Comp. Biochem. PhysioL Vol. 93A, No. 1, pp. 183-193, 1989 0300-9629/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press pie

MINI REVIEW

CYTOCHEMISTRY OF THE TRIGEMINAL AND DORSAL ROOT GANGLIA AND

SPINAL CORD OF THE RAT

M.A. KAI-KAI

Department of Preclinical Veterinary Sciences, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK

(Received 23 August 1988)

AImraet--1. The primary sensory neurones have been classified into large light (LLC), type A, small dark (SDC), type B and type C cells on the basis of size, ultrastructural and immunocytochemical characteristics.

2. Subclassifications have been described according to the configuration and spatial organization of cytoplasmic organelles.

3. Furthermore, the LLC are immunoreactive with a monoclonal antibody, RT97, directed against a neurofilament protein and the SDC are positive with anti-arginine vasopressin (AVP).

4. The majority of the neurochemical substances including substance P (SP), somatostatin (SOM), fluoride resistant acid phosphatase (FRAP), 5-hydroxytryptamine (5-HT) and glutamate were localized to the small and intermediate diameter neurones measuring 9-40 lam.

5. The cytochemistry of the dorsal horn was similar to the dorsal root ganglia (DRG). 6. There is good evidence that substance P (SP) and somatostatin (SOM) are transmitters for a

proportion of nociceptive neurones but the neurotransmitters utilized by the rest of the subtypes are unknown.

7. 5-hydroxytryptamine (5-HT) and glutamate may be putative transmitters of the primary sensory neurones as they are localized in 28-30% of the SDC.

8. The wider distribution and extensive coexistence of the neuropeptides is incompatible with neurotransmitter function, but some may be neuromodulators whereas others such as arginine vasopressin (AVP) are useful markers for idenifying type B neurones.

CLASSIFICATION OF NEURONES IN THE CRANIAL AND SPINAL GANGLIA

Morphology

The perikarya of sensory neurones have been classified into type A and B cells (review Lieberman, 1976). The majority of type A neurones are large (35--50/~m) with translucent cytoplasmic organelles that have low affinity for toluidine blue, whereas type B cells have a small diameter (9-35/~m) and dense granular endoplasmic reticulum that stain intensely with toluidine blue (Fig. IA). Hence, the cells have been appropriately termed large light (LLC) and small dark cells (SDC, Lieberman, 1976).

Another scheme of classification is based on differ- ences in the configuration and spatial organization of cytoplasmic organeUes. Duce and Keen (1977) have used this together with the structure of the Golgi apparatus and its pattern of impregnation with zinc iodide osmium to describe seven subtypes of cells. Subtypes AI_ 3, B 1 and 2 and B 3 form 46%, 56% and 1%, respectively, of the total population of cells (Table 1).

The non-neuronal mast cells are also important in neurotransmitter studies because they have similar diameters with the smallest neurones and it is not always easy to differentiate the two cell populations.

Mast cells are recognized by their high affinity for 1% alcian blue (AB) in an acidic solvent. Using this histochemical criterion we published (Kai-Kai and Keen, 1985) the first report on endoneurial mast cells in lumbar DRG. Further studies have revealed that they are common in DRG from the different spinal levels. The mast cells measure 4-8 #m in diameter and form 6-8% and 14% of the total cells in the DRG and trigeminal ganglion (TG), respectively (Table 2).

Ultrastructure and cytochemistry

Sommer et al. (1985) have proposed a classifi- cation that combined ultrastructure with the cyto- chemistry to describe six (Al_a, B1-3) subpopulations of cells that are similar to the subclasses of previous authors (Duce and Keen, 1977; Rambourg et al., 1985). Subclass A~ were characterized by a strong carbonic anhydrase (CA) activity and by accumula- tion of radiolabelled glutamine. Subclass B formed 63% of the total cell population and was devoid of CA activity in both neurones and gila as well as the inability to accumulate exogenous [3H]- glutamine. Subclass C measured less than 20 #m in diameter and had similar structure to B 3 (Rambourg et al., 1985). This cell type was characterized by the presence of acid phosphatase (AP) and high affinity

183

184 M.A. KAI-KAI

C

15'

10

5

%

15

lO

lO

2

20 3O 40

C e l diameter (jJm)

5b

Fig. I. (A) One #m araldite sections through the fourth lumbar DRG showing the small dark cells (SDC) s.tained intensely with toluidine blue and the large light cells (LLC) with low affinity for the dye. (B) Endoneurial mast cells stained with alcian blue in a 1 #m araldite section through the trigeminal ganglion. (C) Size distribution histogram of AVP-IR neurones in the trigeminal (1) and lumbar five ganglion (2). (D) and (E) Alternate mirror-image of 6/~m paraffin sections through L5 DRG reacted with antisera to RT97 (D) and AVP (E). The patterns of immunoreactivity in both sections are complementary. RT97-positive cells (A-C, white) are negative for AVP (A42, black) and RT97-negative (d-h, black) are

positive for AVP (a-h, white).

