2024.01.22.576584v1.full-html.html

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Abstract

Vasoactive intestinal polypeptide (VIP) is known to be present in a subclass of cortical

interneurons that are involved in the disinhibition of excitatory pyramidal neurons. Here, using

three different antibodies, we demonstrate that VIP is also present in the giant layer 5 pyramidal

(Betz) neurons which are characteristic of the limb and axial representations of the marmoset

primary motor cortex (cytoarchitectural area 4ab). No VIP staining was observed in smaller

layer 5 pyramidal cells present in the primary motor facial representation (cytoarchitectural

area 4c), or in premotor cortex (e.g. the caudal subdivision of the dorsal premotor cortex,

A6DC), indicating the selective expression of VIP in Betz cells. The most intense VIP staining

occurred in the largest Betz cells, located in the medial part of A4ab. VIP in Betz cells was

colocalized with neuronal specific marker (NeuN) and a calcium-binding protein parvalbumin

(PV). PV also intensely labelled axon terminals surrounding Betz cell somata. Whereas Betz

cells bodies were located in layer 5, VIP-positive (VIP

) interneurons were more abundant in

the superficial cortical layers. They constituted about 5-7% of total cortical neuronal

population, with the highest density observed in area 4c. Our results demonstrate the expression

of VIP in the largest excitatory neurons of the primate cortex, which may offer new functional

insights into the role of VIP in the brain, and also provide opportunities for genetic

manipulation of Betz cells.

Keywords:

Vasoactive intestinal polypeptide, Layer 5 pyramidal cells, Betz cells, Primary

motor cortex, inhibitory neurons, Marmoset

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1. INTRODUCTION

Similar to other primates (e.g. Vogt and Vogt, 1919; Matelli et al., 1985; Watanabe-Sawaguchi

et al., 1991) in the marmoset monkey the primary motor cortex (M1, Brodmann Area 4) is

formed by distinct cytoarchitectural subdivisions. According to the contemporary view the

representations of the limb and body axial musculatures are encompassed within

cytoarchitectural area 4ab, located medially, whereas the representations of the head

musculature (including facial and oral) are located laterally, in cytoarchitectural area 4c

(Burman et al., 2008: Paxinos et al., 2012). In particular, A4ab contains the unique

“gigantopyramidal”

neurons, known as Betz cells in primates (Bakken et al., 2021; Betz, 1874;

Jacobs et al., 2018). In contrast, in A4c layer 5 is populated by smaller pyramidal neurons

(Burman et al., 2008). Betz cells are characterised by their pyramidal shape, large size, and

prominent apical dendrites that are oriented along a vertical axis. Although more sparsely

distributed, they can be as 20 times larger in volume when compared to other pyramidal cells

(in humans; Rivara et al., 2003).

Betz cells have specialised molecular and physiological characteristics (Bakken et al., 2021).

Given their distinctive action-potential properties (Spain et al.,1991; Vigneswaran et al, 2011)

and direct connectivity onto the ventral horn of the spinal cord (Lemon, 2008), they are well

placed for initiation and modulation of fine movement. Betz cells can be evidenced using

markers of pyramidal neurons, such as antibodies against non-phosphorylated neurofilament

(e.g. SMI-32; Tsang et al., 2000; Szocsics et al., 2021), as well as general markers of neuronal

nuclei (e.g. NeuN; Szocsics et al., 2021). In addition, some of these cells express specifically

nitric oxide synthase (NOS, Wallace et al., 1996) and/ or the calcium binding protein

parvalbumin (PV, Szocsics et al., 2021).

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There is a large body of evidence to indicate that the local vasoactive intestinal polypeptide-

positive (VIP

) interneurons exert inhibition onto excitatory pyramidal cells, either directly or

indirectly, to shape their outputs (Lee et al., 2013; Garcia-Junco- Clemente et al. 2017; Pi et

al., 2013: Zhou et al., 2017; Yetman et al. 2019; Szadai et al, 2022). Here, we present evidence

that staining of M1 with antibodies against VIP reveals not only a subset of GABAergic

interneurons, as expected from previous studies (Gabbott and Bacon 1997; Peters et al., 1987;

Prönneke et al 2015; Kim et al.., 2017), but also Betz cells. The spatial and laminar distribution

of VIP

interneurons and Betz cells are compared.

2. MATERIALS AND METHODES

2.1 Animals

Materials were sourced from nine healthy adult marmosets (

Callithrix. jacchus

), as detailed in

Table 1. The experiments were conducted in accordance with the Australian Code of Practice

for the Care and Use of Animals for Scientific Purposes. All procedures were overseen by the

Animal Ethics Experimentation Committee at Monash University. The animals had no

veterinary record of serious acute, or chronic health conditions. Some of the animals were also

used anatomical tracing experiment unrelated to the present study (Majka et al., 2016, 2020;

for details of the tracer experiments, see www.marmosetbrain.org).

Table 1.

Details of sex, age, and staining.

Subject (Sex) Neuronal marker

Staining method

Age at perfusion
(months)

CJ195 (M)

IHC

CJ200 (F)

PV, CB, CR, NeuN IHC

CJ205 (M)

CB, CR

IHC

CJ226 (F)

VIP, NeuN

IHC/IF

CJ227 (M)

VIP, NeuN

IHC/IF

CJ232 (F)

VIP, PV

CJ240 (M)

VIP, NeuN

IHC/IF

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CJ249 (F)

VIP, GABA

F1741 (F)

CB, CR, NeuN

IHC/IF

2.2 Tissue perfusion and processing

The marmosets were euthanized with an overdose of sodium pentobarbitone (100 mg/kg, i.m.)

and transcardially perfused with 0.1 M heparinized phosphate buffer (PBS; pH 7.2), followed

by 4% paraformaldehyde in 0.1 PBS. The brains were dissected and post-fixed in the same

medium for one hour prior to immersion in phosphate buffered saline, with increasing

concentrations of sucrose (10%, 20% and 30%), over several days. Using a cryostat, frozen

40μm coronal sections were obtained

. In cases also used for anatomical tracing, the sections

used for the present analyses were sourced from the hemisphere contralateral to the tracer

injections. Five sequential series were collected, with the first two being stained for myelin

(Gallyas, 1979; modified by Worthy et al., 2017), and for cell bodies (cresyl violet Nissl stain).

