THE JOURNAL OF COMPARATIVE NEUROLOGY 2831-12 (1989)

Light and Electron Microscopic Study of Gds.nin-Immunoreactive Nerve Fibers in

the Rat Posterior Thalamus

ISABEL BLASCO, FRANCISCO J. ALVAREZ, ROSA M. VILLALBA, MARIA L. SOLANO, RICARDO MARThEZMURILLO, AND JOSfi RODRIGO

Department of Neuroanatomy, Cajal Institute (C.S.I.C), Madrid 28006, Spain (I.B., R.M.V., M.L.S., R.M.-M., J.R.); Department of Physiology, St. Thomas's Hospital Medical School,

London SE17EH, England (F.J.A.)

ABSTRACT Light and electron microscopic immunocytochemistry was used to study

certain cell groups in the posteromedial thalamus which contain galanin- immunoreactive (GAL-IR) fibers. The nuclei subparafascicularis pars par- vicellularis (SPFpc) and parafascicularis (PF) contain a dense network of GAL-IR fibers which form basketlike structures around unstained cells. The periventricular area also contains numerous GAL-IR fibers and these also occasionally form basketlike structures. The GAL-IR terminal fields continue caudally in the mesodiencephalic junction and merge with other GAL-IR fibers in the dorsal aspects of the substantia nigra and around the dorsolateral tip of the medial lemniscus.

Ultrastructural .analysis of the GAL-IR basketlike structures revealed that GAL-IR terminals make numerous synapses with the cell bodies and proximal dendrites of SPFpc neurones. These results suggest that the activity of cells in the SPFpc and PF nuclei may be strongly influenced by galanin- containing nerve fibers probably originating in the spinal cord.

Key words: spinothalamic, electron microscopy, subparafascicular parvicel- lular, parafascicular

The spinothalamic tract is assumed to be one of the main ascending routes of nociceptive and thermal input to the diencephalon (Price and Dubner, '77; Berkley, '85). The tract is composed of several fiber systems arising from somata of various dorsal horn laminae and ending in dif- ferent nuclei of the thalamus (Giesler et al., '79, '81; Berkley, '85). This variability may indicate the existence of various subsystems of the spinothalamic tract which may subserve different aspects of the pain response. Spinothalamic fibers ending in medial thalamic nuclei have been related mainly with aversive responses to nociceptive stimulation, while lateral spinothalamic fibers ending in the ventrobasal com- plex may be involved in some cognitive aspects of pain (Berkley, '85).

Using classical neuroanatomical techniques it has proved difficult to study selective components of the spinothalamic system such as the subsystems which arise from neurons located in different spinal cord laminae or which end in dif- ferent thalamic nuclei. Recently, however, several neuro- peptides have been found in spinal ascending systems (Leah et al., '88), and immunocytochemistry can therefore be used to recognize certain spinal afferents in their target thalamic nuclei. Thus, Ju et al. ('87) have recently reported that gal-

anin (GAL) may be a reliable marker for some medial spino- thalamic fibers. These fibers originate from cell bodies located in lamina x (LX) of the lumbar spinal cord and end in some medial areas of the posterior thalamus where they form basketlike structures. The neuroanatomical tech- niques used by these authors enabled them to locate exactly the cells of origin of this tract but not the thalamic nuclei in which the fibers terminate. To overcome this problem we have used peroxidase-antiperoxidase immunocytochemical techniques at the light microscopic level in combination with Nissl staining in order to determine the precise distri- bution of galanin-immunoreactive (GAL-IR) fibers in the posterior thalamus. In addition, we have employed corre- lated light and electron microscopic immunocytochemical techniques to study the characteristics of GAL-IR terminals located in the SPFpc and to identify the types of synaptic contacts which they establish.

Accepted November 29,1988. Address reprint requests to Dr. Franco J. Alvarez, Department of Physiol-

ogy, St. Thomas's Hospital Medical School, London SE1 7EH, United King- dom.

0 1989 ALAN R. LISS. INC.

2

MATF3RIALS AND METHODS Experimental animals

Ten male Wistar albino rats (200-250 g) were used in this study. They were all intraperitoneally injected with 1 ml/ 100 g of a mixture of chloral hydrate, magnesium sulphate, and pentobarbital (Equithesin, Jensen Salsberg lab.).

