%) preclinical and 2/13 (15%) recent-onset IDDM subjects contained antibodies that precipitated GAD but inhibited its activity. Thus, overall, 21/26 (81%) preclinical and 5/13 (38%) recent-onset IDDM subjects had antibodies that precipitated GAD activity. Antibodies to GAD were not detected in sera from subjects with other autoimmune diseases (n = 29) or healthy controls (n = 14). GAD affinity-purified to homogeneity (specific activity, 58 units/mg) was specifically immunoprecipitated as a single 60-kDa species by the IDDM sera. In an ELISA incorporating whole mouse brain GAD captured by the GAD-6 monoclonal antibody the frequencies of GAD antibodies for all subject groups were indistinguishable from those found by precipitation of human brain enzymatic activity. We conclude that (i) GAD is an (auto)antigen in a majority of subjects operationally defined as having preclinical IDDM, (ii) pancreatic islet and brain GAD are likely to be cross-reactive, and (iii) the majority of GAD antibodies are directed away from the catalytic site of the brain enzyme. The lower frequency of GAD antibodies in recent-onset IDDM subjects indicates either that immunoreactivity is lost with near-total beta-cell destruction or that GAD antibodies denote a low risk of progression to clinical disease.