ISRAEL JOURNAL OF
VETERINARY MEDICINE
THE DISTRIBUTION OF NEUROKININ 1 RECEPTORIMMUNOREACTIVE NEURONES IN THE RAT SPINAL CORD AND
TRIGEMINAL NUCLEUS CAUDALIS
M. Nazl‫ץ‬
University of Kafkas, Veterinary Faculty, Department of Histology-Embryology, Kars/ TURKEY.
Summary
Neuropeptide substance P (SP), which binds to the neurokinin 1 (NK1) receptor, has been implicated in
many functions such as mediation of pain. The population of neurones that expresses NK1 receptor,
and which are likely to respond to SP, has not been completely characterized. In this report, the
distribution and function of the NK1 receptor in the spinal cord and caudal brainstem neurones was
investigated. NK1 receptor is expressed on neurones; in laminae (L) I and III having a specific
morphology, sections cut parallel of the cervical cord show NK1 receptor-labelled neurones which give
rise to several branches and arborized dendrites in all directions in LI, at the thoracic and lumbar levels
on clusters of intermediolateral cell column (IML) neurones and at the sacral level on some cells
having large dendrites which are distributed bilaterally. These results indicate that the NK1 receptor is
expressed by several distinctly different morphological types of spinal neurones. In LI, neurones
resembling the flattened aspiny or pyramidal neurones were stained and many of these were shown to
be spinothalamic neurones. LI neurones are clear candidates for mediating signals, which normally lead
to perception of pain. Large numbers of IML and central canal (LX) neurones express the neurokinin 1
receptor indicating a primary role for SP in autonomic regulation.
Key Words:
Neurokinin, nociception, pain, spinal cord, substance P, tachykinins.
Introduction
The neuropeptide SP is present in two distinct populations of fine-diameter primary afferent axons
which terminate in the superficial dorsal horn of the spinal cord (1,2), and can be released following
different types of peripheral noxious stimulation (3,4). SP is also present in interneurones within the
dorsal horn (5), and axons descending from the brainstem and terminating within the spinal cord (6).
SP-containing axons form a dense plexus in LI and dorsal part of LII which is derived mainly from
primary afferents, but in addition significant numbers of SP-containing axons are present in the deeper
laminae of the dorsal horn, in LX and in the motor nuclei of the ventral horn.
Recently, following cloning of the NK1 receptor (7,8), it is now possible to map the distribution of this
receptor throughout the central nervous system (CNS). NK1 receptor antibody (9) allowed localization
at the cellular and subcellular level. In the spinal cord, NK1 receptor is expressed in several specific
neuronal groups, including neurones in LI, LIII-V, LX and in the IML (10-15). Numerous NK1
receptor-immunoreactive (-IR) neurones that are located in LI and which possess the receptor are likely
targets for SP-containing primary afferents (14). NK1 receptor-IR neurones are also present in
moderate numbers throughout the remainder of the dorsal horn, with the exception of LII where they
are infrequent (10,11,14). In LIII-IV strongly staining cells are seen, some of these have distinctive
long dendrites passing through LII and then branched extensively in LI (10,11,14,16). The low density
of NK1 receptor-IR neurones in LII is surprising, since this lamina contains many SP-containing
primary afferents. This suggests that some degree of mismatch between receptor and ligand is present
in LII (16). Labelled cells, which express NK1 receptor, were found mainly in LI and III and in the
lateral spinal nucleus (LSN) (17). Thus, it seems certain that a significant proportion of SP-containing
primary afferents terminate directly on spinothalamic neurones. Many neurones in the IML, which
contain sympathetic preganglionic neurones (SPNs), express NK1 receptor or mRNA for this receptor
(9,11,18). An autoradiographic study has shown that many SPNs are responsive to SP and selective
NK1 receptor agonists. However, these studies also revealed that not all preganglionic neurones
respond to SP (19).