Preparation of tissue Animals were perfused via the ascending aorta with a vas-

cular rinse solution, pH 7.4, followed either by a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for light microscopic (LM) purposes or a mixture of 4% paraformaldehyde and 0.05 % glutaraldehyde in PB for electron microscopy (EM). The brains were quickly re- moved, cut into blocks containing the selected areas, and then postfixed in a 4 % paraformaldehyde solution in PB for 3 hours at room temperature. Thereafter, blocks were im- mersed in 0.1 M PB containing 30% sucrose and then stored overnight at 4°C. For LM studies frontal 40 pm frozen sec- tions were cut on a sliding microtome. For EM, tissue blocks were rapidly frozen by immersing them for a few seconds in liquid nitrogen followed by a rapid thaw in cold (4°C) PB; 40 pm frontal sections were then cut on a Lancer vibratome and collected free floating in phosphate-buffered saline (PBS).

Immunohistochemical procedure For LM; sections were incubated in nonimmune goat

serum (NGS) diluted 1 : l O and then in rabbit anti-GAL serum at a 1:2,000 dilution for 48 hours at 4°C. After wash- ing, sections were incubated in goat antirabbit IgG antise- rum (Sigma Chemical Co., diluted 150) for 1 hour at room temperature and then incubated in rabbit PAP complex (Sigma Chemical Co., diluted 1:1,000) for 1 hour 30 minutes at room temperature. All incubations were carried out with PBS containing 0.2% Triton X-100. The PAP complex was localized by using 0.06 % 3,3' diaminobenzidine tetrahy- drochloride (DAB, Sigma Chemical Co) and 0.03% H,02 Finally, sections were washed in PBS and mounted in two serial groups. One series was counterstained with toluidine blue. Sections were dehydrated through graded alcohols, cleared in xylene or toluene, and covered with Dammar resin (Merk). For SM; a similar protocol was used except that no Triton X-100 was employed in the procedure. A 1:1,000 dilution of rabbit anti-GAL serum was used for 12 hours at 4°C. After DAB incubation, sections were incu- bated in 1 % osmium tetroxide (Os04, Sigma Chemical Co.) in PB for 45 minutes at room temperature. They were then dehydrated in graded ethanols and contrasted in block with 1% uranyl acetate in 70 % alcohol. Sections were then flat- embedded on slides in Durcupan ACH resin (FLUKA), plastic coverslipped, and cured for 2 days a t 56°C. The flat- embedded sections were then examined by using a Leitz Dialux 22 and areas of interest were photographed, dis- sected out, and glued onto prepolymerized Durcupan blocks. Ultrathin sections were cut on a Reichert Ultracut. Grids were contrasted with lead citrate (Reynolds, '63) and examined by using a Jeol lOOB electron microscope.

I. BLASCO ET AL.

Antisera characteristics A highly specific polyclonal rabbit antisera against GAL,

kindly donated by Dr. J.M. Polak, was used in this investi- gation. Characteristics and specificity of this antiserum (de-

veloped in the Hammersmith Hospital of London, ref. 1125) have been previously described (Ch'ng et al., '85).

RESULTS Numerous galanin-immunoreactive (GAL-IR) varicose fi-

bers were found in the posterior medial thalamus and mesodiencephalic junction. The section levels studied corre- spond approximately to levels 4.5-5.5 mm posterior to bregma in the Paxinos atlas (Paxinos and Watson, '86) (map in Fig. 1).

Distribution of GALIR fibers in the posterior thalamus: LM

GAL-IR fibers constitute well-defined terminal fields in the posterior thalamus of the rat. The mediolateral location of these fibers and terminals varies at different rostrocaudal levels (Figs. la,b, 2a,b). Rostrally GAL-IR fibers are distrib- uted medially around the third ventricle and extend later- ally under the fasciculus retroflexus (FR) (Fig. la). Cau- dally, the majority of GAL-IR fibers are located laterally over the medial lemniscus (ML); however, some GAL-IR fibers follow the wall of the third ventricle up to mesence- phalic levels. (ML) (Figs. lb , 2a,b).

Some GAL-IR fibers form basketlike structures in which the GAL-IR varicose fibers surround cell bodies and proxi- mal dendrites of nonimmunoreactive neurons (Fig. 3a-d). This characteristic of GAL-IR fibers allows us to determine the shape of the ensheathed unstained cells. Two different groups of GAL-IR terminal fields were distinguished on the basis of this feature.

