Beulah Leitch

GABAergic synapses in the antennal lobe and mushroom body of the locust olfactory system

To help elucidate the role of inhibitory feedback in the genesis of odour‐evoked synchronisation of neural activity, we investigated the distribution of γ‐aminobutyric acid (GABA)ergic synaptic terminals in the antennal lobes (AL) and mushroom bodies (MB) of the locust olfactory system. Electron‐microscopy, intracellular horseradish peroxidase labelling, and immunocytochemistry were combined to assess the distribution of GABAergic synapses, using established methods (Leitch and Laurent [1993] J. Comp. Neurol. 337:461–470).

Snowdrop lectin (GNA) has no acute toxic effects on a beneficial insect predator, the 2-spot ladybird (Adalia bipunctata L.).

Two-spot ladybird (Adalia bipunctata L.) larvae were fed on aphids (Myzus persicae (Sulz.)) which had been loaded with snowdrop lectin (Galanthus nivalis agglutinin; GNA) by feeding on artificial diet containing the protein. Treatment with GNA significantly decreased the growth of aphids. No acute toxicity of GNA-containing aphids towards the ladybird larvae was observed, although there were small effects on development. When fed a fixed number of aphids, larvae exposed to GNA spent longer in the 4th instar, taking 6 extra days to reach pupation; however, retardation of development was not observed in ladybird larvae fed equal weights of aphids. Ladybird larvae fed GNA-containing aphids were found to be 8-15% smaller than controls, but ate a significantly greater number of aphids (approx. 40% to pupation). GNA was shown to be present on the microvilli of the midgut brush border membrane and within gut epithelial cells in ladybird larvae fed on GNA-dosed aphids, although disruption of the brush border was not observed. It is hypothesised that GNA does not have significant direct toxic or adverse effects on developing ladybird larvae, but that the effects observed may be due to the fact that the aphids fed on GNA are compromised and are thus a suboptimal food.

SUBCELLULAR DISTRIBUTION OF L-TYPE CALCIUM CHANNEL SUBTYPES IN RAT HIPPOCAMPAL NEURONS

L-type calcium channels play an essential role in synaptic activity-dependent gene expression and are implicated in long-term alterations in synaptic efficacy underlying learning and memory in the hippocampus. The two principal pore-forming subunits of L-type Ca2+ channels expressed in neurons are the Ca(v)1.2 (alpha(1C)) or Ca(v)1.3 (alpha(1D)) subtypes. Experimental evidence suggests that calcium entry through Ca(v)1.2 and Ca(v)1.3 Ca2+ channels occurs in close proximity to key signalling molecules responsible for triggering signalling pathways leading to transcriptional responses. Determining the subcellular distribution of Ca(v)1.2 and Ca(v)1.3 L-type channels in neurons is clearly important for unravelling the molecular mechanisms underlying long-term alterations in neuronal function. In this study, we used immunogold-labelling techniques and electron-microscopy (EM) to analyse the subcellular distribution and density of both Ca(v)1.2 and Ca(v)1.3 Ca2+ channels in rat hippocampal CA1 pyramidal cells in vivo. We confirm that both Ca(v)1.2 and Ca(v)1.3 channel subtypes are predominantly but not exclusively located in postsynaptic dendritic processes and somata. Both Ca(v)1.2 and Ca(v)1.3 are distributed throughout the dendritic tree. However, the smallest (distal) dendritic processes and spines have proportionally more calcium channels inserted into their plasma membrane than located within cytoplasmic compartments indicating the potential targeting of calcium channels to microdomains within neurons. Ca(v)1.2 and Ca(v)1.3 Ca2+ channels are located at the postsynaptic density and also at extra-synaptic sites. The location of L-type Ca(v)1.2 and Ca(v)1.3 channels in distal dendrites and spines would thus place them at appropriate sites where they could initiate synapse to nucleus signalling.

Spatial learning-induced accumulation of agmatine and glutamate at hippocampal CA1 synaptic terminals.

Agmatine, the decarboxylated metabolite of l-arginine, is considered to be a novel putative neurotransmitter. Recent studies have demonstrated that endogenous agmatine may directly participate in the processes of spatial learning and memory. Agmatine-immunoreactivity has been observed within synaptic terminals of asymmetric excitatory synapses in the hippocampal CA1 stratum radiatum (SR), suggesting that agmatine may be colocalized with glutamate. In the present study we demonstrate, using immunofluorescence confocal microscopy, that agmatine is colocalized with glutamate within CA1-CA3 hippocampal pyramidal cell bodies, in young Sprague-Dawley rats. Subcellular investigation, using postembedding electron microscopy-immunogold cytochemistry, has also revealed that agmatine is colocalized with glutamate in most synaptic terminals in the SR region of CA1. Ninety-seven percent of all agmatinergic profiles were found to contain glutamate, and 92% of all glutamatergic profiles contained agmatine (n=6; 300 terminals). Alterations in colocalized agmatine and glutamate levels in the SR synaptic terminals, following 4 days Morris water maze training, were also investigated. Compared with swim only control rats, water maze-trained rats had statistically significant increases in both agmatine (78%; P<0.01) and glutamate (41%; P<0.05) levels within SR terminals synapsing onto CA1 dendrites. These findings provide the first evidence that agmatine and glutamate are colocalized in synaptic terminals in the hippocampal CA1 region, and may co-participate in spatial learning and memory processing.