The current investigations of the distribution of the NK1 receptor were initiated contemporaneously
with those of several other groups. The present study was particularly targeted on identifying patterns
of organisation within specific neurone groups expressing this receptor. Sections were cut in all planes
to see if repeated patterns of neuronal organisation could be observed. Regional differences in the
spinal cord and caudal brainstem were also investigated to determine whether any functional
specialization in these neurones might exist.
Materials and Methods
Twenty-five adult Wistar rats, weighing approximately 250 g of either sex were used. The rats were
deeply anaesthetized and perfused with a fixative containing 4% paraformaldehyde in 0.1M phosphate
buffer saline (PBS). Cervical, thoracic, lumbar and sacral spinal cord and caudal brainstem segments
were removed, postfixed for 4-6 h and then cryoprotected with 30% sucrose in 0.1M PBS overnight at
40C. Tissue blocks were cut on a freeze knife microtome into 40µm transverse, parasagittal
and dorsal planes sections and processed free floating. The endogenous peroxidase and
non-specific binding sites for antibodies were suppressed by treatment with 1% hydrogen
peroxide for 30 minutes and 10% normal donkey serum for one hour at room temperature
respectively. Sections were processed for immunocytochemistry by the avidin-biotinperoxidase complex (ABC) method (20).
The sections were incubated in primary antiserum to NK1 receptor (gift of Dr. Vigna) diluted 1:20000,
in PBS containing bovine serum albumin (2.5%) and TritonX-100 (2%), for 3 days at room
temperature. Subsequently the binding of primary antiserum was detected using biotinylated anti-rabbit
antiserum (1:200) and streptavidin-conjugated horseradish peroxidase (1:1000) (Amersham). Finally
the chromagen protocol of Shu et al. (21) was used to reveal the distribution of bound peroxidase.
Results
General overview; The labelling pattern and subpopulation of neurones expressing NK1receptor-IR, had similar characteristics at all segmental levels of the spinal cord. However,
there were significant segmental differences with respect to both the density of labelling and
numbers of the cell bodies labelled. In general, LI contained the highest density of NK1
receptor throughout the spinal cord. In contrast, there were few stained neurones in the LII at
all segmental levels (Fig 1). The pattern of NK1 receptor staining in the deeper dorsal horn
and ventral horn varied at different segmental levels. The dendrites of the same neurones in
the deeper laminae of the sacral level extended dorsally into the LI and medio-laterally to the
contralateral side (Fig 2).
Figure 1.
View of the NK1 receptor
staining pattern in the dorsal
horn. LI and LIII-IV contained
heavily stained neurones and
their dendrites. These
photographs also illustrate the
lack of staining in LII.
Figure 2.
Typical NK1 receptor neurone
in LIV-V of the sacral spinal
cord (arrow). Soma was
located in LIV-V and
dendrites radiate dorsolaterally and dorso-medially.
One dendrite runs medially
across the midline passing
through to the contralateral
site. Bar: 50µm.
Lumbar cord and caudal thoracic cord
Sections were cut in transverse, parasagittal and dorsal planes. The densest
labelling in the cord was found in the LI and consisted of a strongly stained neuropil
with many heavily labelled soma and dendrites (Fig 1). The majority of the staining
was located on dendritic processes that arborized rostrocaudally in the marginal zone,
these neurones were best seen in transverse and longitudinal sections. Sections were
cut parallel to the dorsal surface of the lumbar and caudal thoracic spinal cord,
however those cut from the cervical cord were particularly valuable for the
appreciation of the dendritic structure and spatial relationships of NK1-receptor
expressing cells in this lamina. Neurones with dendrites parallel to the dorsal surface
were observed in these sections (Fig 3) show similar cells in the cervical cord. It can
be seen that their soma (approximately soma diameter; 26µm) and principal dendrites
which radiated medially, laterally, rostrally and caudally were typically confined to
LI. These principal dendrites gave rise to their first branches 42µm from the soma.
The distance between their soma was typically 103µm (measured on the basis of the
distance to their nearest neighbour). No pattern was apparent within their
organisation, and they did not seem to form rows or lattice arrangements. LI neurones
occasionally had dendrites extended ventrally into LII and LIII (Fig 1). In the
Lamina I;
transverse sections, LI neurones had cell bodies (soma diameter; 16µm, mean inter
soma distance; 38µm) and three dendrites (from soma to the first branch of dendrites
22µm). The dendrites show only occasional spines.