Neuronal cytochemistry

Table 1. Classification and percentage of cell type occurring in dorsal root ganglia (DRG) neurones

R.efcrcnces

Andros Duce and Keen Rambourg et al. Sommer et al. (1961) (1977) (1983) (1985)

Cell type (Rat %) (Rat %) Rat (diameter) (Mouse %)

A l 25 31 41)-75 #m 12 A 2 2 8 16 A 3 8 5 20-50 # M 8 B t 56 46 B 2 10 10 63 B 3 l I C < 2 0 # m

185

uptake for exogenous [3H]-glutamine, similar to B 2

and A2.

Immunocytochemistry

Class A and B neurones have distinct immunocyto- chemical properties. The 25-30% DRG neurones that constitute the large type A cells react with a mono- clonal antibody, RT97 directed against the neuro- filament protein (NP) subunits, 68, 145 and 200 kDa (Anderton et al., 1983) while the remaining (70-75%) are non-immunoreactive. The majority of these, about 49% and 62% in the trigeminal (TG) and fifth lumbar ganglia, respectively, express arginine vaso- pressin (AVP, Kai-Kai et al., 1985). The size distribu- tion histogram shows the positive cells measuring 7-35 #m in diameter in both the cranial and spinal ganglia (Fig. 1C). AVP-containing cells stain in- tensely with TB and the large AVP-negative neurones had low affinity for TB. Analysis of alternate mirror- image sections showed RT97 ÷ neurones negative for AVP whereas the negative RT97 cells were immuno- reactive for AVP (Fig. 1D and E). The two groups of cells did not seem to overlap, thereby confirming that the majority of AVP-IR neurones belong to class B. The size distribution histogram of cells positive with both antisera appears to be complementary.

The staining of type A and B cells by RT97 and AVP, respectively, is consistent with the ultra- structure of these cells. Type A is characterized by numerous neurofilament bundles and class B by an abundance of ribosomes that indicate hormonal synthesis.

To support this scheme of neuronal marking, RT97 and AVP staining should be compared to other cytochemical markers. AVP has been shown to coexist with some substances demonstrated in the type B neurones, such as oxytocin (OXT), SP, fluoride-resistant acid phosphatase (FRAP, Kai-Kai et al., 1986) and somatostatin (Che and Kai-Kai,

1988). A Comparable study has not been reported for RT97.

NEUROCHEMISTRY OF SENSORY NEURONES

A variety of neuroactive substances have been demonstrated in the neurones of the cranial and spinal ganglia by histochemistry and immunocyto- chemistry. Some occur singly but the majority coexist and some primary afferent neurones are now known to contain as many as four neuropeptides (e.g. Ching et al., 1985). The DRG from all spinal levels seem to contain equal proportions of immunoreactiv¢ sub- stances but some peptides tend to concentrate in specific regions as is shown with the high levels of the vasointestinal peptide (VIP) in the lumbosacral ganglia of the rat (Anand et al., 1983).

Neuropeptides

The neuropeptides in sensory ganglia of all the species investigated so far are located in subpopula- tions of small diameter neurones. There is agreement about the subtypes containing specific neuropeptides but there is disparity about the actual proportion of cells immunoreactive with specific antisera. This can be attributed to experimental procedures rather than a reflection of interspecies differences. One important factor is the in vivo or in vitro application of colchicine or vinblastine which blocks axonal transport result- ing in accumulation of chemical substances in the soma and the subsequent visualization of antigens that would otherwise not have been detected.

Neurohypophysial hormones. The neurohypo- physial hormones, AVP, OXT and their precursor neurophysins detected in extracts of the cranial and spinal ganglia colute with the synthetic molecules on reversed phase high performance liquid chroma- tography (Kai-Kai et al., 1985). The significance of AVP-IR neurones has already been discussed. OXT is also contained in cells of similar size to AVP. In

Table 2. Percentage distribution of mast cells and neurotransmitter substances in the TG and D R G neurones of the rat

Ganglia

Median cell Substance Trigeminal Cervical Thoracic Lumbar Sacral diameter

AVP 49 _+ 2 53.7 ± l 56.07 ___ 3 52 _+ 3 52.67 + 1 OXT 59.8 _ 8 41.52 ± 2 39 ± 4 38 _+ 2 39 ± 4 SP 4 2 . 5 ± 3 31,9 ± 2 3 8 ± 2 36.2__. 1 21.6 ± 0.8 SOM 39.28 _+ 2 30.8 ± 1 32,9 ± 3 32.5 ± 2 19.6 ± l FRAP 35,2 ± 3 32.8 ± 2 20.4 ± 1.3 5-HT 38.53 ± 2 33,5 ± 2 36.78 ± 1 41,98 __. 4 21.8 ± 1.1 Glutamate 30 ± 2 18.5 ± l

186 M.A. KAI-KAi

both the TG and DRG, 49-51% of neurones are positive with antisera to OXT (Table 2) and nearly all of these show coexistence with AVP (Kai-Kai et al., 1986). This is contrary to the situation in the hypo- thalamo-neurohypophysial system where AVP and OXT are contained in separate cells (Vanderhaeghen et al). Furthermore, the neuropeptides are not con- tained in secretory vesicles (Che and Kai-Kai, 1988) unlike the hypothalamo-neurohypophysial system where AVP and OXT occur in distinctive granules. The high proportion of AVP and OXT-positive neurones is incompatible with a neurotransmitter function.

