Dedicated C-fiber vagal sensory afferent pathways to the paraventricular nucleus of the hypothalamus
Graphical abstract
Introduction
The nucleus of the solitary tract (NTS) integrates viscerosensory information from vagal afferents including arterial baroreceptor and chemoreceptor inputs that regulate autonomic outflow. Craniosensory axons, primarily from the vagus, converge to form the solitary tract (ST) and synapse directly on 2nd order NTS neurons (Andresen and Kunze, 1994). Approximately 80–90% of afferents have unmyelinated axons (Coleridge and Coleridge, 1984, Douglas and Ritchie, 1962). These C-fiber afferents express the Transient Receptor Potential Vanilloid receptor (TRPV1) from their peripheral sensory endings to their central synaptic terminals (Kline et al., 2019, Sun et al., 2009) but thinly myelinated A-fiber afferents lack TRPV1 (Andresen et al., 2012). A- and C-fiber vagal afferents transmit distinctly different information (Andresen et al., 2012), have distinctly different reflex characteristics (Fan and Andresen, 1998, Fan et al., 1999), and segregate to different 2nd order neurons (McDougall et al., 2009, Peters et al., 2011).
NTS neurons innervate a wide range of central nervous system structures including the paraventricular nucleus (PVN) of the hypothalamus (Loewy, 1990, Saper et al., 2016). Neurons within the PVN are highly diverse and neuroanatomical evidence indicates that these neurons both receive and send projections to many other brain areas (Geerling et al., 2010, Loewy, 1990, Standish et al., 1995). The PVN has essential roles in neuroendocrine function through projections to the pituitary and median eminence, while neuropeptide and glutamatergic projections to the brainstem and spinal cord regulate autonomic function and regulation (Chen and Toney, 2003, King et al., 2015, Rinaman, 2010). A large array of stress-related neuroendocrine mediators has been associated with the NTS and PVN including: corticotropin-releasing hormone, arginine vasopressin, glucocorticoids, and catecholamines, to name a few (Charmandari et al., 2005). PVN neurons themselves are also neurochemically distinct (Gasparini et al., 2020, Myers et al., 2017). Dense PVN-NTS projections thus support potentially complex network interactions between these two integrative brain nuclei (Sawchenko et al., 1996). However, far less is known about the organization of synaptic transmission through such connections, especially related to vagal afferent activation. Our present studies shed new light on both the organization of pathways within NTS projecting to PVN and characterize some of the functional characteristics of these pathways activated by vagal afferents.
Previous studies identified that most PVN-projecting NTS neurons receive higher order excitatory connections from vagal ST afferents (Bailey et al., 2006a). Here we sought to systematically detect, classify, and test each ST input for TRPV1 expression as a means of assessing A- compared to C-fiber vagal afferent processing. We used retrogradely transported dye stereotaxically injected into the PVN to identify projection neurons within NTS. We conducted voltage clamp recordings of fluorescently labeled neurons in brainstem slices and examined synaptic responses to ST activation to characterize vagal pathway connections. Synaptic responses to the selective TRPV1 agonist resiniferatoxin (RTX) identified vagal C-fiber pathways. The TRPV1 receptor complex is a cation channel with a high conductance to calcium. Activation of TRPV1 thus has two actions: first it is excitatory and depolarizes neurons (indicated by increased sEPSCs) and if sustained results in a secondary depolarization block of conducted processes (consistent with block of ST-EPSCs), likely through inactivation of voltage gated sodium channels (Hofmann et al., 2014). A-fiber responses persisted unchanged in the presence of RTX (as do reflex and whole peripheral nerve conduction) (Fan and Andresen, 1998). PVN-projecting NTS neurons received 3 patterns of inputs; ones that exclusively received only monosynaptic EPSCs, those that received only indirect EPSCs, or those that received both. Our studies indicate that monosynaptic vagal pathways to PVN-projecting NTS neurons were rare and that even convergent ST afferent inputs segregate by TRPV1 expression. However, vagal afferent activity arrives at these higher order NTS neurons with variable timing and unreliable synaptic transmission. This result parallels the A-/C-fiber pathway segregation and predominance of higher order ST connections described for amygdala-projecting NTS neurons (McDougall et al., 2017). Together the studies suggest that segregation of A- and C-fiber ST afferent information may be a common tenant amongst projections to forebrain neurons that follow a labeled-line pattern of viscerosensory signaling in the NTS to effectively transmit to other brain regions.
Section snippets
Stereotaxic placement of rhodamine in the PVN retrogradely transports to NTS.
Small, microinjected volumes of rhodamine Retrobeads (LumaFluor Inc.) into the PVN resulted in retrograde labeling of neurons within NTS (Fig. 1A). Examinations of coronal sections of the forebrain confirmed successful dye placement within the PVN (Paxinos and Watson, 1998) (Fig. 1, B1-8). Successful injections labelled neurons in the ipsilateral NTS (Fig. 1C). Using fluorescence (FITC) and differential interference contrast live cell imaging, we identified individual rhodamine positive neurons
Discussion
The organization and processing of vagal afferent information within the CNS have important implications for our understanding of the regulation of neuroendocrine and autonomic responses across vital organs (Chen and Toney, 2003, King et al., 2015, Rinaman, 2010). Our new studies indicate that C-type vagal afferents commonly drive PVN-projecting NTS neurons predominantly through polysynaptic pathways which converge substantially within the NTS. Even when neurons only had indirect connections,
Ethica statements
The Institutional Animal Care and Use Committee at Oregon Health and Science University approved all animal procedures, and they conform to the guidelines of the National Institutes of Health publication “Guide for the Care and Use of Laboratory Animals”.
Experimental animals
Male Sprague-Dawley rats (270 ± 30 g, Charles Rivers Laboratory) were housed under a 12 h/12 h light/dark cycle.
Stereotaxic retrograde tracer from PVN to NTS
Adult male Sprague–Dawley rats were anaesthetized (isoflurane 5% induction and 2% maintenance) and placed in a stereotaxic frame
CRediT authorship contribution statement
Jessica A. Fawley: Investigation, Formal analysis, Data curation, Writing - original draft. Deborah M. Hegarty: Investigation, Data curation, Writing - review & editing. Sue A. Aicher: Investigation, Writing - review & editing. Eric Beaumont: Conceptualization, Funding acquisition, Writing - review & editing. Michael C. Andresen: Conceptualization, Funding acquisition, Writing - original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We would like to acknowledge Dennis Nguyen for his contribution to tissue sectioning.
Funding
This study was supported by funding from the National Institutes of Health HL133505 (MCA) and HL141560 (EB).
Availability of data and material
The datasets of the current study are available from the corresponding author on reasonable request.
Ethics approval
The Institutional Animal Care and Use Committee at Oregon Health and Science University approved all animal procedures, and they conform to the guidelines of the National Institutes of Health publication “Guide for the Care and Use of Laboratory Animals”.
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