A vibrissa pathway that activates the limbic system

  1. Michaël Elbaz
  2. Amalia Callado Perez
  3. Maxime Demers
  4. Shengli Zhao
  5. Conrad Foo
  6. David Kleinfeld  Is a corresponding author
  7. Martin Deschenes  Is a corresponding author
  1. CERVO Research Center, Laval University, Canada
  2. Department of Physics, University of California, San Diego, United States
  3. Department of Neurobiology, Duke University Medical Center, United States
  4. Section of Neurobiology, University of California, San Diego, United States

Abstract

Vibrissa sensory inputs play a central role in driving rodent behavior. These inputs transit through the sensory trigeminal nuclei, which give rise to the ascending lemniscal and paralemniscal pathways. While lemniscal projections are somatotopically mapped from brainstem to cortex, those of the paralemniscal pathway are more widely distributed. Yet the extent and topography of paralemniscal projections are unknown, along with the potential role of these projections in controlling behavior. Here, we used viral tracers to map paralemniscal projections. We find that this pathway broadcasts vibrissa-based sensory signals to brainstem regions that are involved in the regulation of autonomic functions and to forebrain regions that are involved in the expression of emotional reactions. We further provide evidence that GABAergic cells of the Kölliker-Fuse nucleus gate trigeminal sensory input in the paralemniscal pathway via a mechanism of presynaptic or extrasynaptic inhibition.

Editor's evaluation

Elbaz and colleagues show that the interpolaris subdivision of the trigeminal brainstem innervates not only somatosensory thalamus but also other midbrain and hindbrain structures. A key target of interpolaris appears anatomically to be excitatory projection neurons of the Kolliker-Fuse nucleus. Physiological recordings from the dorsal medullary reticular nucleus, Kolliker-Fuse nucleus, and the central amygdala reveal responses to vibrissa deflection, suggesting a role for this pathway in limbic system behaviors.

https://doi.org/10.7554/eLife.72096.sa0

Introduction

Most sensory systems comprise parallel pathways of sensory information that encode different features of a stimulus and also take part in the control of sensor motion (Merigan and Maunsell, 1993; Lomber and Malhotra, 2008; Nassi and Callaway, 2009; Niu et al., 2013; Igarashi et al., 2012). The vibrissa system of rodents is no exception (reviewed in Kleinfeld and Deschênes, 2011). Ascending signals in the vibrissa system travel along two main trigeminothalamic pathways (reviewed in Prescott et al., 2016): (1) a lemniscal pathway that arises from the trigeminal nucleus principalis (PrV), transits through the ventral posterior medial nucleus (VPM) of the thalamus, and projects to the primary somatosensory cortex; (2) a paralemniscal pathway that arises from the rostral part of trigeminal nucleus interpolaris (SpVIr), transits through the posterior group (Po) of the thalamus, and projects to the somatosensory cortical areas and to the vibrissa motor cortex.

In contrast with PrV cells, which innervate principally VPM thalamus, SpVIr cells that project to Po thalamus also innervate a number of additional regions by means of branching axons (Pierret et al., 2000; Veinante et al., 2000; Bellavance et al., 2017). These include the superior colliculus, the anterior pretectal nucleus, the ventral division of zona incerta, and the dorsal lateral sector of the facial nucleus. Tract-tracing studies by means of classic tracers also reported interpolaris projections to the brainstem and spinal cord (Jacquin et al., 1986; Phelan and Falls, 1991). Whether the latter projections also arise from Po-projecting cells remains an open question.

While the lemniscal pathway conveys tactile information, as well as information about the relative phase of the vibrissae in the whisk cycle (Yu et al., 2006; Curtis and Kleinfeld, 2009; Khatri et al., 2010; Moore et al., 2015; Isett and Feldman, 2020), the role of the paralemniscal pathway remains puzzling. It was proposed that this pathway conveys information about whisking kinematics (Yu et al., 2006; Golomb et al., 2006), but later studies found that encoding of whisking along the paralemniscal pathway is relatively poor (Moore et al., 2015; Urbain et al., 2015). It was also proposed that the paralemniscal pathway is specifically activated upon noxious stimulation (Masri et al., 2009; Frangeul et al., 2014), but it has never been shown that interpolaris cells that respond to vibrissa deflection are also activated by noxious stimuli. Thus, the general function of the paralemnical pathway remains unresolved.

Here, we used virus-based tract-tracing methods and electrophysiology to document the full extent of the collateral projections of interpolaris cells that give rise to the paralemniscal pathway. We searched for new pathways of ascending information to forebrain regions, with a focus on pathways that include the Kölliker-Fuse/parabrachial complex (KF/PBc). The KF/PBc were previously described primarily in terms of their role in eliciting changes in respiration and cardiac function (reviewed in Saper and Stornetta, 2015). Yet the role of inhalation in either driving or pacing whisking (Deschênes et al., 2012; Moore et al., 2013) provides motivation to investigate if the KF/PBc has a broader role in orofacial motor actions. Further motivation comes from the projections of the KF nucleus to the trigeminal sensory nuclei (Geerling et al., 2017), which contains multiple pathways for the control of motoneurons involved in whisking (Bellavance et al., 2017). Finally, we searched for feedback projections from targets along the paralemnical pathways to brainstem nuclei as a means to uncover a potential role of this pathway in rat’s behavior.

Results

Collateral projections of interpolaris cells that project to Po thalamus

To map the collateral projections of interpolaris cells that innervate Po thalamus, we injected the retrograde virus G-pseudotyped Lenti-Cre in Po thalamus, and AAV2/8-hSyn-DIO-GFP in the vibrissa-responsive sector of subnucleus SpVIr (five juvenile rats; Figure 1A). This approach revealed an unexpected diversity of axonal projections across all animals (Figure 1B–G). In accordance with prior studies, terminal fields are found in Po thalamus, the ventral division of zona incerta, the anterior pretectal nucleus, the superior colliculus, the perirubral region, the dorsal lateral part of the facial nucleus, and in the other subdivisions of the sensory trigeminal complex (Jacquin et al., 1986; Jacquin et al., 1989; Veinante et al., 2000; Bellavance et al., 2017). As new results, profuse projections are also found in the ipsilateral dorsal medullary reticular (MdD) nucleus and further caudally in the dorsal horn of the cervical cord. An ascending projection terminates ipsilaterally in the intertrigeminal region (ITr) and in the KF/PBc. Whether individual interpolaris cells project to many or all of the above-mentioned target regions remains an open issue. What is clear, however, is that interpolaris cells that project to Po thalamus broadcast sensory messages to multiple midbrain and hindbrain regions.

Figure 1 with 1 supplement see all
Anatomical and electrophysiological evidence that vibrissa-responsive interpolaris cells have widespread axonal projections.

(A) Viral method used for labeling paralemniscal projections. (B) Labeling of interpolaris cells after injection of G-pseudotyped Lenti-Cre virus in Po thalamus, and a Cre-dependent AAV that expresses GFP in the vibrissa-responsive sector of SpVIr. Horizontal section. (C) Anterograde labeling of terminal fields in Po thalamus and zona incerta. Sagittal section. (D) Anterograde labeling in the KF/PBc. Horizontal section. (E) Anterograde labeling in the ITr. Horizontal section. (F) Anterograde labeling in the dorsal sector of the facial nucleus. Horizontal section. (G) Anterograde labeling in the MdD and cervical cord. Horizontal section. (H) Population peristimulus time histogram of spike discharges evoked in KF (22 cells) by air puff deflection of the vibrissae in the anesthetized rat. A representative response is shown in the insert. (I) Example of a vibrissa responsive KF cell labeled by juxtacellular delivery of Neurobiotin. Horizontal section. (J) Location of eight juxtacellularly labeled KF cells. Horizontal brainstem sections in (I) and (J) were counterstained for cytochrome oxidase. Horizontal section. (K) Spectral coherence of spontaneous discharges of KF cells with the respiratory cycle at the respiratory frequency; 1–3 Hz. Note that, in contrast with the respiratory units (blue dots), spontaneous discharges of vibrissa-responsive cells (red triangles) display low coherence with respiration. (L) Population peristimulus time histogram of spike discharges evoked in MdD (33 cells) by air puff deflection of the vibrissae. (M) Recording site in the MdD labeled by an iontophoretic injection of Chicago Sky Blue. This coronal section was counterstained for cytochrome oxidase and a negative image was generated. Coronal section. See Figure 1—figure supplement 1 for additional anatomical data. Abbreviations for all anatomy: 5n, root of the trigeminal motor nucleus; 5N, trigeminal motor nucleus; 5t, trigeminal tract; 7n, facial nerve tract; 7N, facial nucleus; Amb, ambiguus nucleus; APT, anterior pretectal nucleus; BSTL, bed nucleus of the stria terminalis; CeA, central amygdala; Cerv Cord, cervical cord; CM/PC, central medial/paracentral thalamic nuclei; CPu, caudate putamen; DR, dorsal raphe; EW, Edinger-Westphal; Hab, habenula; IML, intermedio-lateral column of the spinal cord; ITr, intertrigeminal region; KF, Kölliker-Fuse nucleus; KF/PBc, Kölliker-Fuse/parabrachial complex; mcp, middle cerebellar peduncle; MdD, dorsal part of the medullary reticular formation; MdV, ventral part of the medullary reticular formation; mt, mammillothalamic tract; NA, nucleus ammbiguus; NTS, nucleus of the solitary tract; opt, optic tract; PAG, periaqueductal gray; PB, parabrachial nuclei; PC, paracentral thalamic nucleus; PCRt, parvicellular reticular formation; PLH, posterior lateral hypothalamus; Po, posterior nuclear group of the thalamus; PrV, principal trigeminal nucleus; RN, red nucleus; Rt, reticular thalamic nucleus; s5, sensory root of the trigeminal nerve; SC, superior colliculus; scp, superior cerebellar peduncle; SpVC, caudalis division of the spinal trigeminal complex; Sol, nucleus of the solitary tract; SpVIc, caudal sector of the interpolaris trigeminal nucleus; SpVIr, rostral division of the interpolaris nucleus; TG, trigeminal ganglion; VLL, ventral nucleus of the lateral lemniscus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posterior medial nucleus; VPCc, parvicellular sector of the ventral posteromedial thalamic nucleus; VPPc, parvocellular part of the ventral posterior thalamic nucleus; VRG, ventral respiratory group; vsc, ventral spinocerebellar tract; ZIv, ventral division of zona incerta.

