Selective transduction and photoinhibition of pre-Bötzinger complex neurons that project to the facial nucleus in rats affects nasofacial activity.

The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases.

While breathing seems to come easy, it is a complex process in which many muscles coordinate to allow air to flow into the lungs. These muscles also control the flow of air we breathe out to allow us to talk, sing, eat, or drink. The brain circuits that control these muscles, can also influence other parts of the brain. The preBötzinger Complex, which is a key region of brainstem circuits that generate and control breathing, contains neurons that also project widely, connecting to other regions of the brain. This helps to modulate the sense of smell, emotional state, heart rate, and even blood pressure. Understanding how the preBötzinger Complex is organized can untangle how breathing can influence these other processes. Melo et al. wanted to learn whether they could manipulate the activity of a subgroup of preBötzinger Complex neurons that project into the facial nucleus ­ a region of the brain that controls the muscles of the face when we breathe ­ without affecting breathing. If this can be done, it might also be possible to affect blood pressure by manipulating selective preBötzinger neurons, and thus the development of hypertension, without having any impact on breathing. To test this hypothesis, Melo et al. used rats in which the activation of preBötzinger Complex neurons that project into the facial nucleus was blocked. This decreased the activity of the muscles around the nose with hardly any effect on breathing. Melo et al. also found that the state of consciousness of the rat (anesthetized or conscious) could affect how preBötzinger Complex neurons control these muscles. Melo et al. also observed that preBötzinger Complex neurons projecting into the facial nucleus had projections into many other regions in the brainstem. This might help to the coordinate respiratory, cardiovascular, orofacial, and potentially other physiological functions. The findings of Melo et al. set a technical foundation for exploring the influence of specific subgroups of preBötzinger Complex neurons on respiratory modulation of other physiological activities, including blood pressure and heart rate and in conditions, such as hypertension and heart failure. More broadly, most brain regions contain complex and heterogeneous groups of neurons and the strategy validated by Melo et. al. could be applied to unravel other brain-function relationships.

Targeted viral vector transduction of relaxin-3 neurons in the rat nucleus incertus using a novel cell-type specific promoter.

Modern neuroscience utilizes transgenic techniques extensively to study the activity and function of brain neural networks. A key feature of this approach is its compatibility with molecular methods for selective transgene expression in neuronal circuits of interest. Until now, such targeted transgenic approaches have not been applied to the extensive circuitry involving the neuropeptide, relaxin-3. Pharmacological and gene knock-out studies have revealed relaxin-3 signalling modulates interrelated behaviours and cognitive processes, including stress and anxiety, food and alcohol consumption, and spatial and social memory, highlighting the potential of this system as a therapeutic target. In the present study, we aimed to identify a promoter sequence capable of regulating cell-type specific transgene expression from an adeno-associated viral (AAV) vector in relaxin-3 neurons of the rat nucleus incertus (NI). In parallel to relaxin-3 promoter sequences, we also tested an AAV vector containing promoter elements for the tropomyosin receptor kinase A (TrkA) gene, as TrkA is co-expressed with relaxin-3 in rat NI neurons. Stereotaxic injection of an mCherry-expressing AAV vector revealed widespread non-specific TrkA promoter (880 bp) activity in and adjacent to the NI at 8 weeks post-treatment. In contrast, mCherry expression was successfully restricted to relaxin-3 NI neurons with 98% specificity using a 1736 bp relaxin-3 promoter. In addition to detailed anatomical mapping of NI relaxin-3 networks, illustrated here in association with GABAergic medial septum neurons, this method for targeted transgene delivery offers a versatile tool for ongoing preclinical studies of relaxin-3 circuitry.

Septal GABA and Glutamate Neurons Express RXFP3 mRNA and Depletion of Septal RXFP3 Impaired Spatial Search Strategy and Long-Term Reference Memory in Adult Mice.

Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 loxP/loxP mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV(1/2)-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.