These series were used to delineate boundaries of motor cortex (Burman et al. 2008). The other

series were used for immunostaining, as detailed in Table 1.

Immunostainings were conducted by incubating sections in blocking solution (10% normal

horse serum and 0.3% Triton-X100 in 0.1 M PBS) for 1 hr at room temperature before

undergoing incubation in primary antibody at 4°C for 42

–

46 h (Table 2). For

immunohistochemistry using DAB (IHC), a biotinylated horse anti-mouse IgG secondary

antibody incubation was applied for 30 min, followed by treatment with Avidin-Biotin

Complex (ABC) reagent and DAB (3,3'-Diaminobenzidine) substrate working solution. Alexa

Fluor secondary antibodies were used for immunofluorescence staining (IF) (Table 2).

Abbreviations:

PV, Parvalbumin; CB, Calbindin; CR, Calretinin; VIP,

Vasoactive intestinal polypeptide; NeuN, Neuronal nuclei; GABA,

Gamma-

aminobutyric acid;

IHC, Immunohistochemistry using DAB; IF,

Immunofluorescence.

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Subsequently, Aperio Scanscope AT Turbo (Leica Biosystems) was used to scan myelin, Nissl

and IHC stained sections. Fluorescent Images from the motor cortex were captured using a

Nikon C1 laser scanning confocal microscope. A comprehensive list of all antibodies used in

this experiment, along with their respective dilution rates, is presented in Table 2.

Table 2.

List of primary and secondary antibodies used for immunostaining.

Staining method

Primary
Antibody

Source and
Identifier

Secondary antibody

Source and
Identifier

Parvalbumin (PV)-
IHC

Mouse
monoclonal
(1:8000)

Swant
Cat# 235
PRID:
AB_10000343

Secondary Antibody
(Mouse IgG, 1:200)
And
Vectastain elite ABC HRP kit
DAB Substrate kit

Vector Laboratories
Cat# PK-6102
RRID:
AB_2336821

Vector Laboratories
Cat# SK-4100
RRID:
AB_2336382

Calbindin-D28k
(CB)-IHC

Mouse
monoclonal
(1:8000)

Swant
Cat# 300
RRID:
AB_10000347

Same as above

Calretinin (CR)-IHC

Mouse
monoclonal
(1:8000)

Swant
Cat# 6B3
RRID:
AB_10000320

Same as above

Neuronal marker
(NeuN)-IHC

Mouse
monoclonal
(1:800)

Millipore
Cat# A60
RRID:
AB_2298772

Same as above

Vasoactive intestinal
polypeptide (VIP)-
IHC

Mouse
monoclonal
(1:500)

Abcam
Cat# ab30680
RRID:
AB_778830

Same as above

VIP-IF

Rabbit
polyclonal
(1:200)

Atlas Antibodies
Cat#
HPA017324
RRID:
AB_1858754

Donkey Anti-Rabbit IgG
H&L (Alexa Fluor® 594)
preadsorbed
(1:600)

Abcam
Cat# ab150064
RRID:
AB_2734146

VIP-IF

Mouse
Monoclonal
(1: 500)

NeuroMab
Cat# L108/92
PRID:
AB_2651163

Donkey Anti-Mouse IgG
H&L (Alexa Fluor® 488)
preadsorbed (1:600)

Abcam
Cat# ab150109
RRID:
AB_2571721

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GABA-IF

Rabbit
polyclonal
(1:500)

Sigma-Aldrich
Cat# A2052
RRID:
AB_477652

Donkey Anti-Rabbit IgG
H&L (Alexa Fluor® 594)
preadsorbed
(1:600)

Abcam
Cat# ab150064
RRID:
AB_2734146

PV-IF

Mouse
monoclonal
(1:500)

Swant
Cat# 235
PRID:
AB_10000343

Donkey Anti-Mouse IgG
H&L (Alexa Fluor® 488)
preadsorbed (1:600)

Abcam
Cat# ab150109
RRID:
AB_2571721

NeuN-IF

Rabbit
polyclonal
(1:700)

Millipore
Cat# ABN78
RRID:
AB_10807945

Donkey Anti-Rabbit IgG
H&L (Alexa Fluor® 594)
preadsorbed
(1:600)

Abcam
Cat# ab150064
RRID:
AB_2734146

2.3 Quantification of VI

interneurons and statistical analysis

The three distinct regions of the primary motor cortex, namely areas 4a, 4b (encompassing

representations of the axial and limb musculatures) and 4c (the representation of the head

musculature), along with the caudal subdivisions of the dorsal premotor area (A6DC), were

delineated using the criteria defined by Burman et al. (2008; 2014a, b).

Four VIP-stained sections were selected from each subdivision of the primary motor cortex

from three cases that underwent IHC (CJ226, CJ227 and CJ240). Using standard features of

the Aperio Image Scope software, square counting frames (150μm × 150μm) were aligned

across the cortical thickness. The uppermost frame was aligned between the border of layers 1

and 2. The frames were

equidistant (10μm apart) and covered all cortical layers of the cortex

up to the white matter (Atapour et al. 2019), the number of counting frames varied depending

on the thickness of the cortex in each section. Each counting frame was bounded by 2 exclusion

lines (left and bottom) and 2 inclusion lines (right and top), as previously described (Atapour

et al., 2019). A cell was counted if it was completely within the counting frame or if the cell

body contacted the inclusion lines.

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The number of counted cells per frame was averaged in each section and used to calculate the

neuronal density. The section thickness (40μm) and a shrinkage factor of 0.801 obtained in a

previous study (Atapour et al., 2017) were considered when calculating the number of cells per

volume unit. For the analysis presented in Fig. 5

, we measured the VIP fluorescence intensity

of several neurons in one section as a relative quantity. We further calculated the corrected total

fluorescence [CTF = integrated density - (area of selected cell

mean fluorescence

background, Fig 5

)] to best access the amount of VIP expression in the two cell types.

Statistical analysis involved the use of unpaired t test or one-way ANOVA followed by post

hoc Tukey’s tests. Statistically significant differences were those with a p

-value < 0.05.

3. RESULTS

Immunostaining against VIP in the motor cortex led to the visualisation of giant Betz cells of

layer 5, as well as a subset of putative inhibitory neurons (Fig 1). While the expression of VIP

in interneurons has been demonstrated in other species (

Gabbott and Bacon 1997; Peters et al.,

1987;

Prönneke et al 2015; Szadai et al, 2022; Tremblay et al., 2016), our data also indicate

VIP expression in Betz cells, which was confirmed using three different antibodies (Table 2).