Injection of the virus AAV1-hSyn-eGFP-WPRE in the vibrissa-responsive sector of subnucleus SpVIr (three adult rats; Figure 1—figure supplement 1) yielded similar results to those above (Figure 1B, G). Yet anterograde labeling is now found in the cerebellum. This is consistent with prior studies, which reported that different populations of SpVI cells project to the thalamus and cerebellum (Steindler, 1985; Jacquin et al., 1986). Of note, in sagittal sections of the brainstem, the terminal field of interpolaris axons in the KF/PBc covers an extensive crescent-shaped territory at the rostral border of the PrV and trigeminal motor nuclei (Figure 1—figure supplement 1).

KF/PBc and MdD cells respond to vibrissa deflection

Interpolaris cells that give rise to the paralemniscal pathway are known to respond to vibrissa deflection (Veinante et al., 2000). Thus, one expects KF/PBc and MdD neurons to also respond to vibrissa deflection. We indeed found vibrissa-responsive neurons in the KF/PBc (latency: 8.0±1.2 ms; mean ± SD; 22 cells across 14 adult rats; Figure 1H K) and in the MdD nucleus (latency: 6.0±1.4 ms; 33 cells across three additional adult rats; Figure 1L and M) of ketamine/xylazine anesthetized animals. The location of recorded units was assessed by single-cell juxtacellular labeling in the KF/PBc (eight cells in 8 of the 14 rats), and by iontophoretic deposit of Chicago Sky Blue in the MdD nucleus (three rats). Like interpolaris cells, the receptive field of KF/PBc and MdD cells includes multiple vibrissae. In the KF/PBc, vibrissa-responsive cells are intermingled with cells that discharge in phase with the respiratory rhythm. Yet the spontaneous activity of vibrissa-responsive cells displays no statistically significant spectral coherence with respiration (mean coherence< C > in the spectral band of breathing is |< C >|=0.16 for vibrissa-responsive cells versus |< C >|=0.72 for respiratory units (Figure 1K)).

We did not investigate the parameters of vibrissa deflection that best drive KF/PBc and MdD cells. Our goal was to cross-check the tract-tracing results (Figure 1D), which indicate that some KF/PBc and MdD cells receive input from vibrissa-responsive interpolaris neurons.

Axonal projections of KF/PBc cells that receive interpolaris input s

Among the targets of the paralemniscal pathway, we focused on the KF/PBc because of the well-documented hodology of its axonal projections (see review by Saper and Stornetta, 2015). As a means to visualize the axonal projections of KF/PBc cells that receive interpolaris input, we injected AAV2/1 that expresses Cre in the vibrissa-responsive sector of subnucleus SpVI to achieve anterograde transsynaptic expression of Cre (Zingg et al., 2017), and AAV1-EF1a-DIO-hChR2-eYFP was injected in the KF/PBc (three adult rats; transsynaptically labeled cells amount to 55, 62, and 68 in each of the rats). We observed transsynaptic anterograde labeling in the lateral part of the facial nucleus, throughout the ventral lateral medulla, the MdD nucleus, the nucleus of the solitary tract, and the cervical cord in all animals (Figure 2A–D). Ascending projections were found in the dorsal raphe, the Edinger-Wesphal nucleus, the periaqueductal gray, the posterior lateral hypothalamus, the paracentral and central medial thalamic nuclei, the parvicellular part of the ventral posterior thalamic nucleus, the ventral division of zona incerta, the central amygdala (CeA), and the lateral part of the bed nucleus of stria terminalis in all animals (Figure 2E–G - Figure 2—figure supplement 1). In summary, KF/PBc cells that receive interpolaris input in turn project to most of the brainstem and forebrain regions previously identified by means of anterograde tracer injections in the KF/PBc (Saper and Stornetta, 2015; Figure 2H). Yet, it is worth noting that no anterograde labeling is observed in the trigeminal sensory nuclei. This negative result is of critical importance. It indicates that KF/PBc cells that receive interpolaris input do not feedback to the relay cells that give rise to the paralemniscal pathway (see below).

Figure 2 with 1 supplement see all
Axonal projections of KF/PBc cells that receive interpolaris input.

See Figure 1 for abbreviations. (A–C) AAV2/1-hSyn-Cre-WPRE was injected in the vibrissa-responsive sector of the SpVIr, and AAV2/1-EF1a-DIO-hChR2-eYFP was injected in the KF/PBc. This parasagittal section shows labeling in the KF/PBc. The section was immunostained for choline acetyltransferase (A). The green and red framed areas are enlarged in (B) and (C). Sagittal section. (D) Terminal labeling in the ventral respiratory group underneath the ambiguous nucleus. Sagittal section. (E) Anterograde labeling in the central amygdala. Coronal section. (F) Anterograde labeling in the paracentral and central medial thalamic nuclei. Coronal section. (G) Anterograde labeling in the parvocellular division of the ventral posterior medial nucleus of the thalamus (see Figure S2 for additional projection sites). Coronal section. (H) Summary of the first-order axonal projections of interpolaris cells and the second-order projections that derive from the KF/PBc. Projection sites in the framed area are from prior studies (see review by Saper and Stornetta, 2015). The classical paralemniscal pathway, that is, structures that receive input from SpVI, is in cyan. Magenta is the brainstem target of KF and green is the target of PG. MdD appears twice as it received input from both SpVI and the KF/PBc. We have added this to the legend.

As PB cells that project to the CeA receive input from vibrissa-responsive SpVI neurons, one expects to find vibrissa-evoked responses in the amygdala. We addressed this issue by recording the local field potential evoked by air jet deflection of the vibrissae (three adult rats; Figure 3A C). These experiments were carried out under urethane instead of ketamine/xylazine anesthesia as α2 agonists, for example, xylazine, strongly depress synaptic transmission in the parabrachial-amygdaloid pathway (Delaney et al., 2007). Vibrissa deflection evoked a clear negative field potential in the CeA (peak latency of 12 ms as an average over all three rats and 50 trials per rat; Figure 3A and B). This response was abolished in all animals following electrolytic lesion of the ipsilateral PB nucleus (Figure 3A and C). Taken together, our anatomical (Figure 2) and electrophysiological (Figure 3A–C) data delineate a pathway of vibrissa information that reaches the CeA and several limbic regions of the forebrain via a relay in the KF/PBc.

Figure 3 with 1 supplement see all
Vibrissa-evoked responses in central amygdala and anatomical evidence that separate cellular populations in the KF/PBc project to the CeA as compared to the facial nucleus.

See Figure 1 for abbreviations. (A) Average response (50 trials) evoked in the CeA by air puff stimulation of the vibrissae before (red trace) and after (black trace) an electrolytic lesion of the PB complex. (B) Recording site in the amygdala labeled by an iontophoretic injection of Chicago Sky Blue (cytochrome oxidase counterstaining). Coronal section. (C) Electrolytic lesion of the PB complex. Coronal section. (D) Injection site of retroAAV-CAG-eGFP in the CeA. Coronal section. (E) Injection site of retroAAV-CAG-mCherry in the facial nucleus. Sagittal section. (F) Retrogradely labeled cells in the KF/PBc. Sagittal section. (G) Sagittal view of a three-dimensional reconstruction showing the distribution of amygdala-projecting cells (green dots) in the medial and lateral PB, and facial-projecting neurons in the KF (magenta dots). Note that a few cells in lateral PB project to the facial nucleus; yet, none of the KF/PBc cells are doubly labeled. (H) Wiring diagram of the projections of KF and PB cells that receive vibrissa input from the interpolaris nucleus (see also Figure S3 for additional evidence).