A more medial group of GAL-IR fibers surround oval neu- rons which measure approximately 23 pm in their major axis and 13 pm in their minor axis. In this cell group, several microns of irregularly oriented proximal dendrites often ap- pear surrounded by GAL-IR fibers (Fig. 3a). At the level of the posterior commissure this group is located over the fas- ciculus retroflexus (FR) in the parafascicular nucleus (Fig. Ib). More rostrally it is poorly defined and situated under the FR (Fig. la). Caudally it is located laterally and ends in the prerubral field (Fig. 2a).

A lateral group of GAL-IR fibers surround small, densely packed, fusiform and horizontally oriented cells (Figs. la,b, 2a,b, 3b). In this location, GAL-IR fibers encircle cell bodies but do not follow their proximal dendrites as far as in the medial group. This lateral group is located within the nucleus subparafascicularis parvicellularis (SPFpc) (Fig. 4a,c). The highest density of GAL-IR fibers and the greatest numbers of GAL-IR basketlike structures were located in the medial part of the nucleus (Figs. 3b, 4c), and this corre- sponds to an area of high cell density observed in Nissl- stained serial sections (Fig. 4a). In the lateral region of the SPFpc, GAL-IR fibers do not establish basketlike arrange- ments, and only horizontal varicose fibers which make sin- gle contacts with cell somata and dendrites were observed (Fig. 4d). All cell somata in the SPFpc have a major axis measuring approximately 23 pm, but the minor axis varies depending on the location of the cell. The minor axes of neu- rons found in the medial area of the SPFpc nucleus measure about 10 pm, while lateral cells have smaller diameters (ap- proximately 7 hm). Caudally the SPFpc group of GAL-IR fibers ends a t mesencephalic levels over the dorsolateral tip of the medial lemniscus (ML) (Fig. 2b). In this location the cells are less closely packed, the GAL-IR fiber density

GALANIN-ir IN THE RAT POSTERIOR THALAMUS 3

I / , /--

Fig. 1. Darkfield micrographs (montages) and map of frontal sec- tions showing the distribution of GAL-IR throughout the posterior thal- amus. The rostrocaudal level of a and b corresponds to sections A and B in the map. At this level GAL-IR fibers were found in the periventri-

cular region (PV), the parafascicularis nucleus (PF), and the subpara- fascicularis nucleus pars parvicellularis (SPFpc). The arrow in b marks the location of a cell seen in Figure 3c. FR, fasciculus retroflexus; ML, medial lemniscus; 111, third ventricle. Scale bar = 200 pm.

increases, and many GAL-IR basketlike structures ensheath round and polygonal cell bodies (Fig. 4e).

Other neighbouring terminal fields were observed in which GAL-IR fibers form only occasional basketlike struc- tures, and these were located in the periventricular thalamic area and lateral mesencephalon (Figs. la,b, 2a,b). GAL-IR fibers in the latter area merge with the caudal extension of the SPFpc group. At this level some GAL-IR fibers are pres- ent between the ML and dorsolateral aspects of the substan- tia nigra (SN) (Fig. 2b).

EM GAL-IR basketlike structures identified at the light mi-

croscopic level in the SPFpc at the posterior commissure level were reexamined at electron microscopic level (Figs. 5- 9). Ultrastructural examination of these basketlike struc- tures confirmed that GAL-IR terminals surround nonim- munoreactive cell bodies in the SPFpc.

All GAL-IR fibers and terminals surrounding cell bodies contained densely packed, round, agranular, synaptic vesi- cles (Fig. 6a-d). The majority of these GAL-IR fibers also contained numerous large and prominent immunostained dense-cored vesicles. Dense-cored vesicles were not espe- cially concentrated near synaptic junctions and often ap- peared in clusters along the GAL-IR axon.

GAL-IR terminals adjoining cell somas exhibited various synaptic arrangements. Some GAL-IR terminals establish symmetrical axosomatic synapses over cell somas (Figs. 6d, 8b). However, not all the GAL-IR terminals surrounding cell bodies make axosomatic contacts. Frequently they es- tablish asymmetric synapses with adjoining dendritic shafts or spines (Fig. 6a-d). Usually, these dendrites also receive synaptic contacts from nonimmunoreactive terminals which have a similar morphology to the GAL-IR terminals.