The VIP antibody used in different analyses is given in the corresponding figure legends.

3.1. Betz cells

VIP staining intensity was strongest in the largest layer 5 pyramidal cells of A4ab, which are

located more medially in this area, as defined using Nissl and myelin staining (Fig 1

a-c

Paxinos et al., 2012). This region corresponds to the representations of the leg and trunk

musculatures (Burman et al., 2008). The locations of stained Betz cells are shown by black

circles in Fig 1

. VIP staining intensity in Betz cells diminishes toward the more lateral parts

of A4b (Fig 1

e-j

). The cell body and apical dendrites of Betz cells were clearly visible in the

VIP staining, similar to the observations with Nissl staining, yet with more pronounced

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delineation of the apical dendrite (Fig 1

e-j

). There was no VIP staining in layer 5 pyramidal

cells of A4c and premotor area 6DC (Fig 2), which lack the gygantopyramidal characteristics

(Burman et al. 2008, 2014).

Figure 1. Vasoactive intestinal polypeptide (VIP) is expressed in giant pyramidal (Betz)
cells of primary motor cortex.

Representative images of primary motor cortex (M1, Area

4a and Area 4b) are shown using myelin (a), nissl (b) and VIP staining (c) in CJ226.
Arrowheads point to the areal boundaries (Paxions et al., 2012). Red lines in b & c identify
layers 2 to 6 while layer 5 is indicated by small black lines. Schematic of stained layer 5
Betz cells is indicated in d. Betz cells are clearly visible within layer 5 (between the two
dashed lines) in e & f representing the area within the dashed rectangles in b & c. Red arrows
point to Betz neurons from A4a (g & i) and A4b (h & j) as marked in b & c. Scale: 1 mm
for a-d, 200

m for e-f & 100

m for g-j. VIP antibody used in production of images is from

Abcam (Cat# ab30680).

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We confirmed the presence of VIP staining for the Betz cells by colocalization with the

neuronal marker (NeuN), and PV (Fig 3 & 4). PV-positive (PV

) axonal endings, which are

known to heavily innervate Betz cells (Szocsics et al., 2021), were very evident in our material,

forming perisomal networks that delineated the Betz cell bodies (Fig 3). We also found that

PV is present in the soma of a subset of Betz cells in marmoset, as reported previously in human

brain (Szocsics et al., 2021). The PV signal was far less intense in Betz cell bodies, compared

to interneurons or PV

terminal nets delineating soma boundaries, but was clearly

distinguishable from the background, and particularly evident by comparison with PV negative

(PV

) Betz cells. Examples of PV

and PV

Betz cells alongside interneurons, are shown in Fig

4. The two other calcium binding proteins tested, calbindin D28-K (CB) and calretinin (CR)

Figure 2. Expression of vasoactive intestinal polypeptide (VIP) is limited to the largest
layer 5 pyramidal cells.

Representative images of Area 4c (a-e) and A6DC (caudal

subdivisions of the dorsal premotor area, f-j) are shown using myelin (a & f), nissl (b & g) and
VIP staining (c & h) in CJ226. Arrowheads point to the areal boundaries (Paxions et al., 2012).
Red lines in b-c & g-h identify layers 2 to 6, while layer 5 is indicated by small black lines.
Large pyramidal cells are only visible within layer 5 (between the two dashed lines) in Nissl
staining (d & i; representing area within the dashed rectangle in b & g, respectively). Scale: 1
mm for a-c & f-h, 200

m for d-e & i-j. VIP antibody used in production of images is from

Abcam (Cat# ab30680).

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Figure 3. Co-localisation of vasoactive intestinal polypeptide (VIP) with
neuronal marker (NeuN) and parvalbumin (PV) in the Betz cells.

(a) Confocal

images of cortical layer 5 showing immunoreactivity to VIP either with neuronal
marker NeuN (top) or PV (bottom). (b) Insets at higher magnification. White arrows
point to the Betz cells in all images. Scale: 100

m (a) and 50

m (b). VIP antibody

used in production of images is from Atlas Antibodies (Cat# HPA017324).

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3.2.VIP

inhibitory neurons

VIP staining revealed a subset of inhibitory neurons in the marmoset motor cortex (Fig 5

a-c

VIP

interneurons had the morphology of dendritic targeting cells, likely including bipolar

cells, double bouquet cells and bitufted cells (Markram 2004, Fig 5

). Co-staining for VIP and

GABA confirmed the inhibitory nature of VIP

interneurons (Fig 5

b-c

). The arbitrary

Figure 4. Giant pyramidal cells of the primary motor cortex are identified by
parvalbumin (PV) but no other calcium binding proteins.

Examples of NeuN (a),

calbindin (b), calretinin (c) and parvalbumin (d) staining of area 4A. The boxed areas
(dashed squares) in a-d are shown in higher magnification (e-h), respectively. White arrows
in e point to Betz cells. Betz cells in i-k are taken from sections not shown here and those
in l & n are magnified from panel h. Asterisks (in panels k & n) point to Betz cells that do
not contain PV in their soma. PV content is also compared to an interneuron visible in m,
highlighted by red arrow in panel h. Scale: 200

m for a-d, 100

m for e-h & 25

m for i-n.

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fluorescence intensity was much higher in the VIP

interneurons than in VIP

Betz cells (Fig

d-e

, unpaired t-test; 3165 ± 281 vs. 1333 ± 125, p<0.0001). However, considering somatic

area, the corrected total fluorescence within the cell bodies was similar (Fig 5

, unpaired t test,

18347 ± 2473 vs. 14404 ± 258, p = 0.16).