Differential projections of KF and PB cells

Prior studies in mice have shown that separate pools of KF/PBc neurons project to the forebrain and to the lower brainstem (Geerling et al., 2017; Barik et al., 2018). We thus examined whether descending and ascending projections of the KF/PBc in rats arise from different or overlapping neurons. We made large injections, i.e., 100 nl, of the retrograde labels retroAAV-CAG-eGFP and retroAAV-CAG-mCherry in the CeA and the facial nucleus, respectively (two rats; Figure 3D–G). Although a number of retrogradely labeled cells were found in the KF/PBc, none was doubly labeled. In detail, amygdala-projecting cells (326 in rat 1 and 312 in rat 2) were concentrated in lateral and medial PB nucleus, while the majority of facial projecting cells (299 in rat 1 and 140 in rat 2) were located in KF nucleus. In complementary experiments, we injected retroAAV expressing Cre in the ventral respiratory group and AAV2/8-hSyn-DIO-eGFP in the KF/PBc (two rats). Anterograde labeling was observed in the facial nucleus, the ventral respiratory group, and the MdD nucleus (Figure 3—figure supplement 1), but not in midbrain and forebrain regions. Taken together, these results confirm that neighboring but separate populations of KF/PBc neurons project to the forebrain and to the lower brainstem (Figure 3H).

GABAergic KF cells gate sensory transmission in subnucleus SpVI

In rodents, KF projections to the brainstem arise from two neuronal populations: glutamatergic cells that project to the autonomic, respiratory, and motor regions of the medulla, and GABAergic neurons, which project principally to subnucleus SpVI (Geerling et al., 2017). To determine whether GABAergic KF cells inhibit interpolaris cells, we made large injections, i.e., 200 nl, of the transsynaptic label anteroAAV-expressing Cre (Zingg et al., 2017) in the KF/PBc and AAV2-Ef1a-DIO-eYFP in subnucleus SpVI (three rats; Figure 4A–D). Unexpectedly, few interpolaris neurons were labeled (44, 48, and 53 cells, respectively, in each of three rats). Anterograde labeling was observed in the KF/PBc and in the parvicellular part of the ventral posterior thalamic nucleus, a region known to process input from the tongue and oral cavity (Ogawa and Nomura, 1988; Verhagen et al., 2003), in all animals. No labeling was observed in the cerebellum, nor in brainstem and midbrain regions that receive input from the paralemniscal pathway (Figure 2H). Yet, cells in the parvicellular reticular formation (PCRt) at the medial border of the trigeminal nuclei were extensively labeled, which indicates that the paucity of cell labeling in subnucleus SpVI is hardly ascribable to a technical pitfall. Taken together, these results indicate that KF projections do not innervate the SpVI interneurons or the projection cells that give rise to the paralemniscal pathway.

Postsynaptic targets of KF projections to the SPVI.

See Figure 1 for abbreviations. (A) The viral method used for transsynaptic labeling of brainstem cells that receive input from the KF. Sagittal section. (B–D) Transsynaptic labeling in SpVI is restricted to a small population of cells (C) that project to VPCC (B). Yet, numerous cells are transsynaptically labeled in the parvicellular reticular formation (D). Panel (B) in a coronal section while (C) and (D) are horizontal sections. (E, F) Viral labeling method for optogenetic stimulation of KF axons. Injection of AAV2/1-hSyn-hChR2-eYFP in KF results in anterograde labeling in SpVI. The framed region in (E) is shown in (F); arrowheads point to labeled cell bodies. Note the dense network of axon collaterals within the KF/PBc. Sagittal section. (G) Representative responses of a monovibrissa-responsive cell (biphasic unit) and a multivibrissa-responsive cell (positive unit) upon optogenetic stimulation of KF axons labeled as in (E). Population peristimulus time histogram shows the responses of 26 monovibrissa-responsive units upon optogenetic stimulation of KF axons along with 10 multivibrissa-responsive units to optogenetic stimulation of KF axons. Only mono-vibrissa and not multi-vibrissa cells are responsive to KF input. (H) Summary of the effect of KF projections on the flow of sensory input through SpVI.

To further assess these anatomical results, we injected AAV-hSyn-ChR2-eYFP in the KF nucleus and used an optrode to concurrently stimulate KF axons and record the activity of SpVI neurons (three adult rats; Figure 4E–G). Interpolaris cells either discharged spontaneously or were driven by jiggling a single vibrissa with a piezoelectric stimulator. Optogenetic stimulation suppressed sensory-evoked discharges in monovibrissa-responsive cells (26 cells in the same three rats, t-test yields p<0.001; lower panel in Figure 4G) but did not affect the firing of multivibrissa-responsive units (32 cells in the same three rats, p=0.84; Figure 4G). Together with the lack of transsynaptic labeling of local circuit cells from the KF nucleus, these results raise the possibility that KF-induced suppression of sensory responses in local circuit interpolaris cells is mediated by presynaptic or extrasynaptic inhibition, as summarized in Figure 4H.

Discussion

Our study reveals a hitherto unsuspected diversity of axonal projections of interpolaris cells that give rise to the paralemniscal pathway. We identified two new collateral projections that target the MdD nucleus and the KF/PBc (Figures 1 and 2). The diagram in Figure 2H summarizes the first-order axonal projections of interpolaris cells that give rise to the paralemniscal pathway, and the second-order projections that derive from the KF/PBc. Furthermore, we show that the KF/PBc contains two populations of vibrissa-responsive neurons. One population gives rise to an ascending pathway, which projects to limbic regions of the forebrain, and the other one projects to the autonomic, respiratory, and motor regions of the medulla (Figure 3); the diagram in Figure 3H summarizes this circuit. Finally, the KF nucleus contains a pool of GABAergic neurons that do not receive interpolaris input, but project locally and to the trigeminal sensory nuclei (Figure 4); the diagram in Figure 4H summarizes this circuit.

Descending projection to the MdD

By far the most abundant collateral projection of vibrissa-responsive interpolaris cells is to the MdD nucleus and the dorsal horn of the cervical cord. The finding of vibrissa-responsive cells in the MdD nucleus is unexpected, as most studies reported that MdD neurons best respond to noxious stimuli (reviewed in Martins and Tavares, 2017; Figure 3A–C). Although there is no reason to believe that vibrissa deflection per se is painful, an unexpected air puff directed toward the head of a rat elicits fear-related behavior such as a startle response (Engelmann et al., 1996), 22 kHz ultrasonic vocalization (Knapp and Pohorecky, 1995), and avoidance behavior (Cimadevilla et al., 2001). As the MdD nucleus sends profuse projections to the cervical cord and the facial nucleus (Bernard et al., 1990; Takatoh et al., 2013; Takatoh et al., 2021), these results suggest that vibrissa messages to the MdD nucleus may elicit avoidance or flight behaviors. This conclusion is in line with that of the recent study, which reported that the MdD nucleus is part of a brainstem-spinal circuit to control escape responses to noxious stimuli (Barik et al., 2018).

The brainstem medullary reticular formation comprises two main divisions: a ventral part (MdV) and a dorsal part (MdD). The MDV nucleus stands out as specifically targeting subpopulations of forelimb-innervating motor neurons. Selective ablation or silencing experiments reveal that the MdV nucleus is critically important for skilled motor behaviors (Esposito et al., 2014). On the other hand, the MdD nucleus receives parabrachial inputs whose activation induces flight and escape behavior. Yet ‘Activation of PB(tac1) neurons does not produce nocifensive responses on its own but has a major effect on responses to sensory cues. It thus seems possible that peripheral inputs provide the context necessary to trigger a specific behavioral response’. (Barik et al., 2018). We propose that the relay of sensory signals in the paralemniscal pathway is normally gated, and that release from gating occurs in behavioral contexts that are alarming or threatening. A recent paper (Sun et al., 2020) lends support for this proposal by showing that GABAergic KF/PBc cells gate the relay of aversive stimuli to limbic forebrain regions.

The trigemino-parabrachial amygdaloid pathway

A number of studies have reported trigeminal projections to the KF and PB nuclei (Cechetto et al., 1985; Slugg and Light, 1994; Feil and Herbert, 1995; Dallel et al., 2004; Rodriguez et al., 2017; see also review by Chamberlin, 2004). Most of these studies focused on ascending projections from subnucleus caudalis, which convey pruriceptive and nociceptive inputs to the KF/PBc (Jansen and Giesler, 2015). Thus, it came as a surprise to find vibrissa-responsive neurons in KF/PBc, and further find that vibrissa signals are conveyed to the limbic regions of the forebrain via the medial and lateral PB. One study previously reported short-latency activation of amygdala neurons, at about 11 ms, upon electrical stimulation of the mystacial pad (Bernard et al., 1992). Such a short latency is consistent with the activation of a trigemino-parabrachial-amygdaloid pathway by Aβ primary vibrissa afferents.

The paralemniscal pathway and threat

Although vibrissa-responsive interpolaris cells do not respond to noxious stimuli, many of the regions they innervate contain cells that process nociceptive or aversive inputs. This is the case for the MdD nucleus (reviewed in Martins and Tavares, 2017), the KF/PBc (Jansen and Giesler, 2015), the superior colliculus (Dean et al., 1989; Redgrave et al., 1996), the zona incerta (Masri et al., 2009), and Po thalamus (Masri et al., 2009; Frangeul et al., 2014; Sobolewski et al., 2015). These anatomical data suggest that the paralemniscal pathway conveys signals from threatening or alarming sensory inputs, perhaps through association with aversive contexts.