Substance P. SP was one of the earliest neuro- peptides described in the sensory ganglia and it soon became the leading putative neurotransmitter for nociceptive neurones (for review, see Salt and Hill, 1983; Hokfelt et al., 1983). There is general agreement that the peptide is contained in small and intermediate-sized cells, but there is inconsistency about the actual proportion of cells that contain SP. While Hokfelt et al. (1976) reported 10-20% in an unspecified DRG of the rat, Ju et al. (1987) demon- strated 30% in the lumbar DRG of the same species. The percentage varies in the TG with colchicine treatment; for example, Lehtosalo (1984) observed 10-30% of SP-immunoreactivity (SP-IR) in un- treated animals compared to an increase of up to 50% after in vivo application of colchicine.

I have used a monoclonal antibody to correlate SP-immunoreactive to the ultrastructure of the pri- mary afferent neurones. SP-positive cells measured 7-47/~m with a median diameter of 21.6 _+ 0.8 and formed 36-38% of neurones in the TG and DRG, respectively. The immunoreactive neurones did not seem to have a preferential topographical distribution but were randomly distributed in the ganglia among the non-immunoreactive cells (Fig. 2A and B). The punctate immunoreactivity showed two patterns of distribution. In one subset of cells activity was concentrated in the perinuclear zone while in another it was evenly distributed throughout the cytoplasm. For convenience of description these two forms are termed immunoreaction ~ and fl, respectively (Fig. 2A). Relating this to the ultrastructure, the immunoreactivity corresponds with the location of the Golgi apparatus in the perinuclear area (Duce and Keen, 1977) and the E-reaction is typical of the distribution of the cytoplasmic organelles which char- acterise cell B~. It therefore appears that subclass Bl~ and ¢ (Duce and Keen, 1977; Rambourg et al., 1985) contain SP as the percentage of SP-positive cells observed in these studies is similar to the percentage of BI. The interpretation of SP-IR in these studies does not agree with the observations of Sommer and colleagues who report that SP is contained in subclass B3, a cell type that is cytologically identical to class C (Rambourg, 1985). The latter group forms less than 1% of the total cells in any DRG and is therefore unlikely to be the only group that contains SP.

Somatostat in ( S O M ) . SOM is less well investi- gated but the neuropeptide is now receiving in- creasing attention because of the probability of it being involved in thermoreceptive neurotransmission. Hokfelt et al., 1976) have reported 10% of DRG neurones containing SOM whereas Tuscherer and

Seybold (1985) described unequal distribution of SOM-IR in the DRG from the different spinal levels with the highest of 15.4% in the lumbar DRG and the least of 1.5% in the sacral ganglia; but Ju et al. (1987) did not show any differences among DRG from the different spinal levels. I have observed SOM-IR in small and intermediate-sized neurones measuring 9-35/~m with median value of 19.6_ 1 forming 32-39% of the trigeminal, sacral, thoracic and cervical ganglia in decreasing order (Table 2). The increase in the proportion of positive SOM-IR ceils is due to the more effective action of colchicine which results from in vitro incubation of ganglia whereas other authors have injected the drug into the subarachnoid space (Tuscherer and Seybold, 1985).

5-Hydroxytryptamine (5-HT)

The distribution of monoamines in somatosensory ganglia has not been well investigated. We published the first report (Bojkowski et al., 1983) on 5-HT in the TG and lumbar DRG, identified by high performance liquid chromatography (HPLC) with electrochemical detection. Biochemical manipulation suggested that the 5-HT was contained in both neurones and mast cells. Shortly afterwards, Lehtosalo et al. (1984) reported 5-HT-IR only in mast ceils of the TG; but our immunocytochemical studies located 5-HT-IR (Fig. 2C) to small diameter cells measuring 3-35/~m (Kai-Kai and Keen, 1985) confirming an earlier observation that 5-HT was contained in two populations of ceils. We therefore used a histochemical reaction specific for mast cells to differentiate this cell type from the neurones. Mast cell granules have a high affinity for alcian blue (AB) at pH 0.8, while neurones remain unstained in this acidic medium. AB-staining was combined with auto- radiographic (ARG) experiments. Both cell types accumulated radiolabelled 5-HT. The mast cells were identified as AB+/ARG ÷ or AB+/ARG -, since some mast cells did not have a high density of silver grains, and the neurones were AB- /ARG ÷ (Fig. 2D). Quan- titative analysis of the double-labelled experiments revealed that the endoneurial mast cells measured 3-9/~m with median value of 7.6/tm and formed 8.2% and 14% of the total cells in the fifth lumbar DRG and the TG, respectively (Kai-Kai and Keen, 1985). The mast cells in DRG from all the spinal levels have similar diameter with little variation in the percentage of cells (Table 2). The AB- /ARG + neurones measured 8-35#m in diameter with a median value of 21.8 #m and formed about 17% and 22% of cells in the DRG (Ls) and TG, respectively (Kai-Kai and Keen, 1985). Further studies have shown a slight variation in the percentage of 5-HT-IR cells in the sacral, cervical, lumbar and thoracic ganglia in decreasing order (Table 2). It is, however, unclear which cell type contributes to the increase because it is difficult to differentiate mast cells from neurones with the immunocytochemical method. 5-HT coexists with SOM (Kai-Kai, 1987) and SP (Fig. 2B and C). The size distribution graph (Fig. 4) also shows overlapping diameters of 5HT-containing cells with oxytocin and glutamate, but coexistence has not yet been proved. The size distribution histo- gram indicates that the 5HT-containing neurones are included in class B; this is also confirmed by the