In all subfields of primary motor cortex, VIP

inhibitory neurons were mostly concentrated in

the supragranular layers, and their density gradually decreased toward layer 6 (Fig 6

a-c

). The

mean average density of VIP

interneurons was significantly higher in the A4c compared to

the other subdivisions of primary motor cortex [Fig 6

, one-way ANOVA: F (2, 33) = 21.26,

p< 0.0001; Tukey's test; A4c (4745±146/mm

) vs. A4a (3065± 224/mm

) or A4b (3410±

199/mm

), p< 0.0001)]. VIP

interneurons corresponded to 4.5, 5.2 and 6.6% of the neuronal

population in cytoarchitectural subdivisions A4a, A4b and A4c, respectively (Fig 6

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Figure 5. Antibody against vasoactive intestinal polypeptide (VIP) stains inhibitory
interneurons as well as Betz cells

. Representative images of different morphologies of VIP-

positive (VIP

) inhibitory neurons of the primary motor cortex (a). White arrowheads point to

the dendritic bifurcations. Inhibitory nature of VIP

interneurons is shown by co-localisation

with gamma aminobutyric acid (GABA) that is missing in Betz cells (b-c). Fluorescence
intensity comparison for a VIP

inhibitory neuron and Betz cell (d). Cyan and yellow arrows

point to VIP

inhibitory neuron and Betz cell respectively in b-d. Mean ± SEM density of

arbitrary fluorescence intensity obtained from variable somatic locations (e) and corrected total
fluorescence for somatic area (f) for VIP

inhibitory and Betz neurons. Scale: 50

m. VIP

antibodies used in production of images are from Atlas Antibodies (Cat# HPA017324, a & d)
and Neuro Mab (Cat# L108/92, b-c).

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Fig 6. Layer profile of vasoactive intestinal polypeptide-positive (VIP

)

interneurons of the primary motor cortex.

Representative VIP-stained strips from

different parts of primary motor cortex including areas A4a, A4b and A4c (a). Small red
lines identify the border with layer 2 and white matter. Limits of layer 5 are shown by
small black lines. Quantification of VIP

interneuron density across cortical depth (b).

Individual cells are indicated by yellow arrows. Some VIP

interneurons are shown at

higher magnifications in c. Scale: 100

m in and 20

m in c. Mean ± SEM density of VIP

interneurons (d) and its percentage (e, as ratio of total neurons obtained by NeuN staining
in our previous study, Atapour et al., 2019). Black circles represent individual sections
in d. VIP antibody used in production of images is from Abcam (Cat# ab30680).

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4. DISCUSSION

The primary motor cortex is not only involved in motor control, but also in sensory integration,

behavioural strategizing, working memory, and decision-making (Ebbesen et al., 2018). Its

susceptibility in several disease including neurodegenerative conditions and ageing (Cleveland

et al., 2001; Hof & Perl 2002; Scheibel et al., 1977; Seeley 2008; Udaka et al., 1986) warrant

further understanding of its neuronal neurochemistry. Here we demonstrate that VIP, a peptide

with widespread distribution in many organs, is expressed in the largest excitatory pyramidal

neurons of the primate neocortex, adding to the intricacy of distinctive gene-expression and

physiological features of the giant layer 5 Betz cells (Bakken et al., 2021). This finding extends

VIP expression to a new class of cortical neurons, raising new questions about the physiological

role of this peptide in the motor cortex.

4.1.VIP expression in Betz cells

VIP is widely distributed in the nervous system as well as in many other peripheral organs,

acting both as a neuromodulator and neuroendocrine releasing factor (Delgado et al., 2004;

Iwasaki et al., 2019; Waschek 2013). In the brain, VIP regulates the secretion and expression

of neurotrophic factors (Brenneman et al., 1990; Pellegri et al., 1998), has neuroprotective and

anti-inflammatory effects (Delgado et al., 2004, Waschek 2013), and is up-regulated in neurons

and immune cells after injury and/or inflammation (Armstrong

et al

., 2004, Waschek 2013).

wing to VIP’s versatile and widespread effects, it

is presently difficult to pinpoint its possible

role in Betz cells without additional functional assessments. However, the particularly large

size of Betz cells may imply unique metabolic/ homeostatic demands, for which VIP may

contribute in terms of neuroprotective effects. Betz cells, with their large body size and long

myelinated axons, are amongst the most vulnerable neurons (Mattson and Magnus 2006),

which are affected in ageing and motor neuron disease (Cleveland et al., 2001, Scheibel et al.,

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1977, Udaka et al., 1986). High energy requirement, and reliance on long distance axonal

transport along with a large cell surface area, increases exposure to undesirable factors

(Morrison et al., 1998), likely rendering Betz cells in need of extra regulatory protection. In

line with this hypothesis, it has been shown that VIP mRNA has been induced in facial motor

neurons of mice following application of an inflammatory stimulus directly to the nerve

(Armstrong

et al

., 2004). Moreover, the expression of PV in Betz cells (Szocsics et al., 2021,

Preuss & Kaas 1996) may add another layer of protection through calcium regulation.

Consistently, the proportion of PV-containing Betz cells decreases with age in humans

(Szocsics et al., 2023), making them more susceptible to degeneration (Scheibel et al., 1977).

VIP induces concentration-dependent glycogenolysis in mouse cortical slices, which may

indicate a possible regulatory mechanism for the local control of energy metabolism

(Magistretti et al., 1981). The presence of VIP in the largest pyramidal cells may also have a

relationship with the high demands of these cells for energy substrate. Such hypothesis is

consistent with the maximal VIP staining in the largest pyramidal cells, which are located more

medially in A4ab. Whether VIP being released from the Betz cells along with the other

neurotransmitters or its functions are limited to the cytoplasm remains to be understood.

4.2.VIP

interneurons in the motor cortex

VIP

interneurons among the main subclasses of GABAergic neurons, being primarily located

the supragranular layers of the cortex in the areas examined in this study, similar to

observations in other species (

Gabbott and Bacon 1997; Peters et al., 1987;

Prönneke et al

2015; Tremblay et al., 2016, Kim et al.., 2017). VIP

neuronal density across cortical layers

showed a sharp decline toward the white matter, with much fewer VIP

interneurons present in

the infragranular layers. Supra- and infragranular VIP

interneurons may have different

functional roles as the two populations are transcriptionally distinct in mice (Wu et al., 2022).

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Although the drop in the density of VIP

inhibitory neurons in the infragranular layers was

consistent in the three examined areas, the mean average density of VIP

interneurons and their

relative proportion to the total neuronal density were higher in the A4c compared to the areas

A4a and A4b. This may reflect regional differences in network organisation for facial versus

limb and body movements. Together with the differences in expression of VIP in Betz cells,

this result further highlights the fact that functionally defined cortical areas are not necessarily

homogeneous, a principle which has been demonstrated in terms of cellular structure (e.g.

present results; Ungerleider and Desimone 1986), connections (e.g. Palmer and Rosa 2006;

Rosa et al. 2009) and physiological properties (Yu et al. 2010; Yu and Rosa 2014).