Lamina II (Substantia gelatinosa); NK1-receptor positive soma were rare in LII and those that
were observed were not typical Islet or Stalked cells (22,23). Despite the paucity of NK1receptor expressing soma, the main NK1 positive elements in LII were dendrites of positive
neurones in deeper laminae (Fig 1).
Very characteristic neurones
were found scattered through these deeper laminae. Their soma typically gave rise to
small numbers of dendrites, which extended dorsally, ventrally and laterally. The
dorsally directed dendrites usually pass through LII without branching and start to
break up into smaller branches in the outer part of LII and in LI. The ventrally
directed dendrites were typically less densely stained. In sacral cord, NK1-receptor
expressing neurones were seen in LI and LIII-V. The medio-lateral extent of the
dorsal dendrites was frequently across the whole of one side of the spinal cord. In the
sacral segment of cord, neurones were observed with dendrites crossing the midline
(Fig 2).
Laminae III-V (Nucleus proprius and the neck of the dorsal horn);
Central canal; In transverse sections of the lumbar cord, a heavy concentration of NK1
receptor-IR neurones was observed surrounding the central canal (Fig 4). Many neurones
(soma diameter; 19µm, mean inter-soma distance; 48µm) had on average four dendrites,
(distance to first dendritic branches; 19µm) passing very close to the ependymal cells lining
the LX but these did not seem to make special contact with these cells. In the rostral lumbar
and caudal thoracic cord where IML neurones were present in many of the NK1 receptor-IR.
These densely stained neurones
gave rise to dendrites which ran dorsally often into the lateral funiculus and ventrally
into the ventral horn and laterally towards the lateral white matter (Fig 4). Mediolaterally directed dendrites of the IML neurones reached the LX (Fig 4). The dendrites
of several closely packed neurones formed closely associated bundles. There were
some labelled neurones located midway between the IML and the LX (Intercalated
nucleus) (Fig 4). Their dendrites extended both medially and laterally, to the IML and
LX, respectively.
Intermedio-lateral horn-Sympathetic preganglionic neurones;
Figure 3.
Section cut parallel to dorsal
surface of the cervical spinal
cord showing NK1 receptor
labelled neurones in LI. These
photographs show the large
size and sparsely branching
dendrites of these cells. Their
dendritic fields reached over
1mm in diameter and
overlapped extensively. Bar:
50µm.
Figure 4.
NK1 receptor neurones are
occasionally located in the
intercalated nucleus (arrow)
and their dendrites run
laterally into IML and
medially towards to the LX.
Neurones with dendrites,
which extended laterally into
the lateral funiculus are seen in
IML. (cc: central canal, IML:
intermediolateral cell column).
Bar: 200µm.
Clustering of NK1-receptor neurones was observed in the SPNs in longitudinal sections of thoracic
spinal cord. Their dendritic processes ran medially and laterally into the lateral funiculus (Fig 5). The
clusters were spaced at ~150-300µm intervals.
Ventral horn; Lightly labelled triangular or multipolar neurones with four dendrites, which
radiated in all directions were found in the ventral horn. In addition, expression of NK1receptor in sacral cord was found in a distinct region in the ventral horn, which was identified
as Onuf’s nucleus (Fig 6`).
Figure 5.
The NK1 receptor neurones in
the IML appeared in cluster in
longitudinal section of the
thoracic cord. Cells had
several dendrites that run
medially and laterally. Bar:
200µm.
Figure 6.
Low power view of transverse
sections of the sacral spinal
cord illustrating the general
pattern of distribution of the
NK1 receptor. In addition,
expression of NK1 receptor is
found in a distinct region in
the ventral horn, which was
identified as Onuf's nucleus
(arrow). Bar: 250µm.