In light of the evidence that interpolaris cells innervate brain regions that process nociceptive or aversive stimuli, we postulate that the KF nucleus is primarily involved in orchestrating defensive reactions to aversive and threatening stimuli. Three additional lines of evidence support this hypothesis. First, glutamatergic KF cells project to the cardio-respiratory medullary centers, the facial, hypoglossal, and ambiguous motor nuclei, the nucleus of the solitary tract, and to the preganglionic neurons of the sympathetic nervous systems (reviewed in Saper and Stornetta, 2015). Second, activation of KF cells elicits changes in respiration, heart rate, blood pressure (Chamberlin and Saper, 1992; Lara et al., 1994; Guo et al., 2002; Chamberlin, 2004). Third, individual KF neurons project to many of the above-mentioned targets by means of branching axons, which might form the anatomical substrate for coordinating autonomic and somatic reactions (Song et al., 2012).

Sensory gating in the paralemniscal pathway

A prior study in the alert head-restrained rat reported that units in trigeminal nucleus principalis fire more spikes when the animal is whisking as opposed to not whisking (Moore et al., 2015). Yet the same study shows that spike rates are not significantly different between whisking and not whisking in subnucleus SpVI. This is an unexpected result, as primary vibrissa afferents discharge during whisking (Severson et al., 2017) and SpVI projection cells are typically driven by many more vibrissae than principalis neurons (Furuta et al., 2006). The observation that whisking tends to reorganize spike times of neurons in interpolaris, rather than increase overall spike rates, supports the notion that the relay of vibrissa messages in the paralemniscal pathway is normally gated as part of fast, inhibitory feedback. The inhibition of the paralemniscal pathway by KF GABAergic neurons, together with the lack of a direct connection from KF neurons to interpolaris projections cells, suggests that proposed gating occurs by a mechanism of presynaptic or extrasynaptic inhibition (Figure 4H).

What drives the KF nucleus, consistent with the postulated involvement in orchestrating defensive reactions to aversive and threatening stimuli? The KF nucleus receives projections from different subdivisions of the periaqueductal gray, themselves downstream of inhibitory input from the amygdala (Tovote et al., 2016). The release of the inhibition from the amygdala to the periaqueductal gray may well lead to a defensive postures. It is of interest the release of inhibition provides a release from pain (Benarroch, 2012), also consistent with presynaptic inhibition of the trigeminus.

Conclusion

Our results clearly indicate that the paralemniscal pathway broadcasts vibrissa-based sensory signals to a number of brainstem and forebrain regions that are involved in the expression of emotional reactions. They provide a different viewpoint on the role of the Kölliker-Fuse nucleus, which is currently considered to control the mechanics of respiratory and cardiac rhythms, but no more. We propose that the Kölliker-Fuse nucleus is involved in the larger role of orchestrating the expression of emotional reactions, for example, facial expressions, autonomic, and somatic reactions, of which respiration is one of many players.

Materials and methods

Subjects

Experiments were carried out in Long Evans rats of both sexes and included five juvenile rats (P30–P35) and 36 adult rats (P80–P100; 250–350 g in mass). All experiments were carried out according to the National Institutes of Health Guidelines. All experiments were approved by the Institutional Animal Care and Use Committees at Laval University and at the University of California at San Diego.

Viruses

G-pseudotyped Lenti-Cre virus was designed and produced as described (Nelson et al., 2013). AAV2/1-EF1a-DIO-hChR2-EYFP-WPRE-HGHpA, AAV2/1-hSyn-Cre WPRE, AAV2/5-hSyn-eGFP-WPRE, retroAAV-CAG-tdTomato, retroAAV-CAG-eGFP, and AAV2/8-hSyn-DIO-eGFP were obtained from Addgene.

Surgery and virus injections

Request a detailed protocol

All but three rats were anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg), and body temperature was maintained at 37°C with a thermostatically controlled heating pad. Three rats were anesthetized with urethane (1.4 g/kg) to record the local field potential evoked in the amygdala by vibrissa deflection. All virus injections were carried out after electrophysiological identification of the target regions. When AAV injections were made in the interpolaris nucleus, we targeted the lateral most sector of the nucleus based on the well-known somatotopic representation of the vibrissae in this nucleus. This precaution proved useful to avoid infecting cells in neighboring regions. After 3–4 weeks of survival, based on the results of Stanek et al., 2016, animals were either perfused with saline and paraformaldehyde (4% (w/v) in PBS), or anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg) for optogenetic stimulation.

In three rats, the vibrissa-responsive sector of the KF/PBc was first located by electrophysiological recording and then lesioned by passing 0.5 mA through a stainless steel electrode with a tip (10 μm) de-insulated over a length of 500 μm.

Electrophysiology

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Single-unit recordings were carried out in the KF/PBc and MdD with micropipettes (tip diameter, 1 µm) filled with either 0.5 M potassium acetate and 2% (w/v) neurobiotin, or 0.5 M potassium acetate and 4% (w/v) Chicago Sky Blue. To maximize the chance of cell recovery after juxtacellular labeling in the KF/PBc, we injected one cell per animal and perfused the rat thereafter.

A hand-held probe was first used to identify the vibrissal receptive field of MdD and KF/PBc neurons. Then we used an air jet (1.5 s duration) to deflect the vibrissae in the rostro-caudal direction. Air jets were generated by a Picospitzer (General Valve, Brookshire, TX) connected to a pipette (tip diameter, 500 μm). The pipette was positioned at a distance of 4–5 cm of the vibrissae, in front of the animal, and the air jet was directed away from the rat’s face to avoid as much as possible stimulating other orofacial afferents. This created a cone-shaped air displacement that deflected six to seven whiskers. The delay between the command voltage and the actual motion of the vibrissae was measured by placing a piezoelectric film (Measurement Specialties, Fairfield, NJ) at the same distance from the tip of the pipette (Kleinfeld et al., 2002). This delay was subtracted from the recordings to build PSTHs of sensory-evoked responses (20 responses; bin width, 1 ms).

Respiration was monitored with a cantilevered piezoelectric film (LDT1 028K; Measurement Specialties) resting on the rat’s abdomen just caudal to the torso. All signals were sampled at 10 kHz and logged on a computer using the Labchart acquisition system (AD Instruments).

Optogenetics

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Three weeks after injection of AAV2/1-hSyn-hChR2-eYFP in the KF/PBc (three rats), we used a carbon-tip optrode (Kation Scientific) to simultaneously stimulate labeled axons and record interpolaris cells. As a test for KF-induced inhibition, we drove background spiking by jiggling a single vibrissa with a piezoelectric stimulator.

Histology

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Following perfusion brains were postfixed for 1 hr, and cryoprotected overnight in 30% (w/w) sucrose in PBS. Brains were cut at thickness of 50 µm on a freezing microtome. Labeled material was processed for either fluorescence or brightfield microscopy. For fluorescence microscopy, sections were immunoreacted with a chicken anti-GFP antibody (1:1000; Abcam), and a donkey anti-chicken Alexa 488 IgG (1:500; Jackson ImmunoResearch Labs). NeuN immunostaining was carried out with a mouse anti-NeuN antibody (1:1000; MilliporeSigma) and a goat anti-rabbit IgG conjugated to Alexa 594 (1:500; Abcam). For brightfield microscopy, sections were first counterstained for cytochrome oxidase (MilliporeSigma), and then immunoreacted with a rabbit anti-GFP antibody (1:1000; Novus Biological), a biotinylated horse anti-rabbit IgG (1:200; Vector Labs), the avidin/biotin complex (Vectastain ABC Kit; Vector Labs), and the SG peroxidase substrate (ImmPACT SG Substrate; Vector Labs). In three rats, brainstem sections were first immunoreacted with a goat anti-choline acetyltransferase antibody (1:1000; MilliporeSigma) and a rabbit anti-goat IgG conjugated to horseradish peroxidase (1:500; Abcam), which was revealed with diaminobenzidine (brown reaction product). Sections were then immunoreacted with a mouse anti-GFP antibody (1:2000; Abcam), a biotinylated goat anti-mouse IgG (1:200; Vector Labs), which was revealed with streptavidin horseradish peroxidase conjugate (Vector Labs) and the Ni-DAB substrate (black reaction product) (ImmPACT DAB Substrate; Vector Labs). Finally, the extent of electrolytic lesions was assessed on material stained with Neutral Red. Sections were scanned at a resolution of 0.5 µm/pixel (TissueScope LE; Huron Digital Pathology) and imported in Fiji or Photoshop for color and contrast adjustments.

Data analysis

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Three-dimensional maps of retrogradely labeled KF/PBc cells were constructed with the Neurolucida software (Microbrightfield).

Peristimulus time histograms were built using the LabChart 8.0 spike histogram module (AD Instruments), and the Matlab Chronux toolbox (http://www.chronux.org) was used to compute the spectral coherence between respiration and the firing rate of KF/PBc cells.