Our density estimates for VIP

interneurons in the motor cortex (3,065-4,745/mm

) are 50-

100% higher than those in mouse motor cortex (Kim et al., 2017). Overall, we report a 5-7%

contribution of VIP

interneurons to the neuronal population in the motor cortex in the

marmoset monkeys. B

ased on the 23% contribution of GABAergic interneurons in marmoset’s

motor cortex (Bakken et al., 2021) and total neuronal density (Atapour et al., 2019), we estimate

that VIP

interneurons of the motor cortex constitute around 20-29% of the total interneuron

population. These estimates are higher than those reported in several cortical areas of mice,

which are around 11-15% of total GABAergic neurons (Gonchar et al., 2007, Pfeffer et al.,

2013, Prönneke et al., 2015; Szadai et al., 2022). The observed morphologies of VIP

interneurons in the marmoset motor cortex were similar to those reported in other species

(Apicella et al., 2022;

Gabbott and Bacon 1997, Markram et al., 2004

Considering the soma size of neurons, the arbitrary fluorescence intensities in the VIP

interneurons were similar to the Betz cells, indicating a comparatively similar expression of

VIP. While the modes of connectivity of VIP

interneurons with Betz cells remain to be

elucidated, current literature suggest that VIP

interneurons provide inhibitory inputs to

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pyramidal neurons mainly through forming disinhibitory circuits (Pfeffer et al., 2013; Pi et al.,

2013, Lee et al., 2013). However direct inhibition of pyramidal neurons by this population has

also been reported (Zhou et al., 2017; Garcia-Junco- Clemente et al. 2017; Yetman et al. 2019).

The abundance of studies on VIP

interneurons in rodents has shed lights on the specific roles

of this neuronal population, which include motor integration (Lee et al., 2013; Fu et al., 2014;

Yu et al., 2019) and gain control (Pi et al., 2013). However, we are far from attaining a similar

understanding about their function in primates, where only limited studies have been made

available (e.g. Benson et al., 1991; Gabbott and Bacon 1997; Ong & Garey, 1991).

4.3. Conclusion

The distribution of VIP

neurons in the marmoset motor cortex demonstrates that this

neuropeptide is expressed both in interneurons and large pyramidal cells with putative

projections to the spinal cord (Betz cells). The specific distribution of VIP in Betz cells hints

at a different set of functions for this neuropeptide, as well as differences in neuronal circuitry

involved in generating and controlling different types of body movements.

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

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this version posted January 22, 2024.

https://orcid.org/0000-0002-9713-7518

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ACKNOWLEDGEMENTS

Funding was provided by the National Health and Medical Research Council to Marcello G. P. Rosa

(APP1194206) and Nafiseh Atapour (APP2019011). The authors acknowledge the contributions of

the Monash Micro Imaging facility for providing training and support for confocal imaging and the

Monash Histology platform for slide scanning services.

CONFLIC OF INTEREST STATMENT

The authors have no conflicts of interest to declare.

ORCID

S. Teymornejad,

K. H. Worthy,

https://orcid.org/0000-0001-9539-9744

M.G. P. Rosa,

https://orcid.org/0000-0002-6620-6285

N. Atapour,

https://orcid.org/0000-0002-8773-9283

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted January 22, 2024.

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REFERENCES

Apicella, A. J., & Marchionni, I. (2022). VIP-Expressing GABAergic Neurons: Disinhibitory
vs. Inhibitory Motif and Its Role in Communication Across Neocortical Areas.

Frontiers in

Cellular Neuroscience, 16

, 811484. doi:10.3389/fncel.2022.811484

Armstrong, B. D., Hu, Z., Abad, C., Yamamoto, M., Rodriguez, W. I., Cheng, J., Lee, M.,
Chhith, S., Gomariz, R. P., & Waschek, J. A. (2004). Induction of neuropeptide gene
expression and blockade of retrograde transport in facial motor neurons following local
peripheral nerve inflammation in severe combined immunodeficiency and BALB/C mice.

Neuroscience, 129

(1), 93-99. doi: 10.1016/j.neuroscience.2004.06.085

Atapour, N., Majka, P., Wolkowicz, I. H., Malamanova, D., Worthy, K. H., & Rosa, M. G. P.
(2019). Neuronal Distribution Across the Cerebral Cortex of the Marmoset Monkey
(Callithrix jacchus).

Cerebral Cortex, 29

(9), 3836

–

3863.

https://doi.org/10.1093/cercor/bhy263

Atapour, N., Rosa, M. G. P., Bai, S., Bednarek, S., Kulesza, A., Saworska, G., Worthy, K. H.,
& Majka, P. (2024). Distribution of calbindin-positive neurons across areas and layers of the
marmoset cerebral cortex. bioRxiv. https://doi.org/10.1101/2024.01.05.574341

Atapour, N., Worthy, K. H., Lui, L. L., Yu, H. H., & Rosa, M. G. P. (2017). Neuronal
degeneration in the dorsal lateral geniculate nucleus following lesions of primary visual
cortex: comparison of young adult and geriatric marmoset monkeys.

Brain Structure &

Function

222

(7), 3283

–

3293. https://doi.org/10.1007/s00429-017-1404-4

Bakken, T. E., Jorstad, N. L., Hu, Q., Lake, B. B., Tian, W., Kalmbach, B. E., Crow, M.,
Hodge, R. D., Krienen, F. M., Sorensen, S. A., Eggermont, J., Yao, Z., Aevermann, B. D.,

Aldridge, A. I., Bartlett, A., Bertagnolli, D., Casper, T., Castanon, R. G., Crichton, K., …

Lein, E. S. (2021). Comparative cellular analysis of motor cortex in human, marmoset and
mouse.

Nature

598

(7879), 111

–

119. https://doi.org/10.1038/s41586-021-03465-8

Benson, D. L., Isackson, P. J., & Jones, E. G. (1991). In situ hybridization reveals VIP
precursor mRNA-containing neurons in monkey and rat neocortex.

Brain Research.

Molecular Brain Research

(1-2), 169

–

174. https://doi.org/10.1016/0169-328x(91)90145-n

Betz W. (1874). Anatomischer Nachweis zweier Gehirncentra.