The morphology of cells surrounded by GAL-IR profiles was examined in levels of the vibratome sections which had less immunostaining but better ultrastructural preservation (Fig. 7). The neurons are spindle shaped and horizontally oriented (Fig. 7a). They present an indented nucleus with a prominent nucleolus, contain numerous mitochondria in their cytoplasm, have a well-developed endoplasmic reticu- lum and Golgi apparatus, and contain numerous lysosomes and lipofucsin granules. They receive numerous axosomatic contacts which are derived from a t least two different types of terminals. One type contains clear flattened or pleomor- phic vesicles and the other contains numerous clear round vesicles (Figs. 7b,c). The latter type of terminal occasionally contains numerous dense-cored vesicles. Some of these ter- minals resemble GAL-IR terminals found in superficial sec- tions. Occasional somatic spines were observed and some-

4 I. BLASCO ET AL.

Fig. 2. Darkfield micrographs (montages) of frontal sections show- ing the distribution of GAL-IR fibers through the mesodiencephalic junction. Rostrocaudal levels of a and b correspond to sections C and D of the map in Figure 1. A t these levels the GAL-IR fibers are located

laterally and form two terminal fields representing a continuation of the PF and SPFpc. Other GAL-IR fibers appear in the periventricular region (PV) and between the medial lemniscus (ML) and the substantia nigra (SN). 111, third ventricle. Scale bar = 200 pm.

times they were surrounded by GAL-IR terminals (Fig. 9), although synapses were difficult to detect.

DISCUSSION LM

Our results have demonstrated the existence of well- defined terminal fields of GAL-IR nerve fibers in the poste- rior thalamus of the rat. GAL-IR fibers in the same area were previously described by Skofitsch and Jacobowitz ('85) as a fiber bundle traversing this area, and this was consid- ered to be similar to the dorsal and ventral noradrenergic pathways. In agreement with our results Melander et al. ('86), described a dense GAL-IR terminal field in this tha- lamic area. They reported strongly stained GAL-IR basket- like structures which occupied the periventricular region and some aspects of the parafascicular nucleus. In addition, they described a lateral group of fibers in the ventral aspects of the ventral thalamic nucleus. They employed immuno- fluorescence as the detection method; therefore cellular and nuclear boundaries were difficult to establish. To avoid this problem we have used PAP immunocytochemistry com-

bined with Nissl staining, which has enabled us to specifi- cally locate the nuclear groups where GAL-IR fibers and terminals are distributed. Although GAL-IR fibers do not overlap in all cases with specific nuclei, we have found a high degree of correlation between the lateral group of GAL-IR fibers and the parvicellular subdivision of the subparafasci- cular nucleus (SPFpc). I t is of interest to note that the medial subdivision of the SPFpc displays the heaviest den- sity of GAL-IR fibers. This correlates well with similar dif- ferences observed in the cellular composition of the nucleus. Whether the medial and lateral subdivisions of the SPFpc have similar conectivity and functions is unknown. We have also found GAL-IR fibers in some regions of the PF nucleus, in the dorsolateral portion of the substantia nigra, and in the periventricular region. This locations are in full agree- ment with the results of Melander et al. ('86). The same group has demonstrated the spinal origin of these GAL-IR fibers with degeneration techniques (Ju et al., '87) and this suggests that most of the fibers recognized by GAL antibod- ies in the posterior thalamus have an spinothalamic origin.

Although all GAL-IR basketlike structures degenerate af- ter destruction of the spinothalamic tract (Ju et al., '87),

GALANIN-ir IN THE RAT POSTERIOR THALAMUS 5

Fig. 3. Brightfield micrographs of GAL-IR basketlike structures im- munostained with the PAP method. a-c: Photographed with Nomarski optics. These micrographs show several GAL-IR basketlike structures around nonstained cells of the PF (a), SPFpc (b), and P V (c). The Inca- tion of the cell in c is marked with an arrow in Figure lb . In the SPFpc (b) the higher density of GAL-IR fibers is located in the medial (m) region of the nucleus, while GAL-IR fibers are more sparse in the lateral

region (1). Not all the cells in these areas are surrounded by GAL-IR fibers, as can be seen in d. This micrograph shows some Nissl-stained round cell bodies of the PF nucleus immunostained with GAL antise- rum. Some of the cells are completely surrounded (arrows) with GAL-IR fibers; some are only partly surrounded (double arrow); and others are not surrounded at all (open arrows). Scale bars: a, c, d = 25 pm; b = 50 pm.

many of the GAL-IR fibers in the periventricular and SN region which do not form baskets survive unilateral hemi- sections or complete transections of the spinal cord. This fact, together with the existence of a large number of GAL- IR cell bodies in the hypothalamus (Skofitsch and Jacobo- witz, '85; Melander e t al., '86), suggests that there may be an additional descending hypothalamic galaninergic pathway which is located mainly in the periventricular area. In fact we observed in serial sections a continuous system of GAL- IR fibers which pass along the third ventricle up to the hypothalamus.