Data

‘Data sets (anatomical images and physiological time series) supporting this study, and an associated “read me” file, are deposited at Dryad, doi:10.6076/D15C7X.’.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-GFP (chicken
polyclonal)
AbcamCat#: ab139701:1000
AntibodyAnti-Chicken IgY
(IgG) (H+L)
(donkey polyclonal)(Alexa
Fluor 488)
Jackson ImmunoResearch
Labs
Cat#: 703-545-155; RRID: AB_23403751:500
AntibodyAnti-NeuN (mouse
monoclonal (A60))
MilliporeSigmaCat#: MAB3771:1000
AntibodyAnti-Rabbit IgG –
H&L (Goat polyclonal)(Alexa Fluor 594)
AbcamCat#: ab1500801:500
AntibodyAnti-GFP (rabbit
polyclonal)
Novus BiologicalsCat#: NB600-3081:1000
AntibodyAnti-rabbit IgG
(H+L) (horse)(Biotynilated)
Vector LaboratoriesCat#: BA-11001:200
AntibodyAnti-Choline-
acetyltransferase
(Goat polyclonal)
MilliporeSigmaCat#: SAB25002361:1000
AntibodyAnti-Goat IgG H&L
(Rabbit polyclonal)(Horseradish
peroxidase)
AbcamCat#: ab67411:500
AntibodyAnti-GFP (mouse
monoclonal)
AbcamCat#: ab12181:2000
AntibodyAnti-mouse IgG
(H+L) (Goat)(Biotynilated)
Vector LaboratoriesCat#: BA-92001:200
Recombinant DNA reagentG-pseudotyped-
Lenti-Cre
Fan Wang(MIT McGovern Institute)
Recombinant DNA reagentpAAV-hSyn-DIO-
eGFP (AAV8)
AddgeneAddgene ID: 50457Depositor: Bryan Roth
Recombinant DNA reagentpAAV-Ef1a-DIO-
hChR2-eYFP-
WPRE-HGHpA
(AAV1)
AddgeneAddgene ID: 20298Depositor: Karl
Deisseroth.New version of
this virus is pAAV-
EF1a-double floxed-
hChR2(H134R)-
EYFP-WPRE-HGHpA
Recombinant DNA reagentpENN.AAV.hSyn.
Cre.WPRE.hGH
(AAV1)
AddgeneAddgene ID:
105553UPenn ID: AV-
1-PV-2676
Depositor:
James
M. Wilson
Recombinant DNA reagentpAAV.hSyn.eGFP.WPRE.bGH (AAV5)AddgeneAddgene ID:
105539UPenn ID: AV-
5-PV-1696
Viral service
discontinued
Recombinant DNA reagentpAAV-CAG-tdTomato (AAV retrograde)AddgeneAddgene ID: 59462Depositor:
Edward Boyden
Recombinant DNA reagentpAAV-CAG-eGFP (AAV retrograde)AddgeneAddgene ID: 37825Depositor:
Edward Boyden
Peptide, recombinant proteinHorseradish Peroxidase Streptavidin
(HRP-
Streptavidin conjugate)
Vector
Laboratories
Cat#: SA-5704-100
Commercial assay or kitVectastain Elite ABC-HRP Kit, Peroxidase (Standard)Vector
Laboratories
Cat#: PK-6100
Commercial assay or kitImmPACT
SG Substrate, Peroxidase
(HRP)
Vector
Laboratories
Cat#: SK-4705
Commercial assay or kitImmPACT
DAB Substrate, Peroxidase
(HRP)
Vector
Laboratories
Cat#: SK-4105
Chemical compound, drugChicago Sky
Blue 6B
MilliporeSigmaCat#: C8679Powder
Chemical compound, drugNeutral RedMilliporeSigmaCat#: 861251
Software, algorithmNeurolucidaMBF BioscienceRRID:SCR_001775http://mbfbioscience.com/neurolucida
Software, algorithmLabChart 8.0
Spike Histogram Module
AD Instruments Data Acquisition
Systems for
Life Science
RRID:SCR_001620https://www.adinstruments.com/
Software, algorithmMatlab
Chronux
Toolbox
Chronux
(MATLAB)
RRID:SCR_005547http://chronux.org
OtherNeurobiotin
Tracer
Vector
Laboratories
Cat#: SP-1120
OtherCytochrome c oxidase
(from bovine
heart)
MilliporeSigmaCat#: C5499

Data availability

Datasets (anatomical images and physiological time series) supporting this study, and an associated "read me" file, are deposited at Dryad, https://doi.org/10.6076/D15C7X.

The following data sets were generated
    1. Kleinfeld D
    2. Deschenes M
    (2021) Dryad Digital Repository
    High resolution images and compilation of raw data.
    https://doi.org/10.5061/dryad.D15C7X

References

  1. Book
    1. Saper CB
    2. Stornetta RL
    (2015) Central autonomic nervous system
    In: Paxinos G, editors. The Rat Nervous System (4th ed). San Diego: Academic Press. pp. 369–388.
    https://doi.org/10.1016/C2009-0-02419-2
    1. Steindler DA
    (1985)
    Trigeminocerebellar, trigeminotectal, and trigeminothalamic projections: A double retrograde axonal tracing study in the mouse
    Journal of Comparative Neurology 237:155–175.

Decision letter

  1. Jesse H Goldberg
    Reviewing Editor; Cornell University, United States
  2. John R Huguenard
    Senior Editor; Stanford University School of Medicine, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "A vibrissa pathway that activates the limbic system" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by John Huguenard as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Please increase clarity over some details of the G-pseudotyped Lenti-cre virus

Can the authors expand on aspects of this G-pseudotyped Lenti-cre virus? e.g, in Figure 1, are the authors certain that only monosynaptic projections are labelled? Is there a possibility of secondary jumping? If there is a possibility of secondary jumping, how did the authors choose the survival time of the rats?

Relatedly, Stanek is referenced several times, but there's no entry in the bibliography. If it's the JNeurosci paper from Fan Wang, those authors did not use the term "G-pseudotyped", so I think the authors here need to fully explain what this means and how the virus works.

2) Paper framing and setup of the KF nucleus

While the starting point of this paper is about one pathway, the paper also includes substantial data related to an entirely different pathway and topic: namely, interpolaris and the Kolliker-Fuse nucleus. The background of these nuclei are only hinted at and only at the end. More to the topic, the authors do discuss a whisker-limbic pathway, which is interesting, but their data also suggest additional functions of these pathways other than avoidance and flight behaviors, which could be discussed. Similarly, the authors zero in on their results regarding KF GABA neurons, but never discuss what might even conceivably be driving these cells. Some rounding out of the concepts, both known and hypothetical, would really improve this paper. Separately, this is the first paper to ever study the physiological responses of several of these structures to whisker stimulation, but the manuscript says absolutely nothing about the nature of these stimuli (see point 3 below).

(2a) Intro, 1st paragraph, "…two main trigeminothalamic pathways". While there are anatomical connections between these structures, it is unclear the degree to which the paralemniscal connections serve as a main route for ascending signals and may have other functions. The idea of two parallel pathways is largely speculative and not based on functional studies. The caveat should be noted rather than presented as a consensus or textbook view. It is also not the focus of this study and somewhat distracting.

(2b) The second to last sentence of the Conclusion reflects one of the biggest take home messages of this paper: its implications for the function(s) of the Kolliker-Fuse nucleus. From my point of view, this is also a paper about SpVI-KF paper. I would encourage the authors to bring up the SpVI/KF nuclei earlier in the manuscript to explain their background to the naïve reader who is unlikely to be familiar with them and then in the Discussion better explain the implications.

(2c) pp.7-8 focus on a role of MdD and cervical cord innervation in avoidance or flight behaviors, which seems overly narrow. If the authors are serious about this suggestion, they should explain how this would work. Do the authors really think that certain kinds of whisker stimuli but not others could trigger flight? Which ones? This seems odd and not terribly ethological. Perhaps a more ethological scenario is that some of these subcortical targets are used for shaping on-going movements in response to whisker stimuli rather than triggering whole new behaviors. For instance, subcortical pathways are sufficient for mice to shape their limb movements as they jump over obstacles they detect with their whiskers (Warren and Sawtell 2021 eLife). A Po-MdD or Po-cervical cord pathway would be prime candidates for that kind of sensory-motor behavior and many others.

(3) Please increase clarity around sensory stimuli and responses

(3a) The authors mention on line 5 of page 5 that 'the receptive field of KF/PBc and MdD cells include multiple vibrissae,' but there is no mention of the receptive field size, or a panel showing how responses of KF/PBc and MdD cells change with stimulation of different whiskers. Could the authors clarify this point?