Zbl Med Wiss, 12

(578

–

580):594

–

599.

Brenneman, D. E., Nicol, T., Warren, D., & Bowers, L. M. (1990). Vasoactive intestinal
peptide: a neurotrophic releasing agent and an astroglial mitogen.

Journal of Neuroscience

Research, 25

(3), 386

–

394. https://doi.org/10.1002/jnr.490250316

10.

Burman, K. J., Palmer, S. M., Gamberini, M., Spitzer, M. W., & Rosa, M. G. (2008).
Anatomical and physiological definition of the motor cortex of the marmoset monkey.

The

Journal of Comparative Neurology, 506

(5), 860

–

876. https://doi.org/10.1002/cne.21580

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted January 22, 2024.

bioRxiv preprint

11.

Burman, K. J., Bakola, S., Richardson, K. E., Reser, D. H., & Rosa, M. G. (2014). Patterns of
cortical input to the primary motor area in the marmoset monkey.

The Journal of

Comparative Neurology

522

(4), 811

–

843. https://doi.org/10.1002/cne.23447

12.

Cleveland, D. W., & Rothstein, J. D. (2001). From Charcot to Lou Gehrig: deciphering
selective motor neuron death in ALS. Nature reviews.

Neuroscience, 2

(11), 806

–

819.

https://doi.org/10.1038/35097565

13.

Delgado, M., Pozo, D., & Ganea, D. (2004). The significance of vasoactive intestinal peptide
in immunomodulation.

Pharmacological Reviews

(2), 249

–

290.

https://doi.org/10.1124/pr.56.2.7

14.

Ebbesen, C. L., Insanally, M. N., Kopec, C. D., Murakami, M., Saiki, A., & Erlich, J. C.
(2018). More than Just a "Motor": Recent Surprises from the Frontal Cortex.

The Journal of

Neuroscience, 38

(44), 9402

–

9413. https://doi.org/10.1523/JNEUROSCI.1671-18.2018

15.

Fu, Y., Tucciarone, J. M., Espinosa, J. S., Sheng, N., Darcy, D. P., Nicoll, R. A., Huang, Z. J.,
& Stryker, M. P. (2014). A cortical circuit for gain control by behavioral state.

Cell, 156

(6),

1139

–

1152. https://doi.org/10.1016/j.cell.2014.01.050

16.

Gabbott, P. L., & Bacon, S. J. (1997). Vasoactive intestinal polypeptide containing neurones
in monkey medial prefrontal cortex (mPFC): colocalisation with calretinin.

Brain

Research

744

(1), 179

–

184. https://doi.org/10.1016/s0006-8993(96)01232-2

17.

Gallyas F. (1979). Silver staining of myelin by means of physical development.

Neurological

Research, 1

(2), 203

–

209. https://doi.org/10.1080/01616412.1979.11739553

18.

Garcia-Junco-Clemente, P., Ikrar, T., Tring, E., Xu, X., Ringach, D. L., & Trachtenberg, J. T.
(2017). An inhibitory pull-push circuit in frontal cortex.

Nature Neuroscience

(3), 389

–

392. https://doi.org/10.1038/nn.4483

19.

Gonchar, Y., Wang, Q., & Burkhalter, A. (2008). Multiple distinct subtypes of GABAergic
neurons in mouse visual cortex identified by triple immunostaining.

Frontiers in

Neuroanatomy

, 3. https://doi.org/10.3389/neuro.05.003.2007

20.

Hof, P. R., & Perl, D. P. (2002). Neurofibrillary tangles in the primary motor cortex in
Guamanian amyotrophic lateral sclerosis/parkinsonism-dementia complex.

Neuroscience

Letters

328

(3), 294

–

298. https://doi.org/10.1016/s0304-3940(02)00523-2

21.

Iwasaki, M., Akiba, Y., & Kaunitz, J. D. (2019). Recent advances in vasoactive intestinal
peptide physiology and pathophysiology: focus on the gastrointestinal system.

F1000Res, 8

doi:10.12688/f1000research.18039.1

22.

Jacobs, B., Garcia, M. E., Shea-Shumsky, N. B., Tennison, M. E., Schall, M., Saviano, M. S.,
Tummino, T. A., Bull, A. J., Driscoll, L. L., Raghanti, M. A., Lewandowski, A. H., Wicinski,
B., Ki Chui, H., Bertelsen, M. F., Walsh, T., Bhagwandin, A., Spocter, M. A., Hof, P. R.,
Sherwood, C. C., & Manger, P. R. (2018). Comparative morphology of gigantopyramidal

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted January 22, 2024.

bioRxiv preprint

neurons in primary motor cortex across mammals.

The Journal of Comparative Neurology,

526

(3), 496

–

536. https://doi.org/10.1002/cne.24349

23.

Kim, Y., Yang, G. R., Pradhan, K., Venkataraju, K. U., Bota, M., García Del Molino, L. C.,
Fitzgerald, G., Ram, K., He, M., Levine, J. M., Mitra, P., Huang, Z. J., Wang, X. J., & Osten,
P. (2017). Brain-wide Maps Reveal Stereotyped Cell-Type-Based Cortical Architecture and
Subcortical Sexual Dimorphism.

Cell, 171

(2), 456

–

469.e22.

https://doi.org/10.1016/j.cell.2017.09.020

24.

Kondo, H., Tanaka, K., Hashikawa, T., & Jones, E. G. (1999). Neurochemical gradients along
monkey sensory cortical pathways: calbindin-immunoreactive pyramidal neurons in layers II
and III.

The European Journal of Neuroscience, 11

(12), 4197

–

4203.

https://doi.org/10.1046/j.1460-9568.1999.00844.x

25.

Lee, S., Kruglikov, I., Huang, Z. J., Fishell, G., & Rudy, B. (2013). A disinhibitory circuit
mediates motor integration in the somatosensory cortex.

Nature Neuroscience, 16

(11), 1662

–

1670. https://doi.org/10.1038/nn.3544

26.

Lemon R. N. (2008). Descending pathways in motor control.

Annual Review of Neuroscience,

, 195

–

218. https://doi.org/10.1146/annurev.neuro.31.060407.125547

27.

Magistretti, P. J., Morrison, J. H., Shoemaker, W. J., Sapin, V., & Bloom, F. E. (1981).
Vasoactive intestinal polypeptide induces glycogenolysis in mouse cortical slices: a possible
regulatory mechanism for the local control of energy metabolism.