Neuronal cytochemistry 187

coexistence of 5-HT with other neurochemical sub- stances demonstrated in this class. Furthermore, the majority of 5-HT-IR cells stain intensely with toluidine blue whereas the non-immunoreactive cells have low affinity for the dye. The ultrastructure of the 5HT-containing neurones (Kai-Kai and Keen, 1985) and the percentage of positive cells (Table 2) are characteristic of subtype Bm neurones.

Enzymes

Four enzymes, tyrosine hydroxylase (TH; Price and Mudge, 1983), the extralysosomal fluoride resis- tant acid phosphatase (FRAP), 5-nucleotidase and adenosine deaminase (Doddet al., 1983) have been reported in varying proportions of sensory neurones. FRAP positive cells are the most numerous and also the most extensively investigated to identify the most effective substrate as it is possible that the enzyme is a sensory neurotransmitter. /~-Glycerophosphate produces intense histochemical reaction (Fig. 2E) whereas Bucsics et al. (1988) recently reported that thiamine monophosphate is more sensitive than /~- glycerophosphate in biochemical assay. Figure 2E shows the distribution of FRAP in lumbar four DRG neurones. The positive cells were small and formed 32-35% of the total cell population (Table 2). This figure corresponds closely to that reported by other workers. For example, Dodd et al. (1983) observed FRAP staining in 35-50% of the trigeminal and DRG cultured neurones with very little variation of positive cells in ganglia from the different spinal levels. The enzyme may therefore serve similar func- tion in all the DRG. In earlier studies it was assumed that FRAP and related isoenzymes are characteristic of sensory neurones, but this is not the case as an identical isoenzyme is expressed by human prostatic cells and non-neural tissue (Dodd et al., 1983). It is therefore necessary to differentiate the sensory neuronal FRAP isoenzyme from those expressed by other cells, and Bucsics et al. (1988) have appropri- ately introduced the term SNAP (sensory neurone acid phosphatase) to make the distinction. We have to await the acceptance of this term.

Excitatory amino acids

Glutamate is considered a putative sensory trans- mitter (Johnson, 1977), but the demonstration of glutamatergic neurones has been hindered because radiolabelled glutamate and its analog I)-aspartate are accumulated by satellite gila (Duce and Keen, 1983). Although glia uptake does not prevent pene- tration into neurones, the labelled cells are few and not a true representation of the proportion of neurones that may utilize glutamate. For instance, when lumbar DRG was incubated in [3H]D-aspar- tate, only 2% of neurones accumulated the labelled material but all glial cells contained dense silver grains (Fig. 2F). But the problem seems to be mini- mized by immunohistochemistry because the anti- bodies are directed against the endogenous glutamate, yet there are still discrepancies about the subtype of glutamate-immunoreactive (GIu-IR) cells. Wanaka et al. (1987) has reported GIu-IR in inter- mediate- and large-sized DRG neurones of the rat, whereas Cangro et al. (1985) demonstrated glutaminase-IR exclusively in small cells in similar

ganglia of the same species. I have observed Glu-IR in predominately small diameter neurones and all satellite glia (Fig. 2(3). The immunoreactive neurones formed 30% of the total cells in the ganglia. The size distribution histogram revealed labelling of some intermediate-sized cells since the positive neurones measured 9-38 and 9-45 #m in the TG and thoracic DRG, respectively. Therefore the marking of type B neurones seems more consistent and this is also confirmed by the pattern of distribution of GIu-IR in the dorsal horn (Fig. 3D).

NEUROCHEMISTRY OF THE SUPERFICIAL DORSAL HORN

DRG neurones terminate in the dorsal horn (DH) of the spinal cord. Similarly, neurones from the TG terminate in the subnucleus caudalis (SNC) of the spinal trigeminal nucleus (STN). The SNC is sub- divided into laminae that morphologically resemble laminae I-V of the DH. The DH and its counter- part, the SNC, form the interface between the central and peripheral nervous systems. The majority of unmyelinated C-fibres and myelinated A fibres terminating in the marginal layer (lamina I) and the substantia gelantinosa (SG: laminae II) are derived from nociceptive neurones. Similarly, lamina I and the SG of the SNC receive nociceptive and thermal receptors (Magnusson et al., 1986). This differential projection of primary afferents into the DH and STN explain the high concentrations in these areas of the neurochemical substances present in the cranial and spinal ganglia associated with sensory transmission mechanisms.