(3b) One of the nice aspects of this paper is that the experimenters physiologically checked for whisker-evoked responses in structures not previously examined by the field. However, there are absolutely no details about the parameters of the whisker stimulus whatsoever: the nature of movement (square? sine? sawtooth? ramp-and-hold?), the amplitude, the velocity, the duration. If repetitive, what is the frequency? On a related note, pp.7,22, "jiggling" is a colloquialism and not a standard word for describing the manipulation of the whiskers. "deflecting", "stimulating"? Did the authors use different sets of sensory stimuli (e.g., changes in magnitude, direction, or frequency of stimulus, etc.) while recording KF/PBc or MdD neurons to see whether KF/PBc or MdD responses in the same whisker change with parameters of the stimulus?Reviewer #1:

This work presents an advance over previous studies by providing anatomical evidence for a potential role of the paralemniscal pathway in responses to threat. High quality anatomical studies in these deep brainstem pathways are particularly important for identifying circuit architectures for high stakes sensorimotor processing, such as response to immediate threat.

Below are some issues that, if addressed, would strengthen this study and make it more suitable for publication:

(1) Could the authors provide motivation the use of juvenile rats for the tracing experiments in Figure 1, but adult rats for all other figures provided?

A previous study (Stanek et al., 2014 – eLife) performed similar circuit tracing experiments investigating the diversity of masseter and genioglossus premotor neurons in mice, and provided evidence for transient connections between DCN and XII motor neurons in P1 – P8, but not P8 – P15, age mice. Although Figure 1 – supplemental figure 1 suggests there may not be such differences between age groups, did the authors find any differences in connectivity between juvenile and adult rats?

(2) Can the authors expand on aspects of this G-pseudotyped Lenti-cre virus? e.g, in Figure 1, are the authors certain only monosynaptic projections are labelled? Is there a possibility of secondary jumping? If there is a possibility of secondary jumping, how did the authors choose the survival time of the rats?

(4) The authors mention on line 5 of page 5 that 'the receptive field of KF/PBc and MdD cells include multiple vibrissae,' but there is no mention of the receptive field size, or a panel showing how responses of KF/PBc and MdD cells change with stimulation of different whiskers. Could the authors clarify this point?

(5) Did the authors use different sets of sensory stimuli (e.g., changes in magnitude, direction, or frequency of stimulus, etc.) while recording KF/PBc or MdD neurons to see whether KF/PBc or MdD responses in the same whisker change with parameters of the stimulus?

The authors provide citations and propose that KF/PBc and MdD are involved in processing threatening or noxious stimuli, and projections from interpolaris cells to these regions may be involved in the expression of emotional reactions. The authors also provide evidence that unexpected air puffs directed towards the head of rats elicits fear-related behavior, which may be driven via the vibrassal system.

If interpolaris projections to KF/PBc or MdD are involved in the processing of noxious stimuli, how exactly is a valence, such as threatening or non-threatening, assigned to a particular whisker stimulus, but not others? This may be done through multiple mechanisms, but one possibility is that the magnitude, direction, or frequency of a stimulus may elicit different responses. For example, a rat may respond very differently to an airpuff that acts at a single whisker versus a gust of wind that acts on all.

(6) The projections from interpolaris cells to MdD are interesting. A recent study (Ruder et al., 2021 – Nature) found that optogenetic activation of MdD-projecting, but not MdV- or spinal cord-projecting, lateral rostral medulla (LatRM) neurons elicited grooming sequences or hand-to-mouth movements in mice. Although the authors of the current study focus on the role of MdD in fear and threat responses in their Discussion, one possibility is that the projections from SpVI to MdD may provide a short latency pathway to elicit a grooming sequence in response to potentially aversive stimuli on the whisker pad (e.g., dirt/mud on the whiskers). The authors may want to add a few sentences in their discussion regarding this point.

(7) While the authors' data suggest sensory gating in the paralemniscal pathway is by presynaptic inhibition, and provide further citations in the Discussion to strengthen this point, there is no discussion as to what this may mean for the animal's behavior or the computations performed in this circuit. Could the authors expand in the Discussion on what gating of the paralemniscal pathway might suggest? In other words, why might this pathway need to be gated, and what does this mean with respect to behavior (for example, the response to noxious/threatening stimuli)?

While the authors' findings suggest gating of the paralemniscal pathway is via presynaptic inhibition, could the authors expand on why gating via presynaptic inhibition from KF to interpolaris cells might be computationally advantageous in this circuit, relative to other mechanisms such as postsynaptic inhibition?Reviewer #2:

This Short Report from Elbaz and coauthors investigated the connectivity of one of the vibrissal brainstem nuclei (interpolaris, SpVI), thought by some to belong to a "paralemniscal" pathway from the sensory periphery to the cerebral cortex. Using sophisticated viral tracing methods, they show that individual interpolaris cells can diverge to target multiple midbrain and hindbrain structures, including MdD and the cervical spinal cord. They then show that Kolliker-Fuse neurons receiving interpolaris inputs project to numerous targets but do not feedback onto interpolaris. Physiological recordings from MdD, KF, and the central amygdala reveal responses to vibrissa deflection. Using optogenetics, they show that KF GABAergic interneurons, however, do project back to interpolaris and may gate sensory responses. They conclude that this circuitry may allow the whisker system to engage the limbic system.

The viral tracing methods are state-of-the-art, and the authors put them to good use not only confirming known interareal connections but also revealing novel ones. The physiology data are nice complements to the anatomical experiments, demonstrating that these connections can relay sensory signals and that several targets are vibrissa responsive. They also suggest that the KF GABA neurons may participate in a feedback inhibitory circuit or descending inhibitory circuit, rather than a feedforward one.

The main weaknesses of the paper are largely related to interpretation and presentation, which often leaves the reader confused. While the starting point of this paper is about one pathway, the paper is really about an entirely different pathway and topic: namely, interpolaris and the Kolliker-Fuse nucleus. The background of these nuclei are only hinted at and only at the end. More to the topic, the authors do discuss a whisker-limbic pathway, which is interesting, but their data also suggest additional functions of these pathways other than avoidance and flight behaviors, which could be discussed. Similarly, the authors zero in on their results regarding KF GABA neurons, but never discuss what might even conceivably be driving these cells. Some rounding out of the concepts, both known and hypothetical, would really improve this paper. Separately, this is the first paper to ever study the physiological responses of several of these structures to whisker stimulation, but the manuscript says absolutely nothing about the nature of these stimuli.

1. Intro, 1st paragraph, "…two main trigeminothalamic pathways". While there are anatomical connections between these structures, it is unclear the degree to which the paralemniscal connections serve as a main route for ascending signals and may have other functions. The idea of two parallel pathways is largely speculative and not based on functional studies. The caveat should be noted rather than presented as a consensus or textbook view. It is also not the focus of this study and somewhat distracting.

2. The second to last sentence of the Conclusion reflects one of the biggest take home messages of this paper: its implications for the function(s) of the Kolliker-Fuse nucleus. From my point of view, this is really an SpVI-KF paper, not a Po/paralemniscal pathway. I would encourage the authors to bring up the SpVI/KF nuclei earlier in the manuscript to explain their background to the naïve reader who is unlikely to be familiar with them and then in the Discussion better explain the implications.

3. One of the nice aspects of this paper is that the experimenters physiologically checked for whisker-evoked responses in structures not previously examined by the field. However, there are absolutely no details about the parameters of the whisker stimulus whatsoever: the nature of movement (square? sine? sawtooth? ramp-and-hold?), the amplitude, the velocity, the duration. If repetitive, what is the frequency? On a related note, pp.7,22, "jiggling" is a colloquialism and not a standard word for describing the manipulation of the whiskers. "deflecting", "stimulating"?

4. pp.7-8 focus on a role of MdD and cervical cord innervation in avoidance or flight behaviors, which seems overly narrow. If the authors are serious about this suggestion, they should explain how this would work. Do the authors really think that certain kinds of whisker stimuli but not others could trigger flight? Which ones? This seems odd and not terribly ethological. Perhaps a more ethological scenario is that some of these subcortical targets are used for shaping on-going movements in response to whisker stimuli rather than triggering whole new behaviors. For instance, subcortical pathways are sufficient for mice to shape their limb movements as they jump over obstacles they detect with their whiskers (Warren and Sawtell 2021 eLife). A Po-MdD or Po-cervical cord pathway would be prime candidates for that kind of sensory-motor behavior and many others.

5. Stanek is referenced several times, but there's no entry in the bibliography. If it's the JNeurosci paper from Fan Wang, those authors did not use the term "G-pseudotyped", so I think the authors here need to fully explain what this means and how the virus works.

6. What-either known or hypothetically-could activate the KF GABA neurons (e.g., shown in Figure 4H and discussed several times)? If state-dependent GABAergic gating is an idea the authors wish to put forward here, they could elaborate a bit more.

Reviewer #3:

The authors address the important problem of defining the functional role of the paralemniscal pathway for ascending somatosensory information. This major pathway's function remains relatively obscure, at least in comparison to the lemniscal pathway. The authors' focus is on the whisker system. Their main experimental objective therefore was to anatomically and physiologically map the collaterals of SpVi neurons that respond to whisker inputs and project to the PO thalamus. They use viral and conventional labeling methods together with electrophysiology in anesthetized rats.