Proceedings of the

National Academy of Sciences of the United States of America, 78

(10), 6535

–

6539.

https://doi.org/10.1073/pnas.78.10.6535

28.

Majka, P., Bai, S., Bakola, S., Bednarek, S., Chan, J. M., Jermakow, N., Passarelli, L., Reser,
D. H., Theodoni, P., Worthy, K. H., Wang, X. J., Wójcik, D. K., Mitra, P. P., & Rosa, M. G.
P. (2020). Open access resource for cellular-resolution analyses of corticocortical connectivity
in the marmoset monkey.

Nature Communications, 11

(1), 1133.

https://doi.org/10.1038/s41467-020-14858-0

29.

Majka, P., Chaplin, T. A., Yu, H. H., Tolpygo, A., Mitra, P. P., Wójcik, D. K., & Rosa, M. G.
(2016). Towards a comprehensive atlas of cortical connections in a primate brain: Mapping
tracer injection studies of the common marmoset into a reference digital template.

The

Journal of Comparative Neurology, 524

(11), 2161

–

2181. https://doi.org/10.1002/cne.24023

30.

Markram, H., Toledo-Rodriguez, M., Wang, Y., Gupta, A., Silberberg, G., & Wu, C. (2004).
Interneurons of the neocortical inhibitory system.

Nature Reviews. Neuroscience, 5

(10), 793

–

807. https://doi.org/10.1038/nrn1519

31.

Matelli, M., Luppino, G., & Rizzolatti, G. (1985). Patterns of cytochrome oxidase activity in
the frontal agranular cortex of the macaque monkey.

Behavioural Brain Research

(2),

125

–

136. https://doi.org/10.1016/0166-4328(85)90068-3

32.

Mattson, M. P., & Magnus, T. (2006). Ageing and neuronal vulnerability.

Nature Reviews.

Neuroscience

(4), 278

–

294. https://doi.org/10.1038/nrn1886

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted January 22, 2024.

https://doi.org/10.1002/ar.a.10015

bioRxiv preprint

33.

Morrison, B. M., Hof, P. R., & Morrison, J. H. (1998). Determinants of neuronal vulnerability
in neurodegenerative diseases.

Annals of Neurology

(3 Suppl 1), S32

–

S44.

https://doi.org/10.1002/ana.410440706

34.

Ong, W. Y., & Garey, L. J. (1991). Ultrastructural characteristics of human adult and infant
cerebral cortical neurons.

Journal of Anatomy

175

, 79

–

104.

35.

Palmer, S. M., & Rosa, M. G. (2006). A distinct anatomical network of cortical areas for
analysis of motion in far peripheral vision.

The European journal of neuroscience

(8),

2389

–

2405. https://doi.org/10.1111/j.1460-9568.2006.05113.x

36.

Paxinos G, Watson C, Petrides M, Rosa M., & Tokuno H. (2012).

The marmoset brain in

stereotaxic coordinates.

Elsevier.

37.

Pellegri, G., Magistretti, P. J., & Martin, J. L. (1998). VIP and PACAP potentiate the action
of glutamate on BDNF expression in mouse cortical neurones.

The European Journal of

Neuroscience

(1), 272

–

280. https://doi.org/10.1046/j.1460-9568.1998.00052.x

38.

Peters, A., Meinecke, D. L., & Karamanlidis, A. N. (1987). Vasoactive intestinal polypeptide
immunoreactive neurons in the primary visual cortex of the cat.

Journal of

Neurocytology

(1), 23

–

38. https://doi.org/10.1007/BF02456695

39.

Pfeffer, C. K., Xue, M., He, M., Huang, Z. J., & Scanziani, M. (2013). Inhibition of inhibition
in visual cortex: the logic of connections between molecularly distinct interneurons.

Nature

Neuroscience

(8), 1068

–

1076. https://doi.org/10.1038/nn.3446

40.

Pi, H. J., Hangya, B., Kvitsiani, D., Sanders, J. I., Huang, Z. J., & Kepecs, A. (2013). Cortical
interneurons that specialize in disinhibitory control.

Nature

503

(7477), 521

–

524.

https://doi.org/10.1038/nature12676

41.

Preuss, T. M., & Kaas, J. H. (1996). Parvalbumin-like immunoreactivity of layer V pyramidal
cells in the motor and somatosensory cortex of adult primates.

Brain Research

712

(2), 353

–

357. https://doi.org/10.1016/0006-8993(95)01531-0

42.

Prönneke, A., Scheuer, B., Wagener, R. J., Möck, M., Witte, M., & Staiger, J. F. (2015).
Characterizing VIP Neurons in the Barrel Cortex of VIPcre/tdTomato Mice Reveals Layer-
Specific Differences.

Cerebral Cortex

(12), 4854

–

4868.

https://doi.org/10.1093/cercor/bhv202

43.

Rivara, C. B., Sherwood, C. C., Bouras, C., & Hof, P. R. (2003). Stereologic characterization
and spatial distribution patterns of Betz cells in the human primary motor cortex.

The

Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary
Biology

270

(2), 137

–

151.

44.

Rosa, M. G., Palmer, S. M., Gamberini, M., Burman, K. J., Yu, H. H., Reser, D. H., Bourne,
J. A., Tweedale, R., & Galletti, C. (2009). Connections of the dorsomedial visual area:
pathways for early integration of dorsal and ventral streams in extrastriate cortex.

The Journal

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted January 22, 2024.

bioRxiv preprint

of neuroscience : the official journal of the Society for Neuroscience

(14), 4548

–

4563.

https://doi.org/10.1523/JNEUROSCI.0529-09.2009

45.

Scheibel, M. E., Tomiyasu, U., & Scheibel, A. B. (1977). The aging human Betz
cell.

Experimental Neurology

(3), 598

–

609. https://doi.org/10.1016/0014-4886(77)90323-5

46.

Seeley W. W. (2008). Selective functional, regional, and neuronal vulnerability in
frontotemporal dementia.

Current Opinion In neurology

(6), 701

–

707.

https://doi.org/10.1097/WCO.0b013e3283168e2d

47.

Spain, W. J., Schwindt, P. C., & Crill, W. E. (1991). Post-inhibitory excitation and inhibition
in layer V pyramidal neurones from cat sensorimotor cortex.