Lateral spinal nucleus; The LSN is a region of the dorsal funiculus lateral to the main dorsal
horn, which contains a small number of relatively large neurones. Processes of NK1 receptor
neurones were seen in the dorsal lateral funiculus.
Trigeminal (Vth nerve) nucleus caudalis; NK1 receptor-expressing neurones in Vnc were found
to resemble the distribution with the spinal cord dorsal horn. Many densely stained NK1
receptor-IR neurones with dendrites, which arborized laterally and medially were present in
the marginal layer (Fig 7). LII was significantly devoid of the NK1 receptor expressing
neurones (Fig 7). Lamina II contained only dendritic processes of neurones that were usually
derived from laminae III or I.
In trigeminal nucleus caudalis (Vnc), a similar pattern of the distribution of
the NK1 receptor was seen to that seen in the dorsal horn of the spinal cord. Bar:
50µm.
Figure 7.
Most NK1 receptor-IR neurones in the spinal cord are relatively large. Their
soma give rise to several principal dendrites which are not densely covered in spines.
Their dendrites branch occasionally and do not form tufts or glomerular structures.
They show little rostral-caudal or medio-dorsal flattening. Neurones in LI do,
however, have a very restricted dorsal-ventral extent. Only occasional fine processes
were stained which could be axons or long terminal dendrites. The NK1-receptor is
probably not expressed on most axons. Some regional differences in receptor
expression in the dendrites in a single neurone were also apparent.
Summary;
Discussion
The distribution of the NK1 receptor-immunoreactivity in the spinal cord
found in this study confirmed previous findings using the same antibody raised
against the C-terminal sequence of the receptor (10-16).
Spinal cord;
Superficial dorsal horn (LI-II); In transverse section, NK1 receptor expressing neurones in LI
resembled the flattened aspiny and pyramidal cells described by Lima and Coimbra 1989 in
Golgi stained material (24,25). Many of these morphological types of LI neurone project into
the thalamus (26). Many spinothalamic neurones in LI and the LSN were also NK1 receptorIR (15). It now appears as though there is one major target for SP-containing afferents is this
group of spinothalamic neurones and hence, a mono-synaptic relay pathway between the
periphery and the thalamus is a major route by which SP-containing afferents relay
information to the cortex. If the morphology of these LI neurones is considered it is
immediately apparent that they are large and have a considerable dendritic spread in both the
medio-lateral and rostro-caudal dimensions. It would be predicted that each of these
neurones receives input from a relatively large peripheral receptive field. Hence, they would
be more suited to integrating input from large tissue areas and encoding the extent of injury
rather than specifically pinpointing the precise site of an injury. Some LI neurones projecting
to the parabrachial nucleus also express NK1 receptor-IR. It is probable that projections to the
parabrachial nucleus arise as collaterals of axons LI neurones that project to the thalamus
(12).
A few NK1 receptor-expressing marginal neurones had dendrites, which arborized ventrally through
the LII and LIII. LI, NK1 receptor expressing neurones with ventrally directed dendrites have been
observed in other studies (11,12,14). However, neurones described in Golgi studies (24), which have
ventrally directed dendrites appear to be different as they are covered in spines. SP-containing primary
afferent fibers largely terminate in both LI and in LII0 of the dorsal horn (1,2,27) and these descending
dendrites would provide termination sites for SP-containing afferents in LIIo.
The intrinsic Islet cells (22) of LII did not express the NK1 receptor. These neurones are the target for
unmyelinated afferents containing FRAP which terminate in glomerular synapses. The Stalked cells
(23) which have soma mainly at the LI-LII boundary also do not resemble any of the NK1 receptor
neurones as they are covered in spines which are almost absent from the NK1 neurones.