Neuropeptides

The distribution of AVP-IR is consistent with its role as a chemical marker for class B neurones. The density of AVP-IR in laminae I-III shows a rostro- caudal gradient which reflects the presence of the peptide in nociceptive neurones. SP-IR distribution in the superficial DH (Fig. 3B) overlaps with AVP-IR. Both AVP and SP are also present in the dorsal grey commissure (DGC) and the dorsolateral funiculus (DLF) of the white matter.

5-Hydroxytryptamine

The punctate appearance of the 5HT-IR in the spinal cord (Fig. 3C) may be associated with positive terminals. Intense immunoreactive occurs in laminae I, II and X and to a lesser extent in laminae III; hence the distribution overlaps with the neuropeptides although light microscopic immunocytochemistry is inadequate to resolve colocalization at the terminals. The lateral funiculus was also positive for 5-HT, but the highest density of immunoreactive-fibres was observed in the autonomic area, the nucleus inter- mediolateralis of the thoracic and lumbar segments. The 5-HT in the spinal cord has been assumed to be derived from the supraspinal descending fibres (Dahlstrom and Fuxe, 1965), but the high concen- tration of 5-HT in the DRG (Bojowski et al., 1983) and the transport in the central branch suggests that the primary sensory neurones do contribute to the 5-HT in the DH.

188 M.A. KAI-KA1

Fig. 2

Neuronal cytoehemistry

Table 3. Distribution and coexistence of neuropeptides and FRAP in the TG and DRG

189

Species Ganglia Cell type Substance % Coexistence References

Rat small SP 30-50 Lehtosalo et al. (1984) Guinea pig Rat SOM 10-15 Hokfelt et al. (1976)

trigeminal.

DRG

DRG

small/ intermediate small/ intermediate

Rat SP +CCK-8 Tuseherer and Seybold SOM (1985) CCK-8

Rat DRG (culture) small FRAP 30-50 Dodd et al. (1983) small and SP 16 Dodd et al. (1983) large SOM 15 Dodd et al. (1983)

Cat DRG CCK-8 SP Leah et al. (1985) Rat DRG SP SOM Leah et al. (1985)

VIP Rat DRG Small and CGRP SP or Ju et al. (1987)

large SOM or galanin Rat TG

DRG CRF Jozsa et al. (1987)

G l u t a m a t e

The distribution of Glu-IR in the thoracic spinal cord is shown in Fig. 3D. The micrograph reveals two sources of glutamate in the DH. A group of intensely stained glutamatergic spinal interneurones and weak immunoreactive fibres observed in laminae I-III is consistent with the localization of glutamate in small diameter primary sensory neurones. The inter- neurones are generally round or spindle-shaped and small, measuring 3-6 #m in diameter and concen- trated in the lateral and medial regions of the dorsal horn.

PHARMACOLOGICAL STUDIES

The heterogeneity in structure, cytochernistry and function of the primary sensory neurones poses a problem in the elucidation of specific putative neuro- transmitters. Similarly, the spinal cord and its counterpart, the subnucleus caudalis, receive inputs from different systems, such as neurones in the cranial and spinal ganglia, the interneurones and from the bulbospinal system. All of these contain identical transmitter substances. Pharmacological and surgical lesions are used to study the influence of any one system on the whole. Of particular importance are the selective neurotoxin effects of capsaicin on putative nociceptive neurotransmitters. Capsaicin (8-methyl-N-Vanillyl-6-noneamide), the active ingre- dient of red peppers, has proved a useful tool in the identification of putative nociceptive transmitter sub- stances. The effect of neonatal capsaicin treatment on sensory neurones has been reviewed (Fitzgerald,

1983). It seems that the majority of neurochemical substances depleted and the neurones destroyed are associated with polymodal nociceptive transmission (for review, see Russell and Burchiel, 1984), but little is known about the physiochemical properties that make this class of sensory neurones susceptible to destruction by capsaicin. All the neuropeptides and FRAP occurring in the sensory ganglia and spinal cord are sensitive to systemic administration of capsaicin, either to neonates or adults of several mammalian species (Fitzgerald, 1983; Bucsics e t a l . ,

1988). 5-HT-containing sensory neurones are simi- larly sensitive to capsaicin. Neonatal Wistar rats were first injected with capsaicin on day 2 of life and on the subsequent 3 days to a cumulative value of 70 mg/kg. At 10 weeks old the sensory ganglia from capsaicin treated and control animals were processed by immunohistochemistry, light microscopic auto- radiography and transmission electron microscopy. Quantitative analysis revealed destruction of 55% and 80% of TG and DRG (Ls) respectively. The capsaicin-resistant 5HT-neurones showed deteriora- tion of cytoplasmic organelles but this did not impair the ability to accumulate exogenous [3H]-HT (Kai-Kai, 1988).

While the ICC and biochemical data provide good evidence for using capsaicin to differentiate the noci- ceptive transmitting from the innocuous neurones, the physiological effects are less convincing as several authors have questioned the specificity of capsaicin on sensory modalities (Russell and Burchiel, 1984) but the drug will continue to be a valuable tool in neuropharmacology for the elucidation of sensory, particularly nociceptive, neurotransmitters.

