Elsevier

Neuroscience

Volume 494, 1 July 2022, Pages 140-151
Neuroscience

Research Article
Layers 3 and 4 Neurons of the Bilateral Whisker-Barrel Cortex

https://doi.org/10.1016/j.neuroscience.2022.05.018Get rights and content

Highlights

  • Bilateral whisker representations in the somatosensory thalamus and cortex.

  • Smaller barrels and more focalized, simpler dendritic fields.

  • Bilateral whisker stimulation activates neighboring groups of layer 3 neurons.

Abstract

In Robo3R3-5cKO mouse brain, rhombomere 3-derived trigeminal principal nucleus (PrV) neurons project bilaterally to the somatosensory thalamus. As a consequence, whisker-specific neural modules (barreloids and barrels) representing whiskers on both sides of the face develop in the sensory thalamus and the primary somatosensory cortex. We examined the morphological complexity of layer 4 barrel cells, their postsynaptic partners in layer 3, and functional specificity of layer 3 pyramidal cells.

Layer 4 spiny stellate cells form much smaller barrels and their dendritic fields are more focalized and less complex compared to controls, while layer 3 pyramidal cells did not show notable differences. Using in vivo 2-photon imaging of a genetically encoded fluorescent [Ca2+] sensor, we visualized neural activity in the normal and Robo3R3-5cKO barrel cortex in response to ipsi- and contralateral single whisker stimulation. Layer 3 neurons in control animals responded only to their contralateral whiskers, while in the mutant cortex layer 3 pyramidal neurons showed both ipsi- and contralateral whisker responses. These results indicate that bilateral whisker map inputs stimulate different but neighboring groups of layer 3 neurons which normally relay contralateral whisker-specific information to other cortical areas.

Introduction

An essential feature of the sensory systems is the formation of a neural map of the sensory periphery with disproportionately larger areas devoted to areas with high density of sensory receptors in the periphery. A prominent example is the “whisker-barrel” tactile sensory system of nocturnal rodents. The precise array of the whiskers on the snout is represented with topographic precision first in the brainstem, next in the contralateral somatosensory thalamus and finally in the primary somatosensory cortex (reviewed in Erzurumlu et al., 2010, Erzurumlu and Gaspar, 2012, Iwasato and Erzurumlu, 2018). These maps are relayed sequentially from the periphery to the brainstem and all the way to the neocortex during early postnatal development (Erzurumlu and Gaspar, 2012, Erzurumlu and Gaspar, 2020). During this process, several axon guidance molecules direct correct targeting, and midline crossing of trigeminal lemniscal axons between the brainstem and the thalamus (Iwasato and Erzurumlu, 2018). Defects or absence of such guidance cues result in abnormal sensory maps in the brain. Unlike the visual and auditory sensory systems, the tactile somatosensory system is a mostly crossed pathway similar to the main motor pathway, the corticospinal tract, in mammals. The right primary sensory cortex thus receives information from the left side of the body and the left primary sensory cortex from the right side of the body.

Developmental regulation of netrin and slit proteins and attracting/repulsing receptors (e.g., Deleted in Colorectal Cancer -DCC-, Roundabout -Robo-) on the axonal growth cones play an essential role in contralateralization of sensory and motor pathways (Chédotal, 2014, Kennedy et al., 1994, Kidd et al., 1998). Genetic and developmental defects in midline crossing have serious consequences, such as uncrossed or partially crossed descending motor tracts resulting in involuntary movements of the hands and forearm on one side of the body that mirror intentional movements on the opposite side (Méneret et al., 2015). Horizontal gaze palsy with progressive scoliosis (HGPPS), a human condition that results from mutations in the ROBO3 gene affecting hindbrain axon midline crossing in sensory-motor circuits (Jen et al., 2004). Altered nociceptive topognosis occurs in mice and in humans due to mutations in midline crossing gene DCC (da Silva et al., 2018). Humans with this mutation experience bilateral pain sensations following unilateral noxious stimulation (da Silva et al., 2018).