Through difficult experiments, the authors show that these SpVi neurons have broad collateral projections to numerous midbrain and hindbrain targets, including to the dorsal medullary reticular nucleus (MdD) and the Kollicker-Fuse/parabrachial complex (KF/PB). They show that neurons in the MdD and KF/PB respond to whisker input at relatively short latency, implying drive from the projections from SpVi. They further trace projections emanating from the KF/PB neurons that receive input from SpVi, following the circuit downstream, and find that the areas previously known to receive KF/PB input do in fact receive such input from the SpVi-recipient neurons. Interestingly, they do not observe KF/PB feedback to the trigeminal nuclei. The authors show that the central amygdala responds to whisker input in rats, consistent with a projection from the PB and a possible role in mediating aversive responses to whisker input. The authors also find that KF/PB neurons are segregated into those projecting to forebrain and to lower brainstem targets. Finally, the authors provide physiological and anatomical evidence that KF neurons can gate sensory input flowing through the SpVi nucleus via a presynaptic or extrasynaptic mechanism.

The experiments and analyses are carefully performed and the overall quality of evidence for the authors' conclusions is strong. This is a substantial set of observations of value to the field. The authors succeed in their objective of mapping functional pathways originating from the SpVi neurons of the paralemniscal pathway. In so doing they shed light on the function of the paralemniscal pathway.

It is sometimes not clear how many rats the data shown in figures comes from, leading to a bit of a mismatch between the text, which describes findings across a population of rats, and the figures.

https://doi.org/10.7554/eLife.72096.sa1

Author response

Essential revisions:

1) Please increase clarity over some details of the G-pseudotyped Lenti-cre virus

Can the authors expand on aspects of this G-pseudotyped Lenti-cre virus? e.g, in Figure 1, are the authors certain that only monosynaptic projections are labelled? Is there a possibility of secondary jumping? If there is a possibility of secondary jumping, how did the authors choose the survival time of the rats?

The pseudotyped retrograde lenti virus-cre was devised and supplied by our colleague and collaborator Prof. Fan Wang (MIT). It was first described in Nelson et al., (2013).

Nelson A, Schneider DM, Takatoh J, Sakurai K, Wang F, Mooney R. 2013. A circuit for motor cortical modulation of auditory cortical activity. J. Neurosci. 33:14342-14353.

Lenti virus are enveloped viruses. Viral fusion glycoproteins (FuG proteins) are essential for enveloped virus infection as these proteins mediate fusion between the virus envelope and host cellular membrane to release the viral genome into cells. The G protein is not replicated upon viral infection. Thus the virus cannot jump across synapses. As a practical issue, we did not observe cell body labeling outside the interpolaris nucleus. Survival time was based on prior studies using this virus (Stanek et al., 2016).

Stanek, E., Rodriguez, E., Zhao, S., Bao-Xia Han, B.-X., and Wang, F. (2016) Supratrigeminal bilaterally projecting neurons maintain basal tone and enable bilateral phasic activation of jaw-closing muscles. J. Neurosci. 36, 7663-7675.

To cross-check results obtained with G-pseudotyped lentivirus we injected the anterograde virus AAV1-hSyn-eGFP-WPRE in the vibrissa-responsive sector of the SpVIr (3 adult rats). We found similar results.

Relatedly, Stanek is referenced several times, but there's no entry in the bibliography. If it's the JNeurosci paper from Fan Wang, those authors did not use the term "G-pseudotyped", so I think the authors here need to fully explain what this means and how the virus works.

Thank you for noting this error. We now include Nelson et al., 2013 and Stanek et al., (2015) as references.

2) Paper framing and setup of the KF nucleus

While the starting point of this paper is about one pathway, the paper also includes substantial data related to an entirely different pathway and topic: namely, interpolaris and the Kolliker-Fuse nucleus. The background of these nuclei are only hinted at and only at the end.

We agree with the reviewer that the studies on the Kölliker-Fuse/parabrachial complex constitute an important and novel part of our findings. We now motivate these studies in the Introduction with the text.

"with a focus on pathways that include the Kölliker-Fuse/parabrachial complex (KF/PBc). The KF/PBc were previously described primarily in terms of their role in eliciting changes in respiration and cardiac function (reviewed in Saper and Stornetta, 2015). Yet the role of inhallation in either driving or pacing whisking (Deschênes et al., 2012; Moore et al., 2013) provides motivation to investigate if KF/PBc has a broader role in orofacial motor actions. Further motivation comes from the projections of the KF to the trigeminal sensory nuclei (Geerling et al., 2017), which contains multiple pathways for the control of motoneurons involved in whisking (Bellavance et al., 2017). Lastly, we searched for feedback projections from targets along the paralemnical pathways to brainstem nuclei as a means to uncover a potential role of this pathway in rat’s behavior.”

More to the topic, the authors do discuss a whisker-limbic pathway, which is interesting, but their data also suggest additional functions of these pathways other than avoidance and flight behaviors, which could be discussed. Similarly, the authors zero in on their results regarding KF GABA neurons, but never discuss what might even conceivably be driving these cells.

We now add material to the Discussion relevant to autonomic "avoidance and flight behaviors" and consider projections from different subdivisions of the periaqueductal gray that themselves are downstream of inhibitory input from the amygdala (Tovote et al., 2016). If is important to note that driving KF by release of inhibition from amygdala provides release from pain as well as defensive postures (Benarroch 2012). Both possibilities are consistent with presynaptic inhibition of the trigeminus following disinhibition, i.e., turning off, to the amygdala.

The new text is

“What drives the Kölliker-Fuse, consistent with the postulated involvement in orchestrating defensive reactions to aversive and threatening stimuli? Kölliker-Fuse receives projections from different subdivisions of the periaqueductal gray, themselves downstream of inhibitory input from the amygdala (Tovote et al., 2016). The release of the inhibition from the amygdala to the periaqueductal gray may well lead to a defensive postures. It is of interest the release of inhibition provides a release from pain (Benarroch 2012), also consistent with presynaptic inhibition of the trigeminus.”

Tovote, P., Esposito, M.S., Botta, P., Chaudun, F., Fadok, J.P., Markovic, M., Wolff, S.B.E., Ramakrishnan, C., Fenno, L., Deisseroth, K., Herry, C., Arber, S., Lüthi, A. (2016) Midbrain circuits for defensive behaviour. Nature 534, 206-216.

Benarroch, E. E. (2012) Periaqueductal gray: An interface for behavioral control. Neurology 78, https://doi.org/10.1212/WNL.0b013e31823fcdee

Some rounding out of the concepts, both known and hypothetical, would really improve this paper. Separately, this is the first paper to ever study the physiological responses of several of these structures to whisker stimulation, but the manuscript says absolutely nothing about the nature of these stimuli (see point 3 below).

We have attempted to "round out the concepts" as noted above. We apologize for not including details of stimulation parameters, etc., and have added them to the Methods as noted below.

(2a) Intro, 1st paragraph, "…two main trigeminothalamic pathways". While there are anatomical connections between these structures, it is unclear the degree to which the paralemniscal connections serve as a main route for ascending signals and may have other functions. The idea of two parallel pathways is largely speculative and not based on functional studies. The caveat should be noted rather than presented as a consensus or textbook view. It is also not the focus of this study and somewhat distracting.

The idea of two parallel pathways is not based on functional studies. We state in the first paragraph of the Introduction that the anatomical evidence, based on tract tracing, is unequivocal for two pathways. We then address the unresolved issue of function, as requested by the reviewer, in chapter three of the Introduction.

That the paralemniscal pathway exerts other function(s) than coding for stimuli parameters is precisely the point of our paper, and we appreciate the reviewer's enthusiasm for this point. We originally wrote:

"While the lemniscal pathway conveys tactile information, as well as information about the relative phase of the vibrissae in the whisk cycle (Yu et al., 2006; Curtis and Kleinfeld, 2009; Khatri et al., 2010; Moore et al., 2015; Isett and Feldman, 2020), the role of the paralemniscal pathway remains puzzling. It was proposed that this pathway conveys information about whisking kinematics (Yu et al., 2006; Golomb et al., 2003), but later studies found that encoding of whisking along the paralemniscal pathway is relatively poor (Moore et al., 2015; Urbain et al., 2015). It was also proposed that the paralemniscal pathway is specifically activated upon noxious stimulation (Masri et al., 2009; Frangeul et al., 2014), but it has never been shown that interpolaris cells that respond to vibrissa deflection are also activated by noxious stimuli. Thus the general function of the paralemnical pathway remains unresolved."

We conclude by showing that the paralemniscal pathway carries both ascending and descending signals to brain regions that are involved in the expression of emotional reactions; KF/PB in particular; and the MdD; please see papers by Barik et al., (2018) and Palmiter (2018).

Barik et al., (2018) A brainstem-spinal circuit controlling nocifensive behavior. Neuron 100, 14911503.

Palmiter RD (2018) The parabrachial nucleus: CGRP neurons function as a general alarm. Trends in Neurosci. 41, 280-293.

(2b) The second to last sentence of the Conclusion reflects one of the biggest take home messages of this paper: its implications for the function(s) of the Kolliker-Fuse nucleus. From my point of view, this is also a paper about SpVI-KF paper. I would encourage the authors to bring up the SpVI/KF nuclei earlier in the manuscript to explain their background to the naïve reader who is unlikely to be familiar with them and then in the Discussion better explain the implications.