The gap junction-like form of a vacuolar proton channel component appears not to be an artifact of isolation: An immunocytochemical localization study☆

Gap junctional structures containing a 16-kDa intrinsic membrane protein have been isolated from the hepatopancreas of the crustacean Nephrops norvegicus. These structures are double membranes 14-15 nm thick and composed of hexagonal arrays of particles which have a central pore that is penetrated by a cationic negative stain. Membrane preparations have also been isolated from the hepatopancreas and these contain similar gap junctional regions of uniform width. Affinity purified antibodies to the 16-kDa protein bind principally to these gap junctional regions. Antiserum raised against the isolated gap junctional structures binds strongly to the lateral surfaces of the columnar epithelial cells and in particular to gap junction-like regions.

Spatial learning-induced increase in agmatine levels at hippocampal CA1 synapses

Agmatine, a metabolite of L‐arginine, is considered as a novel putative neurotransmitter. It has been detected in axon terminals that synapse with pyramidal cells in the hippocampus, a brain region that is critically involved in spatial learning and memory. However, the role of agmatine in learning and memory is poorly understood. Recently, we demonstrated water maze training‐induced increases in tissue levels of agmatine in the CA1 subregion of the hippocampus. This finding has raised an issue whether an endogenous agmatine could directly participate in learning and memory processes as a neurotransmitter. In the present study, quantitative immunogold‐labeling and electron‐microscopical techniques were used to analyze the levels of agmatine in CA1 stratum radiatum (SR) terminals (n = 600) of male Sprague–Dawley rats that had been trained to find a hidden escape platform in the water maze (WM) task or forced to swim (SW) in the pool with no platform presented. Agmatine levels were significantly increased by ∼85% in the synaptic terminals of SR of trained WM group compared with the SW control group (all P < 0.001). These results, for the first time, demonstrate spatial learning‐induced elevation in agmatine levels at synapses in the hippocampus and provide evidence of its participation in learning and memory processing as a novel neurotransmitter. Synapse, 2011.

Selective loss of AMPA receptors at corticothalamic synapses in the epileptic stargazer mouse

Absence seizures are common in the stargazer mutant mouse. The mutation underlying the epileptic phenotype in stargazers is a defect in the gene encoding the normal expression of the protein stargazin. Stargazin is involved in the membrane trafficking and synaptic targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at excitatory glutamatergic synapses. Thus, the genetic defect in the stargazer results in a loss of AMPARs and consequently, excitation at glutamatergic synapses. Absence seizures are known to arise in thalamocortical networks. In the present study we show for the first time, using Western blot analysis and quantitative immunogold cytochemistry, that in the epileptic stargazer mouse, there is a global loss of AMPAR protein in nucleus reticularis (RTN) and a selective loss of AMPARs at corticothalamic synapses in inhibitory neurons of the RTN thalamus. In contrast, there is no significant loss of AMPARs at corticothalamic synapses in excitatory relay neurons in the thalamic ventral posterior (VP) region. The findings of this study thus provide cellular and molecular evidence for a selective regional loss of synaptic AMPAR within the RTN that could account for the loss of function at these inhibitory neuron synapses, which has previously been reported from electrophysiological studies. The specific loss of AMPARs at RTN but not relay synapses in the thalamus of the stargazer, could contribute to the absence epilepsy phenotype by altering thalamocortical network oscillations. This is supported by recent evidence that loss of glutamate receptor subunit 4 (GluA4) (the predominant AMPAR-subtype in the thalamus), also leads to a specific reduction in strength in the cortico-RTN pathway and enhanced thalamocortical oscillations, in the Gria4(-/-) model of absence epilepsy. Thus further study of thalamic changes in these models could be important for future development of drugs targeted to absence epilepsy.