Fig. 2. (A) The figure shows SP-IR in two types of DRG (L5) neurones. In one subset of cells the reaction is concentrated in the perinuclear region (~t), while in another the immunoreactivity is evenly distributed in the cytoplasm. This pattern of ~t and fl localization is characteristic of type B~ and B~p respectively. (B) and (C) Alternate mirror-image sections reacted with antisera to SP(B) and 5-HT(C), revealing coexistence of the two substances. Cells A-C (white) are positive with both antisera whereas A and B reacted with neither. (D) Light microscopic autoradiogram viewed by dark-background microscopy. The dense silver grains overlay small diameter neurones in the DRG (L3). These cells have accumulated exogenous radiolabclled 5-HT. (E) Distribution of FRAP positive neurones among the negative cells in 14 #m frozen sections through the sixth thoracic DRG. (F) Light microscopic autoradiogram showing accumulation of [3H]D-aspartate by the satellite glia surrounding the sensory neurones in the fifth lumbar DRG. (G) Glutamate-immunofluorescence in small diameter neurones and satellite gila in 8 #m frozen

sections from the cervical (4) DRG.

Fig. 3. (A) AVP-IR in the caudal lumbar spinal cord with positive terminals in laminae I-III. With less intense activity in the dorsal grey commissure (DGC). (B) Intense SP-IR in the DH of the rostral lumbar spinal cord. Immunoreactive fibres are visible in laminae I-III and V and in the dorsal grey commissure (DGC) and dorsal of the lateral funiculus (LF). The dorsal funiculus (DF) is devoid of immunoreactivity. (C) 5-HT-IR in the DH, LF and nucleus intermediolateralis (IML) of the lumbar spinal cord. The punctate appearance of the immunoreactivity suggests localization at terminals. (D) Glutamate-im-

munofluorescence interneurones and fibres in laminae I-III of the thoracic DH.

190

Neuronal cytochemistry

A B 12

'tO

8

%

6

4

2

o lo 20 3o 40 so o lo 20 3o 40 so

Cell diameter qJm)

Fig. 4. Composite graph showing overlapping distribution of oxytocin (square), somatostatin (closed circle), 5-hydroxytryptamine (open circle), substance P (closed triangle) and glutamate (open triangle) in the trigeminal (A) and thoracic (B) ganglia. Most of the positive neurones are small, measuring 5-36 and 7-38 #m in (A) and (B), respectively, but some large cells in (A) are stained with oxytocin or somatostatin. The considerable overlap in cell sizes suggest coexistence, but careful analysis is required to demonstrate

which substances are colocalized.

191

FUNCTIONAL SIGNIFICANCE OF THE NEUROCHEMISTRY OF THE PRIMARY

AFFERENT NEURONES

There are more neurochemcial substances in the sensory ganglia than the stimuli they transmit and the high incidence of coexistence is against the majority of these substances functioning as neurotransmitters. The function of the neuropeptides in the pain path- way has been reviewed (Hokfelt et al., 1983). A parallel article by Salt and Hill (1983) covers a wider range of sensory transmitter candidates to include the distribution and criteria for transmission for the neuropeptides, the acid phosphatases and the excit- atory amino acids. The review by Basbaum (1985) deals with the relevance of cytochemistry to the function of the DH with particular reference to the pharmacology of transmission and modulation of nociceptive stimuli. All the available data provide good evidence for SP as the leading candidate for nociceptive transmission, but there are discrepancies about the stimuli involved. Kuraishi et al. (1985) postulate that SP is released in the DH by noxious mechanical stimuli, but other workers have reported that the peptide is released by thermal and poly- modal nociceptors (Wiesenfeld-Hallin, 1986), while the release of SOM is thermosensitive (Kuraishi et al., 1985). Both SP and SOM induce the biting, licking and scratching (BLS) syndrome. BLS mimics noxious peripheral stimulation (Wiesenfeld-Hallin, 1986) thereby rendering further support for the neurotransmitter function of SP and SOM. But evidence is emerging that questions the direct role of either of the two peptides in nociceptive transmission. For instance, Leah et al. (1985) attempted to correlate the immunocytochemistry of DRG neurones with the sensory modalities they transmit, but SP was not detected in many of the nociceptive neurones whereas it was present in those transmitting innocu- ous stimuli. Also, SP and SOM produce a slow

response of long duration (Wiesenfeld-Hallin, 1985). Furthermore, although the BLS behaviour is induced by intrathecal injection of SP, the desensitization does not result in a change of the baseline potential. The authors have indicated that these observations are more compatible with a neuromodulator than a neurotransmitter function, but other interpretations are conceivable, such as that proposed by Piercey and co-workers (1983). The latter group have suggested that SP could be a transmitter for intense diffuse pain but not for extremely sharp pain. This concept needs further analysis but it is compatible with the transmission of pain by multiple pathways.

Contrary to the neuropeptides, it is the inhibitory role of 5-HT that has been emphasized, since there is good evidence for the inhibitory modulation of primary afferent neurones via enkephalinergic inter- neurones (review Duggan, 1985), but 5-HT excites interneurones in the substantia gelatinosa (Todd and Millar, 1983) and elicits a variety of responses in the somata of bullfrog DRG neurone. Holziv et al. (1985) reported 5-HT depolarization of 47% and 70% of type A and B DRG neurones, respectively. It is therefore probable that 5-HT could function as an excitatory transmitter in the somatosensory pathway.