A notable fraction of trigeminal principal sensory (PrV) nucleus neurons are differentiated from rhombomere 3 (R3). In a conditional mouse mutant (the Robo3R3-5cKO mouse), these PrV neurons project ipsilaterally rather than contralaterally to the ventroposteromedial (VPM) nucleus of the thalamus (Renier et al., 2017). This bilateral trigeminal projection creates a nonoverlapping or nonconvergent map of the whiskers from both sides of the snout. The bilateral whisker “barreloid” map in the thalamus is then conveyed to the primary somatosensory cortex, which normally has a precise map of the distribution of whiskers on the contralateral snout (the barrels), resulting in an ipsilateral whisker distribution map nestled between the dorsal and ventral whisker representation barrels of the contralateral side (Fig. 1). While the areal boundaries and the size of the primary somatosensory and even the whisker barrel area do not change, the whisker barrel area has twice the number of barrels, corresponding to the ipsilateral and contralateral whiskers (Renier et al., 2017). Consequently, the individual barrel sizes are smaller (Fig. 1).

In this study, we investigated the morphology and dendritic complexities of layer 4 spiny stellate cells, which form the barrels, and their primary targets the layer 3 pyramidal cells located superficial to them. These layer 3 pyramidal cells convey whisker sensory information to the motor and association areas of the same hemisphere and via the corpus callosum to the opposite hemisphere. We found that layer 4 spiny stellate neurons arranged into smaller barrels, showed more focalized dendritic orientation towards the barrel hollows and showed altered dendritic complexity. In contrast, layer 3 pyramidal neurons did not show notable changes in their dendritic complexity.

Next, we identified the ipsi- versus contralateral whisker barrels with whisker stimulation and intrinsic optical signal (IOS) imaging. We then used two-photon calcium imaging to investigate whether layer 3 pyramidal neurons receive unilateral whisker inputs or information from both contra- and ipsilateral whiskers. We identified numerous layer 3 pyramidal cells responding to ipsi- or contralateral whiskers. These results indicate that wiring and patterning of cortical circuits, at least in a primary sensory cortical region, are dependent on thalamocortical inputs and peripheral activity-dependent mechanisms.

Section snippets

Animal breeding

We bred Robo3 conditional knockout mice (Robo3R3–5cKO) in our colony at the University of Maryland Baltimore, School of Medicine, in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited laboratory facility. We obtained the original breeding pairs from Dr. A. Chédotal, Institut de la Vision, Paris, France (Renier et al., 2017, Tsytsarev et al., 2017). We previously detailed the generation of this mutant mouse line and whisker-related patterns, bilateral whisker

Bilateral whisker-barrel fields

Whisker barrels are more numerous (almost double the normal) in the Robo3R3–5cKO S1 cortex (Fig. 1). Previously, Renier et al., (2017) measured the number and size of barrels in this conditional knockout mouse line. They found that there were ∼35% more large barrels (corresponding to the five rows of large whiskers) arranged in 8–10 rows rather than the usual five rows but their sizes were reduced in mutants, compared with controls. Our current results are in agreement (Fig. 1), average of

Discussion

The barrels comprise distinct patches of TCAs corresponding to single whiskers on the contralateral face (with preserved spatial organization), and a gathering of layer 4 spiny stellate cell bodies that encircle these patches and orient their dendrites towards these patches. Such asymmetric polarization of stellate cells around barrel walls has been noted since the original studies of T. Woolsey and H. van der Loos in the nineteen seventies (Woolsey and Van der Loos, 1970, Erzurumlu and Gaspar,

Acknowledgement

We thank Dr. S. M. Bentzen and Ms. S. Holt, SOM. U. Maryland, for statistics consultations.

Authorship contributions

RSE and DHO designed the experiments, analyzed results, wrote the paper. VT and SEK performed the in vivo imaging experiments, analyzed data, and participated in writing the paper. SZ performed immunohistochemical and histological staining, CP performed and analyzed morphological data and helped write the paper.

Funding

Research supported by NINDS R01NS84818 (RE) and R01NS089652 (DHO).

Competing interest

The authors declare no disclosure of any financial or ethical interests.

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  • Cited by (0)

    The authors contributed equally to this work.

    Present address: Department of Molecular, Cellular and Developmental Biology University of Michigan, 1105 N. University Ave, Ann Arbor, MI 48108, United States.

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