We agree and, as noted – we have added to the Introduction and Discussion as noted above.

(2c) pp.7-8 focus on a role of MdD and cervical cord innervation in avoidance or flight behaviors, which seems overly narrow. If the authors are serious about this suggestion, they should explain how this would work. Do the authors really think that certain kinds of whisker stimuli but not others could trigger flight? Which ones? This seems odd and not terribly ethological.

We expand on this point in response to the reviewer. The brainstem medullary reticular formation ventral part (MdV – not MdD) stands out as specifically targeting subpopulations of forelimbinnervating motor neuronsWe do not suggest that certain kinds of whisker stimuli trigger flight. We have added a section to the Discussion on "Descending Projection to the MdD".

The brainstem medullary reticular formation comprises two main divisions: a ventral part (MdV) and a dorsal part (MdD). The MDV stands out as specifically targeting subpopulations of forelimb-innervating motor neurons. Selective ablation or silencing experiments reveal that MdV, is critically important for skilled motor behaviors (Esposito et al., 2014). On the other hand the MdD receives parabrachial inputs whose activation induces flight and escape behavior. Yet "Activation of PB(tac1) neurons does not produce nocifensive responses on its own but has a major effect on responses to sensory cues. It thus seems possible that peripheral inputs provide the context necessary to trigger a specific behavioral response." (Barik et al., 2018). We propose that the relay of sensory signals in the paralemniscal pathway is normally gated, and that release from gating occurs in behavioral contexts that are alarming or threatening. A recent paper (Sun et al., 2020) lends support for this proposal by showing that GABAergic KF/PBc cells gate the relay of aversive stimuli to limbic forebrain regions.

Barik, A., Thompson, J.H., Seltzer, M., Ghitani, N., and Chesler, A.T. (2018). A brainstem-spinal circuit controlling nocifensive behavior. Neuron 100, 1491-1503.

Esposito, M.S., Capelli, P. and Arber, S. (2014) Brainstem nucleus MdV mediates skilled forelimb motor tasks. Nature 508: 351-356.

Sun, L., Liu, R., Guo, F. Wen, W.-q., Ma, X.-l., Li, K.-y., Sun, H. Xu, C.-l., Li, Y.-y.. Wu, M.-y., Zhu, Z-g., Li, X.-j.,Yu, Y.-q., Chen, Z., Li, X.-y., and Duan, S. (2020) Parabrachial nucleus circuit governs neuropathic. Nat. Comm. 11, e5974.

Perhaps a more ethological scenario is that some of these subcortical targets are used for shaping on-going movements in response to whisker stimuli rather than triggering whole new behaviors. For instance, subcortical pathways are sufficient for mice to shape their limb movements as they jump over obstacles they detect with their whiskers (Warren and Sawtell 2021 eLife). A Po-MdD or Po-cervical cord pathway would be prime candidates for that kind of sensory-motor behavior and many others.

We thank the reviewer for her/his suggestion. Feedback to SpVI would modulate the pathway from SpVI to the facial motor nucleus that control the contact time within a whisk cycle (Sachdev et al., 2003; Deutsch et al., 2012). However, we feel that discussion of this possibility and other potentially ethological issues are outside the scope of the current work.

Sachdev, R.H.S., Berg, R.W., Chompney, G., Kleinfeld, G. and Ebner, F.F. (2003) Somatosensory and Motor Research 20:162-169.

Deutsch, D., Pietr, M., Knutsen, P.M., Ahissar, E., and Schneidman, E. (2012). Fast feedback in active sensing: Touch-induced changes to whisker-object interaction. Public Library of Science ONE 7, e44272.

(3) Please increase clarity around sensory stimuli and responses

(3a) The authors mention on line 5 of page 5 that 'the receptive field of KF/PBc and MdD cells include multiple vibrissae,' but there is no mention of the receptive field size, or a panel showing how responses of KF/PBc and MdD cells change with stimulation of different whiskers. Could the authors clarify this point?

We did not investigate the parameters of vibrissa deflection that best drive KF/PBc and MdD cells. Our goal was to cross-check the tract tracing results which indicate that some KF/PBc and MdD cells receive input from vibrissa-responsive interpolaris neurons.

(3b) One of the nice aspects of this paper is that the experimenters physiologically checked for whisker-evoked responses in structures not previously examined by the field. However, there are absolutely no details about the parameters of the whisker stimulus whatsoever: the nature of movement (square? sine? sawtooth? ramp-and-hold?), the amplitude, the velocity, the duration. If repetitive, what is the frequency?

We added this paragraph to the Methods section; the method was previously used in Kleinfeld et al., (2002).

“A hand-held probe was first used to identify the vibrissal receptive field of MdD and KF/PB neurons. Then we used an air jet (1.5 s duration) to deflect the vibrissae in the rostro-caudal direction. Air jets were generated by a Picospitzer (General Valve, Brookshire, TX) connected to a pipette (tip diameter, 500 μm). The pipette was positioned at a distance of 4 – 5 cm of the vibrissae, in front of the animal, and the air jet was directed away from the rat's face to avoid as much as possible stimulating other orofacial afferents. This created a cone-shaped air displacement that deflected six to seven whiskers. The delay between the command voltage and the actual motion of the vibrissae was measured by placing a piezoelectric film (Measurement Specialties, Fairfield, NJ) at the same distance from the tip of the pipette (Kleinfeld et a;. 2002). This delay was subtracted from the recordings to build PSTHs of sensory-evoked responses (20 responses; bin width, 1 ms).”

Kleinfeld, D., Sachdev, R.N.S., Merchant, L.M., Jarvis, M.R., and Ebner, F.F. (2002). Adaptive filtering of vibrissa input in motor cortex of rat. Neuron 34, 1021-1034.

On a related note, pp.7,22, "jiggling" is a colloquialism and not a standard word for describing the manipulation of the whiskers. "deflecting", "stimulating"? Did the authors use different sets of sensory stimuli (e.g., changes in magnitude, direction, or frequency of stimulus, etc.) while recording KF/PBc or MdD neurons to see whether KF/PBc or MdD responses in the same whisker change with parameters of the stimulus?

We did not investigate the parameters of vibrissa deflection that best drive KF/PB and MdD cells. Here our goal was to cross-check the tract tracing results which indicate that some KF/PB and MdD cells receive input from vibrissa-responsive interpolaris neurons.

https://doi.org/10.7554/eLife.72096.sa2

Article and author information

Author details

  1. Michaël Elbaz

    CERVO Research Center, Laval University, Québec City, Canada
    Contribution
    Investigation
    Contributed equally with
    Amalia Callado Perez
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6223-7359
  2. Amalia Callado Perez

    1. CERVO Research Center, Laval University, Québec City, Canada
    2. Department of Physics, University of California, San Diego, San Diego, United States
    Contribution
    Investigation
    Contributed equally with
    Michaël Elbaz
    Competing interests
    No competing interests declared
  3. Maxime Demers

    CERVO Research Center, Laval University, Québec City, Canada
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  4. Shengli Zhao

    Department of Neurobiology, Duke University Medical Center, Durham, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  5. Conrad Foo

    Department of Physics, University of California, San Diego, San Diego, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  6. David Kleinfeld

    1. Department of Physics, University of California, San Diego, San Diego, United States
    2. Section of Neurobiology, University of California, San Diego, San Diego, United States
    Contribution
    Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing
    For correspondence
    dk@physics.ucsd.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9797-4722
  7. Martin Deschenes

    CERVO Research Center, Laval University, Québec City, Canada
    Contribution
    Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review and editing
    For correspondence
    martin.deschenes@fmed.ulaval.ca
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5385-6633

Funding

National Institutes of Health (NS107466)

  • Martin Deschenes
  • David Kleinfeld

National Institutes of Health (NS097265)

  • David Kleinfeld
  • Martin Deschenes

Canadian Institutes of Health Research (MT-5877)

  • Martin Deschenes

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank Fan Wang for critical discussions and for supplying the retrograde-Lenti-Cre virus. This study was supported by grants from the Canadian Institutes of Health Research (Grant MT-5877) and the National Institutes of Health (U19 NS107466 and R35 NS097265).

Ethics

All experiments were carried out according to the National Institutes of Health Guidelines. All experiments were approved by the Institutional Animal Care and Use Committees at Laval University (protocol number: VRR-18-068) and at the University of California at San Diego (protocol numbers S02173M and S03174).

Senior Editor

  1. John R Huguenard, Stanford University School of Medicine, United States

Reviewing Editor

  1. Jesse H Goldberg, Cornell University, United States

Publication history

  1. Received: July 10, 2021
  2. Preprint posted: August 4, 2021 (view preprint)
  3. Accepted: January 24, 2022
  4. Version of Record published: February 10, 2022 (version 1)

Copyright

© 2022, Elbaz et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Michaël Elbaz
  2. Amalia Callado Perez
  3. Maxime Demers
  4. Shengli Zhao
  5. Conrad Foo
  6. David Kleinfeld
  7. Martin Deschenes
(2022)
A vibrissa pathway that activates the limbic system
eLife 11:e72096.
https://doi.org/10.7554/eLife.72096

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