Little is known about the function of glutamate in sensory neurones, but there are suggestions that it may be a transmitter for large A-fibres and that some spinal interneurones are amino acidergic, utilizing either aspartate or glutamate (Hutchinson et al., 1978). The distribution pattern of Glu-IR in the DRG and DH support a hypothesis that glutamate is a putative neurotransmitter in class B neurones.

From the evidence available it seems that the classical neurotransmitters, for example 5-HT, and glutamate may play a direct role in sensory trans- mission. The majority of the neuropeptides are too widely distributed to be neurotransmitters; some could be neuromodulators whereas others may be vestigial peptides of little functional significance.

C.B.P. 93/IA~M

192 M.A. KAI-KA1

Acknowledgements--The preliminary work was done in the Department of Pharmacology, University of Bristol. The Wellcome Unit, Department of Prectinical Veterinary Sciences, University of Edinburgh is also thanked for maintenance of the animals.

REFERENCES

Anand P., Gibson S. S., McGregor G. P., Blank M. A., Bocarese-Hamilton A. J., Polak J. M. and Bloom S. R. (1983) VIP-containing system concentrated in the lum- bosacral region of the human spinal cord. Nature 305, 143-145.

Anderton B., Cookham H. B., Garson J. A., Harper A. A. and Harper E, I. (1983) A monoclonal antibody against neurofilament protein specifically labels the large light cell population in rat dorsal root ganglia. J. Physiol., Lond. 334, 97P-98P.

Andres K. H. (1961) Untersuchungen uber den Feinbau yon spinal ganglion. Z. Zellforsch 55, 1-48.

Basbaum A. I. (1985) Functional analysis of the cyto- chemistry of the spinal dorsal horn. Advances in Pain Research and Therapy (Edited by Fields H.L., Dubner R., Cervero F. and Jones L. E.), Vol. 9, pp. 149-175. Raven Press, New York.

Bojowski C. J., Kai-Kai M. A., Keen P. and Kilpatrick I. C. (1983) On the 5-hydroxytryptamine content of rat sensory ganglia. Br. J. Pharmacol. 80, 674P.

Bucsics A., Sutter D., Jancso G. and Lembeck F. (1988) Quantitative assay of capsaicin-sensitive thiamine mono- phosphatase and fl-Glycerophosphatase activity in rodent spinal cord. J. Neurosci. Methods 24, 155-162.

Cangro C. B., Sweetnam P. M., Wrathall J. R., Hasen W, B., Curthoys N. P. and Neale J. H. (1985) Localization of elevated glutaminase immunoreactivity in small DRG neurones. Brain Res. 336, 158-161.

Che Yan Ming and Kai-Kai M. A. (1988) Distribution of the neurohypophysial hormones in neurones and termi- nals in sensory ganglia. Neurosci. Left. Suppl. 32, $82.

Ch'ng J. L., Christofides N. D., Anand P., Gibson S. J., Allen Y. S., Su H. C., Tatemoto K., Harrison J. F. B., Polak J. M. and Bloom S. R. (1985) Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal path- ways to injury. Neuroscience 16, 343-354.

Dahlstrom A. and Fuxe K. (1985) Evidence of the existence of monoamine containing neurones in the cen- tral nervous system. II. Experimentally induced changes in the intraneuronal levels of bulbospinal neuron system. Acta Physiol. Scand. 64 (Suppl. 247), 5-36.

Dodd J., Jahr C. E., Hamilton P. N., Heath M. S. J., Matthew W. D. and Jessell T. M. (1983) Cytochemical and phsyiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation. Cold Spring Harbor Symposia on quantitative Biology 48, 685~595.

Duce I. R. and Keen P. (1977) An ultrastructural classification of the neuronal cell bodies of the rat dorsal root ganglia using zinc iodide osmium impregnation. Cell Tiss. Res. 185, 263-277.

Duggan A. W. (1985) Pharmacology of descending control systems. Phil. Trans. Roy. Soc. B, Lond. 308, 375-391.

Fitzgerald M. (1983) Capsaicin and sensory neurones--a review. Pain 15, 109-130.

Hokfelt T., Elde R., Johansson O., Lulft R., Nilsson G. and Arimura A. (1976) Immunohistochemical evidence for separate populations of somatostatin-contalning and sub- stance-P-containing primary afferent neurones in the rat. Neuroscience 1, 131-135.

Hokfelt T., Skirboll L., Lundberg J. M., Dalsgaard C. J., Johansson O., Pernow B. and Jancso G. (1983) Neuropeptides and Pain Pathways. In Advances in Pain Research and Therapy (Edited by Bonica J. J., Lindblom U. and Iggo A.), Vol. 5, pp. 227-246.

Holziv G. G., Shelfner S. A. and Anderson E. G. (1985) Serotonin depolarizes type A and C primary afferents. An intracellular study in bullforg dorsal root ganglion. Brain Res. 327, 71-79.

