N-type calcium channels control GABAergic transmission in brain areas related to fear and anxiety.

N-type (CaV2.2) calcium channels are key for action potential-evoked transmitter release in the peripheral and central nervous system. Previous studies have highlighted the functional relevance of N-type calcium channels at both the peripheral and central level. In the periphery, the N-type calcium channels regulate nociceptive and sympathetic responses. At the central level, N-type calcium channels have been linked to aggression, hyperlocomotion, and anxiety. Among the areas of the brain that are involved in anxiety are the basolateral amygdala, medial prefrontal cortex, and ventral hippocampus. These three areas share similar characteristics in their neuronal circuitry, where pyramidal projection neurons are under the inhibitory control of a wide array of interneurons including those that express the peptide cholecystokinin. This type of interneuron is well-known to rely on N-type calcium channels to release GABA in the hippocampus, however, whether these channels control GABA release from cholecystokinin-expressing interneurons in the basolateral amygdala and medial prefrontal cortex is not known. Here, using mouse models to genetically label cholecystokinin-expressing interneurons and electrophysiology, we found that in the basolateral amygdala, N-type calcium channels control ~50% of GABA release from these neurons onto pyramidal cells. By contrast, in the medial prefrontal cortex N-type calcium channels are functionally absent in synapses of cholecystokinin-expressing interneurons, but control ~40% of GABA release from other types of interneurons. Our findings provide insights into the precise localization of N-type calcium channels in interneurons of brain areas related to anxiety.

Characterization of cachexia in the human fibrosarcoma HT-1080 mouse tumour model.

BACKGROUND: Cancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms. METHODS: Female severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined. RESULTS: The HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model. CONCLUSIONS: The subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Cacna1b alternative splicing impacts excitatory neurotransmission and is linked to behavioral responses to aversive stimuli.

Presynaptic CaV2.2 channels control calcium entry that triggers neurotransmitter release at both central and peripheral synapses. The Cacna1b gene encodes the α1-pore forming subunit of CaV2.2 channels. Distinct subsets of splice variants of CaV2.2 derived from cell-specific alternative splicing of the Cacna1b pre-mRNA are expressed in specific subpopulations of neurons. Four cell-specific sites of alternative splicing in Cacna1b that alter CaV2.2 channel function have been described in detail: three cassette exons (e18a, e24a, and e31a) and a pair of mutually exclusive exons (e37a/e37b). Cacna1b mRNAs containing e37a are highly enriched in a subpopulation of nociceptors where they influence nociception and morphine analgesia. E37a-Cacna1b mRNAs are also expressed in brain, but their cell-specific expression in this part of the nervous system, their functional consequences in central synapses and their role on complex behavior have not been studied. In this report, we show that e37a-Cacna1b mRNAs are expressed in excitatory projection neurons where CaV2.2 channels are known to influence transmitter release at excitatory inputs from entorhinal cortex (EC) to dentate gyrus (DG). By comparing behaviors of WT mice to those that only express e37b-CaV2.2 channels, we found evidence that e37a-CaV2.2 enhances behavioral responses to aversive stimuli. Our results suggest that alternative splicing of Cacna1b e37a influences excitatory transmitter release and couples to complex behaviors.

Tissue- and cell-specific expression of a splice variant in the II-III cytoplasmic loop of Cacna1b.

Presynaptic CaV 2.2 (N-type) channels are fundamental for transmitter release across the nervous system. The gene encoding CaV 2.2 channels, Cacna1b, contains alternatively spliced exons that result in functionally distinct splice variants (e18a, e24a, e31a, and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a-Cacna1b and ∆e18a-Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a-Cacna1b splice variants are expressed in monoaminergic regions of the midbrain. We expanded these studies and identified +e18a-Cacna1b mRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a-Cacna1b is enriched in cholecystokinin-expressing interneurons. Our results provide key information to understand cell-specific functions of CaV 2.2 channels.