Deeper dorsal horn; NK1 receptor expressing neurones are large and in some cases, a single
neurone had a dendritic arborization extending mediolateral across the whole of the dorsal
horn of one side of the spinal cord. Parasagittal sections revealed that they also had dendrites
extending rostrocaudally. The morphology of these NK1 receptor cells is similar to the
antenna cell described in Golgi study (28). The dendrites which are observed crossing LII will
belong mainly to these neurones with cell bodies in LIII-IV (10,11,14) whilst a few will belong
to LI neurones with ventrally directed dendrites (12,14). Again the large size of the dendritic
spread of these neurones suggests that they receive input from large receptive fields. The
ventrally directed dendrites would also be optimally placed for input from myelinated afferents.
In a study by De Koninck et al. (29) in the cat, three wide dynamic range neurones in LIII-IV
were shown to have many SP-containing synaptic contact upon them, and assuming that
these expressed the NK1 receptor. Hence, it is highly probable that some if not all, of these
large LIII-IV NK1 receptor-expressing neurones are wide-dynamic range neurones receiving
input from both high- and low-threshold afferents. Potentially this population of NK1 receptor
expressing neurones could have axons projecting to the diencephalon, midbrain, dorsal
column nuclei, reticular formation or lateral cervical nucleus (30).
NK1 receptor in the neck of the dorsal horn; It is not clear that these neurones receive a
nociceptive SP input, although scattered SP terminals are located in the neck of the dorsal
horn (1,2). Consistent with detailed light microscopic analysis of the distribution of NK1
receptor (11), a few NK1 receptor-expressing neurones in LV had dendrites that extended
into the LII. In most of the cases however, their dendrites did not extend into LII. Retrograde
labelling studies have shown that several LV NK1 receptor expressing neurones project to the
thalamus (15) and a few also project to the parabrachial nucleus (12). Interestingly however,
many of these neurones in the neck of the dorsal horn can be labelled from injection of the
cerebellum (31). It is well established that SP is present in descending pathways from the
brainstem and this projection accounts for much of the diffuse SP-containing fibre plexus in
the deeper dorsal horn, around the central canal, in the IML and the ventral horn (1,32).
Around the central canal (LX); The findings in this study confirmed previous
observations (11). At least some fibres of somatic origin and also visceral afferent
fibres terminate in LX bilaterally. It is probable that these inputs come from visceral
C-fibre primary afferents, some of which may be concerned with visceral pain (33).
The terminals of some afferents to LX clearly had a spatial relationship to locations of
dendrites and somata of preganglionic neurones (34). Some of the NK1 receptor-IR
neurones in LX are spinothalamic neurones (15). Electrophysiological studies of the
neurones in this region revealed that they received primary afferent inputs from high
threshold mechanical nociceptors, some of which may contain SP (33).
Intermediolateral horn-Sympathetic preganglionic neurones; The majority of the NK1
receptor expressing neurones which have been identified in this study were similar to those
described previously in IML (11,13,35). In longitudinal sections it could be seen that these
neurones were arranged in groups. These groups were spaced fairly irregularly at intervals of
approximately 200 µm. This does not correspond to segmental spacing.
Their morphology and subsequent retrograde labelling studies identify many of these IML
NK1 receptor expressing neurones as SPNs (35-37). However, less than one-third (29.9%) of
the total number of SPNs in the IML of adult rat express NK1 receptor (36). This suggests that
SPNs do not form a homogeneous population and that they could have diverse functions. The
studies of NOS expressing neurones in the IML clearly reinforce this view of several distinct
populations of SPNs. In fact there is considerable evidence that SP contributes to the
regulation of sympathetic function. A high level of SP-IR has been found close to SPNs
neurones and small number of SP-containing terminals contacted SPNs (38). In an electronmicroscopic study in the rat it was shown that in the IML of midthoracic spinal cord, only 37%
of SP-IR varicosities made synapses on SPNs that expressed NK1 receptor. Thus it was
suggested that SP might be co-localised with another tachykinin, which binds to a different
neurokinin receptor. SP released from these terminals could also diffuse from its site of
release to influence SPNs that have NK1 receptor on their surfaces by volume transmission
(13,37). NK1 receptor is found predominantly in thoraco-lumbar SPNs projecting to adrenal
medulla suggesting a target-related distribution of the receptor (37).
