In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in >90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Local translation in primary afferents and its contribution to pain.

ABSTRACT: Chronic pain remains a significant problem due to its prevalence, impact, and limited therapeutic options. Progress in addressing chronic pain is dependent on a better understanding of underlying mechanisms. Although the available evidence suggests that changes within the central nervous system contribute to the initiation and maintenance of chronic pain, it also suggests that the primary afferent plays a critical role in all phases of the manifestation of chronic pain in most of those who suffer. Most notable among the changes in primary afferents is an increase in excitability or sensitization. A number of mechanisms have been identified that contribute to primary afferent sensitization with evidence for both increases in pronociceptive signaling molecules, such as voltage-gated sodium channels, and decreases in antinociceptive signaling molecules, such as voltage-dependent or calcium-dependent potassium channels. Furthermore, these changes in signaling molecules seem to reflect changes in gene expression as well as posttranslational processing. A mechanism of sensitization that has received far less attention, however, is the local or axonal translation of these signaling molecules. A growing body of evidence indicates that this process not only is dynamically regulated but also contributes to the initiation and maintenance of chronic pain. Here, we review the biology of local translation in primary afferents and its relevance to pain pathobiology.

Mechanical Allodynia Circuitry in the Dorsal Horn Is Defined by the Nature of the Injury.

The spinal dorsal horn is a major site for the induction and maintenance of mechanical allodynia, but the circuitry that underlies this clinically important form of pain remains unclear. The studies presented here provide strong evidence that the neural circuits conveying mechanical allodynia in the dorsal horn differ by the nature of the injury. Calretinin (CR) neurons in lamina II inner convey mechanical allodynia induced by inflammatory injuries, while protein kinase C gamma (PKCγ) neurons at the lamina II/III border convey mechanical allodynia induced by neuropathic injuries. Cholecystokinin (CCK) neurons located deeper within the dorsal horn (laminae III-IV) are important for both types of injuries. Interestingly, the Maf+ subset of CCK neurons is composed of transient vesicular glutamate transporter 3 (tVGLUT3) neurons, which convey primarily dynamic allodynia. Identification of an etiology-based circuitry for mechanical allodynia in the dorsal horn has important implications for the mechanistic and clinical understanding of this condition.

Animal Models: Challenges and Opportunities to Determine Optimal Experimental Models of Pancreatitis and Pancreatic Cancer.

At the 2018 PancreasFest meeting, experts participating in basic research met to discuss the plethora of available animal models for studying exocrine pancreatic disease. In particular, the discussion focused on the challenges currently facing the field and potential solutions. That meeting culminated in this review, which describes the advantages and limitations of both common and infrequently used models of exocrine pancreatic disease, namely, pancreatitis and exocrine pancreatic cancer. The objective is to provide a comprehensive description of the available models but also to provide investigators with guidance in the application of these models to investigate both environmental and genetic contributions to exocrine pancreatic disease. The content covers both nongenic and genetically engineered models across multiple species (large and small). Recommendations for choosing the appropriate model as well as how to conduct and present results are provided.

Layer- and cell type-selective co-transmission by a basal forebrain cholinergic projection to the olfactory bulb.

Cholinergic neurons in the basal forebrain project heavily to the main olfactory bulb, the first processing station in the olfactory pathway. The projections innervate multiple layers of the main olfactory bulb and strongly influence odor discrimination, detection, and learning. The precise underlying circuitry of this cholinergic input to the main olfactory bulb remains unclear, however. Here, we identify a specific basal forebrain cholinergic projection that innervates select neurons concentrated in the internal plexiform layer of the main olfactory bulb. Optogenetic activation of this projection elicits monosynaptic nicotinic and GABAergic currents in glomerular layer-projecting interneurons. Additionally, we show that the projection co-expresses markers for GABAergic neurotransmission. The data thus implicate neurotransmitter co-transmission in the basal forebrain regulation of this inhibitory olfactory microcircuit.Cholinergic neurons innervate multiple layers in the main olfactory bulb but the precise circuitry of this input is not known. Here the authors show that VGLUT3+ cholinergic neurons selectively innervate deep short axon cells in specific layers and elicit robust monosynaptic GABAergic and nicotinic postsynaptic currents.

Dorsal Horn Circuits for Persistent Mechanical Pain.

Persistent mechanical hypersensitivity that occurs in the setting of injury or disease remains a major clinical problem largely because the underlying neural circuitry is still not known. Here we report the functional identification of key components of the elusive dorsal horn circuit for mechanical allodynia. We show that the transient expression of VGLUT3 by a discrete population of neurons in the deep dorsal horn is required for mechanical pain and that activation of the cells in the adult conveys mechanical hypersensitivity. The cells, which receive direct low threshold input, point to a novel location for circuit initiation. Subsequent analysis of c-Fos reveals the circuit extends dorsally to nociceptive lamina I projection neurons, and includes lamina II calretinin neurons, which we show also convey mechanical allodynia. Lastly, using inflammatory and neuropathic pain models, we show that multiple microcircuits in the dorsal horn encode this form of pain.