Hutchinson G. B., McLennan H. and Wheal H. V. (1978) The responses of Renshaw cells and spinal interneurones of the rat to L-glutamate and L-aspartate. Brain Res. 141, 129-136.

Jozsa R., Korf H. W. and Merchenthaler I. (1987) Growth hormone-releasing factor (GRF)-like immunoreactivity in sensory ganglia of the rat. Cell Tiss. Res. 247, 441444.

Ju G., Hokfelt T., Brodin E., Fahrenkrug J., Fischer J. A., Frey P., Elde R. and Brown J. C. (1987) Primary sensory neurones of the rat showing calcitonin gene-related pep- tide immunoreactivity and their relation to substance P, somatostatin, galanin, vasoactive intestinal polypeptides and cholecystokinin-immunoreactive ganglion cells. Cell Tiss. Res. 247, 417-431.

Kai-Kai M. A. (1987) Immunohistochemical colocalization of 5-hydroxytryptamine and somatostatin in primary afferent neurones of the rat. J. Anat. 155, 225.

Kai-Kai M. A. (1988) Response of 5-hydroxytryptaminergic neurones to capsaicin. J. Anat. 158, 218.

Kai-Kai M. A. and Keen P. M. (1985) Localization of 5-hydroxytryptamine to neurones and endoneurial mast- cells in sensory ganglia. J. Neurocytol. 14, 63-78.

Kai-Kai M. A., Anderton B. H. and Keen P. (1986) A quantitative analysis of the interrelationships between subpopulations of rat sensory neurones containing arginine vasopressin or oxytocin and those containing substance P, fluoride-resistant acid phosphatase or neuro- filament protein. Neuroscience 18, 475-486.

Kai-Kai, M. A., Swann R. W. and Keen P. (1985) Local- ization of chromatographically characterized oxytocin and arginine-vasopressin to sensory neurones in the rat. Neurosci. Lett. 55, 83-88.

Kuraishi Y., Hirota N., Sato Y., Hino Y., Satoh M. and Tagaki H. (1985) Evidence that substance P and somato- statin transmit separate information related to pain in the spinal dorsal horn. Brain Res. 325, 294-298.

Leah J. D., Cameron A. A. and Snow P. J. (1985) Neuropeptides in physiologically identified mammalian sensory neurones. Neurosci. Lett. 56, 257-263.

Lehtosalo J. I. (1984) Substance P-like immuno- reactive trigeminal ganglion cells supplying the cornea. Histochemistry 80, 273-276.

Lieberman A. R. (1976) Sensory ganglia. In The Peripheral Nerve (Edited by Landon D. N.), pp. 188-278. Chapman & Hall, London.

Magnusson K. R., Larson A. A., Hadl J. E., Altschuler R. A. and Beitz J. (1986) Co-localization of fixative modified glutamate and glutaminase in neurones of the spinal trigeminal nucleus of the rat. An immunohisto- chemical and immunoradiochemical analysis. J. comp. Neurol. 247, 477-490.

Nagy J. I. and Daddona P. E. (1985) Anatomical and cytochemical relationships of adenosine deaminase con- taining primary afferent neurones in the rat. Neuroscience 15, 799-813.

Price J. (1985) An immunohistochemical and quantitative examination of dorsal root ganglion neuronal sub- populations. J. Neurosci. 5, 2051-2059.

Price J. and Mudge A. W. (1983) A subpopulation of rat dorsal root ganglion neurones is catecholaminergic. Nature 301, 241-243.

Rambourg A., Clermont Y. and Beaudet A. (1983) Ultra- structural features of six types of neurones in rat dorsal root ganglia. J. Neurocytol. 12, 47<66.

Russell L. C. and Burchiel K. J. (1984) Neurophysiological effects of capsaicin. Brain Res. Rev. 8, 165-176.

Salt T. E. and Hill R. G. (1983) Neurotransmitter candidates of somatosensory primary afferent fibres. Neuroscience 10, 1083-1103.

Neuronal cytochemistry 193

Sommer E. W., Kazimierzak J. and Dorz B. (1985) Neuronal subpopulations in the DRG of mouse as characterized by combination of ultrastructural and cytoehemical features. Brain Res. 346, 310-326.

Tuscherer M. M. and Seybold V. S. (1985) Immuno- histochemical studies of substance P, cholecystokinin- octapeptide and somatostatin in dorsal root ganglia of the rat. Neuroscience 14, 593-605.

Vanderhaeghen J. J., Lotstra F., Vandesande F. and Dierickx K. (1981) Coexistence of cholecystokinin and oxytocin-neurohypophysin in some magnocellular

hypothalamo-hypophysial neuron. Cell Tiss. Res. 271, 227-231.

Wanaka A., Shiotani Y., Kiyama H., Matsuyama T., Kamada T., Shiosak S. and Tohyama M. (1987) Glutamate-like immunoreactivity structures in primary sensory neurones in the rat detected by a specific anti- serum against glutamate. Exp. Brain Res. 65, 691-694.

Wiesenfeld-Hallin Z. (1985) Intrathecal somatostatin modu- lates spinal sensory and reflex mechanisms, behavioural and electrophysiologlcal studies in the rat. Neurosci. Lett. 62, 69-74.