Lateral spinal nucleus; LSN was first described as "a nucleus in the dorsolateral funiculus of the
rat" (39). In this region, neurones project mainly to mesencephalic areas, including the
periaqueductal grey and reticular formation (25). A major projection of the axons to the
hypothalamus from the LSN has recently been demonstrated by Giesler et al. (40). The LSN
contains many SP-IR fibres and terminals (2). In the present study, intensely labelled
dendrites in the dorso-lateral funiculus were seen that arose from the labelled neurones in the
IML (11).
Ventral horn; Occasional staining was seen in the ventral horn of the spinal cord. In the sacral
region, immunostaining was seen in the lateral part of the ventral horn and Onuf's nucleus. This
nucleus was also seen to bind radiolabelled SP in an autoradiographic study (19). Hemisection of the
spinal cord does not alter the level of SP around the motoneurones in Onuf's nucleus (41). Small
diameter primary afferent fibres do not arborize in the ventral horn and hence it is probable that SP
input to this region derives from intra- or supra-spinal sources. There is however, a direct primary
afferent-derived SP input to NK1 receptor-IR parasympathetic preganglionic neurones of the sacral
spinal cord.
NK1 receptor-immunoreactivity in the Vnc was similar
to that seen in the dorsal horn of spinal cord. A dense network of NK1 receptor cell
bodies and dendrites was observed in LI of the Vnc in an agreement with a previous
study (11). SP-IR in the Vnc was decreased following lesion of the trigeminal
ganglion or trigeminal rhizotomy (42), suggesting that SP-IR fibres originate
predominantly from primary afferent neurones whose cell bodies are located in the
trigeminal ganglion. Hence, it is reasonable to assume that these LI and LIII NK1
receptor neurones in the Vnc are the target of SP-containing afferents from the head.
Trigeminal nucleus caudalis;
SP/ NK1 receptor mismatch and volume transmission; In spinal cord and in substantia
nigra, the mismatch between the distribution of SP and NK1 receptor has been emphasised
by some authors (16,43). This is particularly apparent in LII where the intrinsic neurones do
not express this receptor but are in a region with a rich SP innervation, which is a site of SP
release (44). This leads to two alternative interpretations. Firstly, that not all SP acts at the
NK1 receptor and secondly that SP diffuses to act on distant receptors by a process of
"volume transmission". It seems likely that SP acts at a different receptor in the case in the
substantia nigra and it is known that SP will act on the NK2 and NK3 receptors. Many NK1
receptors are not located in post-synaptic membranes and are distributed over the plasma
membrane of extensive regions of the dendrites and soma. These extra-junctional receptors
can be internalised when they bind to SP and hence, would be assumed to be active. Hence,
it is probable that in the dorsal horn SP acts by volume transmission.
Conclusion; There
is a tendency to equate SP exclusively with nociception even to refer
to it as a “pain” transmitter. Clearly the distribution of the NK1 receptor indicates its
involvement in several other processes besides nociception. However, the LI neurones
expressing the NK1 receptor are clearly a major candidate neurone group for
signalling responses initiated by noxious intensity stimuli which would under most
circumstances lead to pain. In this respect they are of major interest for the targeting
of analgesic compounds. The involvement of LIII-IV NK1 receptor expressing
neurones in relaying information leading to pain perception is not so clear as they do
not typically have axons projecting to the thalamus. They are however responsive to
noxious intensities of stimuli and may relay this information to the thalamus by a
multi-synaptic pathway.
In considering other spinal neurones expressing this receptor it is probable that they could be receiving
SP-containing inputs from other sources. This is particularly clear in the case of SPNs, which express
this receptor and probably do not have a primary afferent input from SP-containing afferents but do
clearly get descending connections from axons containing this peptide. The specific function of LX
neurones expressing the NK1 receptor or the origin of the SP activating these receptors requires further
study.
Acknowledgement: This study was supported by Kafkas University, Kars, Turkey.
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