Cerebellar Golgi, Purkinje, and basket cells have reduced γ-aminobutyric acid immunoreactivity in stargazer mutant mice

The stargazer mutant mouse has characteristic ataxia and head‐tossing traits coupled with a severe impairment in the acquisition of classical eye‐blink conditioning (Qiao et al. [ 1996 ] J. Neurosci. 16:640–648; Qiao et al. [ 1998 ] J. Neurosci. 18:6990–6999). These phenotypes are thought to be cerebellar mediated and have been attributed to the specific reduction in brain‐derived neurotrophic factor (BDNF). The granule cells in the cerebellum of the stargazer mouse exhibit a near‐total and exclusive ablation of BDNF mRNA expression and a consequent defect in TrkB receptor signalling. To investigate whether the stargazer mutation and lack of availability of BDNF in the granule cells compromise the phenotype of the cerebellar inhibitory neurons, specifically their immunoreactivity for γ‐aminobutyric acid (GABA); the levels of GABA neurotransmitter expressed in Golgi, Purkinje, and basket cells; and the density of their synaptic contacts were compared in stargazer and wild‐type controls using electron microscopy and quantitative immunogold labelling. The data presented in this study clearly show that, in the spontaneous ataxic mutant mouse stargazer, the cerebellar inhibitory neurons have significantly reduced levels of GABA immunoreactivity indicative of a significant decrease in their GABA content compared with wild‐type controls. Furthermore, the density of inhibitory synapses between Golgi interneurons and granule cells and also between basket and Purkinje cells in stargazer mutants is reduced to approximately half that in wild‐type controls. Whether this reduction in GABA content and inhibitory synapse density is directly attributable to the lack of BDNF in the cerebellum of the stargazer mutant is yet to be proved. J. Comp. Neurol. 453:85–99, 2002.

Octopaminergic modulation of synaptic transmission between an identified sensory afferent and flight motoneuron in the locust.

The role of the biogenic amine octopamine in modulating cholinergic synaptic transmission between the locust forewing stretch receptor neuron (fSR) and the first basalar motoneuron (BA1) was investigated. The amines 5‐hydroxytryptamine (5‐HT, serotonin) and dopamine were also studied. Bath application of octopamine, 5‐HT, and dopamine at concentrations of 10‐4 M reversibly decreased the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in BA1 by electrically stimulating the fSR axon. These effects occurred without any detectable change in either input resistance or membrane potential of BA1. The amines also reversibly decreased the amplitude of responses to acetylcholine (ACh) pressure‐applied to the soma of BA1. The muscarinic antagonist scopolamine (10‐6 M) had no significant effect on the octopamine‐induced decrease in ACh responses. These observations suggest that these amines potentially could physiologically depress cholinergic transmission between fSR and BA1, at least in part, by altering nicotinic rather than muscarinic cholinergic receptor function. Although the octopaminergic agonists naphazoline and tolazoline both mimicked the actions of octopamine, the receptor responsible for octopamine‐mediated modulation could not be characterized since amine receptor antagonists tested on the preparation had complex actions. Confocal immunocytochemistry revealed intense octopamine immunoreactivity in the anterior lateral association center, thus confirming the presence of octopamine in neuropil regions containing fSR/BA1 synapses and therefore supporting a role for this amine in the modulation of synaptic transmission between the fSR and BA1. 5‐HT‐immunoreactivity, conversely, was concentrated within the ventral association centers; very little staining was observed in the dorsal neuropil regions in which fSR/BA1 synapses are located. J. Comp. Neurol. 462:55–70, 2003.

Phenotype of cerebellar glutamatergic neurons is altered in stargazer mutant mice lacking brain-derived neurotrophic factor mRNA expression.

Brain‐derived neurotrophic factor (BDNF) influences neuronal survival, differentiation, and maturation. More recently, its role in synapse formation and plasticity has also emerged. In the cerebellum of the spontaneous recessive mutant mouse stargazer (stg) there is a specific and pronounced deficit in BDNF mRNA expression. BDNF protein levels in the cerebellum as a whole are reduced by 70%, while in the granule cells (GCs) there is a selective and near total reduction in BDNF mRNA expression. Recently, we published data demonstrating that inhibitory neurons in the cerebella of stgs have significantly reduced levels (∼50%) of γ‐aminobutyric acid (GABA) and fewer, smaller inhibitory synapses compared to wildtype (WT) controls. Our current investigations indicate that the stargazer mutation has an even more pronounced effect on the phenotype of glutamatergic neurons in the cerebellum. There is a profound decrease in the levels of glutamate‐immunoreactivity (up to 77%) in stg compared to WT controls. The distribution profile of presynaptic vesicles is also markedly different: stgs have proportionally fewer docked vesicles and fewer vesicles located adjacent to the active zone ready to dock than WTs. Furthermore, the thickness of the postsynaptic density (PSD) at mossy fiber‐granule cell (MF‐GC) and parallel fiber‐Purkinje cell (PF‐PC) synapses is severely reduced (up to 33% less than WT controls). The number and length of excitatory synapses, however, appear to be relatively unchanged. It is possible that at least some of theses changes in phenotype are directly attributable to the lack of BDNF in the cerebellum of the stg mutant. J. Comp. Neurol. 481:145–159, 2005.