The Journal of Physiology

434

609

–

626. https://doi.org/10.1113/jphysiol.1991.sp018489

48.

Szadai, Z., Pi, H. J., Chevy, Q., Ócsai, K., Albeanu, D. F., Chiovini, B., Szalay, G., Katona,
G., Kepecs, A., & Rózsa, B. (2022). Cortex-wide response mode of VIP-expressing inhibitory
neurons by reward and punishment.

eLife

, e78815. https://doi.org/10.7554/eLife.78815

49.

Szocsics, P., Papp, P., Havas, L., Lőke, J., & Maglóczky, Z. (2023). Interhemispheric

differences of pyramidal cells in the primary motor cortices of schizophrenia patients
investigated postmortem.

Cerebral Cortex, 33

(13), 8179

–

8193.

https://doi.org/10.1093/cercor/bhad107

50.

Szocsics, P., Papp, P., Havas, L., Watanabe, M., & Maglóczky, Z. (2021). Perisomatic
innervation and neurochemical features of giant pyramidal neurons in both hemispheres of the
human primary motor cortex.

Brain Structure & Function

226

(1), 281

–

296.

https://doi.org/10.1007/s00429-020-02182-8

51.

Tremblay, R., Lee, S., & Rudy, B. (2016). GABAergic Interneurons in the Neocortex: From
Cellular Properties to Circuits.

Neuron

(2), 260

–

292.

https://doi.org/10.1016/j.neuron.2016.06.033

52.

Tsang, Y. M., Chiong, F., Kuznetsov, D., Kasarskis, E., & Geula, C. (2000). Motor neurons
are rich in non-phosphorylated neurofilaments: cross-species comparison and alterations in
ALS.

Brain Research

861

(1), 45

–

58. https://doi.org/10.1016/s0006-8993(00)01954-5

53.

Udaka, F., Kameyama, M., & Tomonaga, M. (1986). Degeneration of Betz cells in motor
neuron disease. A Golgi study.

Acta Neuropathologica

(3-4), 289

–

295.

54.

Ungerleider LG, Desimone R. (1986). Projections to the superior temporal sulcus from the
central and peripheral field representations of V1 and V2.

J Comp Neurol

. 8;248(2):147-63.

doi: 10.1002/cne.902480202.

55.

Yu, H. H., & Rosa, M. G. (2014). Uniformity and diversity of response properties of neurons
in the primary visual cortex: selectivity for orientation, direction of motion, and stimulus size
from center to far periphery.

Visual neuroscience

(1), 85

–

98.

https://doi.org/10.1017/S0952523813000448

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

The copyright holder for this

this version posted January 22, 2024.

https://doi.org/10.1007/BF00686086

bioRxiv preprint

56.

Yu, H. H., Verma, R., Yang, Y., Tibballs, H. A., Lui, L. L., Reser, D. H., & Rosa, M. G.
(2010). Spatial and temporal frequency tuning in striate cortex: functional uniformity and
specializations related to receptive field eccentricity.

The European journal of

neuroscience

(6), 1043

–

1062. https://doi.org/10.1111/j.1460-

9568.2010.07118.x

57.

Vigneswaran, G., Kraskov, A., & Lemon, R. N. (2011). Large identified pyramidal cells in
macaque motor and premotor cortex exhibit "thin spikes": implications for cell type
classification.

The Journal of Neuroscience

(40), 14235

–

14242.

https://doi.org/10.1523/JNEUROSCI.3142-11.2011

58.

Vogt, C., & Vogt, O. (1919).

Allgemeine ergebnisse unserer hirnforschung

. Verlag von

Johann Ambrosius Barth.

59.

Wallace, M. N., Tayebjee, M. H., Rana, F. S., Farquhar, D. A., & Nyong'o, A. O. (1996).
Pyramidal neurones in pathological human motor cortex express nitric oxide
synthase.

Neuroscience Letters

212

(3), 187

–

190. https://doi.org/10.1016/0304-

3940(96)12809-3

60.

Waschek J. A. (2013). VIP and PACAP: neuropeptide modulators of CNS inflammation,
injury, and repair.

British Journal of Pharmacology

169

(3), 512

–

523.

https://doi.org/10.1111/bph.12181

61.

Watanabe-Sawaguchi, K., Kubota, K., & Arikuni, T. (1991). Cytoarchitecture and intrafrontal
connections of the frontal cortex of the brain of the hamadryas baboon (Papio
hamadryas).

The Journal of Comparative Neurology

311

(1), 108

–

133.

https://doi.org/10.1002/cne.903110109

62.

Worthy, K.H., Burman, K.J., and Rosa, M.G.P. (2017). Gallyas Silver Stain for Myelin.
Melbourne,Australia: The Marmoset Brain Connectivity Atlas Project. Online. https://
doi.org/10.13140/RG.2.2.23807.76968. http://r.marmosetbrain.org/ Gallyasmethod.pdf.

63.

Wu, J., Zhao, Z., Shi, Y., & He, M. (2022). Cortical VIP

Interneurons in the Upper and

Deeper Layers Are Transcriptionally Distinct.

Journal of Molecular Neuroscience

(8),

1779

–

1795. https://doi.org/10.1007/s12031-022-02040-8

64.

Yetman, M. J., Washburn, E., Hyun, J. H., Osakada, F., Hayano, Y., Zeng, H., Callaway, E.
M., Kwon, H. B., & Taniguchi, H. (2019). Intersectional monosynaptic tracing for dissecting
subtype-specific organization of GABAergic interneuron inputs.

Nature Neuroscience

(3),

492

–

502. https://doi.org/10.1038/s41593-018-0322-y

65.

Yu, J., Hu, H., Agmon, A., & Svoboda, K. (2019). Recruitment of GABAergic Interneurons
in the Barrel Cortex during Active Tactile Behavior.

Neuron

104

(2), 412

–

427.e4.

https://doi.org/10.1016/j.neuron.2019.07.027

66.

Zhou, X., Rickmann, M., Hafner, G., & Staiger, J. F. (2017). Subcellular Targeting of VIP
Boutons in Mouse Barrel Cortex is Layer-Dependent and not Restricted to
Interneurons.

Cerebral cortex, 27

(11), 5353

–

5368. https://doi.org/10.1093/cercor/bhx220

preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

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this version posted January 22, 2024.