Although the thalamus is not characterized by a rich con- tent in peptides, several neuropeptides have been described in these same areas of the posterior thalamus. The presence

of GAL-IR fibers is well documented (Skofitsch and Jacobo- witz, '85; Melander e t al., '86; Ju et al., '87) , as is its coexis- tence with cholecystokinin ( Ju et al., '87). In addition, enke- phalin (Wamsley et al., '80; Finley et al., '81), dynorphin-B, and dynorphin,, immunoreactivity have been described in the same areas (Fallon and Leslie, '86). Dynorphin-B has been located in some fusiform cell bodies and in fibers which form dense pericellular baskets, so it is possible that some dynorphin coexists with GAL.

EM Our electron microscopic results in the SPFpc nucleus

support the light microscopic observations of GAL-IR bas-

6 I. BLASCO ET AL.

Fig. 4. a: Brightfield micrograph of the SPFpc in an immunostained section counterstained with Nissl. c: Darkfield of noncounterstained serial section (see blood vessels marked with asterisks) showing the dis- tribution of galaninergic fibers in the SPFpc. In a the nucleus is identi- fied by its spindle-shaped and horizontally oriented neurons. The medial (m) region of the nucleus is characterized by a higher cellular density (a) and a denser galaninergic innervation (c), while the lateral

(1) region displays a smaller cellular density and sparser galaninergic innervation. Cells located in the medial area are completely surrounded with GAL-IR basketlike structures @); in contrast, the lateral area (d) is characterized by smaller cell bodies occasionally contacted by GAL- IR fibers (arrow). Some polygonal cell bodies located over the dorso- lateral tip of the ML are also surrounded by GAL-IR fihers (e ) . Scale bars: a, c = 100 pm; b, d , e = 25 pm.

GALANIN-ir IN THE RAT POSTERIOR THALAMUS 7

Fig. 5. GAL-IR nerve fibers surrounding a cell body in the SPFpc photographed at light microscopic level in a flat-embedded section pro- cessed for electron microscopy (a). (Arrows in a mark the areas with immunostained terminals in b.) The same section was then studied at the ultrastructural level (b). A blood vessel (asterisk) was used as a land-

mark to help with the correlation between light and electron micros- copy. This blood vessel is situated just over the ML. Some GAL-IR ter- minals (T, and T,) were found contacting the cell (arrows in b). Scale bars: a = 10 pm; b = 1 pm.

8 I. BLASCO ET AL.

Fig. 6. Nonadjacent serial electron micrographs (a-c) through a GAL-IR terminal (TI) contacting a cell soma (S). (This terminal also appears in Fig. 5b as T,.) In deeper sections this terminal loses most of the DAB precipitate (c). TI makes asymmetric synaptic contacts (short arrows) over a dendrite (D) (a). In a nonadjacent serial section (b) this dendrite (D) receives another asymmetric synapse (short arrows) from an unstained terminal (T,). The synapse of TI is in opposition to the synapse formed by T, in a region of the dendrite where several postjunc- tional dense bodies (arrowhead) are present. In a further nonadjacent

serial section (c) the dendrite (D) receives a third asymmetric synaptic contact from another unstained terminal (T3). In section c, T, is no longer contacting the cell soma (S). d: Two GAL-IR terminals (T, and T2) contacting a cell soma (S) of the SPFpc. One of them (TI) makes a symmetric synapse (short arrow) with the cell soma, and the other (T,) an asymmetric synaptic contact (open arrow) with a dendrite (D). The dendrite receives another asymmetric synaptic contact (curved arrow) from an unstained terminal. Scale bars: 0.25 rrm.

GALANIN-ir IN THE RAT POSTERIOR THALAMUS 9

Fig. 7. Nonadjacent serial electron micrographs through the cell body shown in Figure 5. These ultrathin sections were obtained from a deep level of the vibratome section; therefore they present a better ultrastructural preservation but poor immunostaining. The cell (a) has an indented nucleus (N) and shows a prominent nucleolus. Numerous lipofucsin granules (open arrows) can be observed in its cytoplasm. The cell is surrounded by several types of terminals contacting the cell soma;

some of them abut in the somatic plasma membrane (arrow in a). Sev- eral types of terminals can be distinguished a t higher magnification. b, c: Some are filled with clear, round, synaptic vesicles (T, and T2), and may contain numerous dense-cored synaptic vesicles (T, arrowheads), Other terminals have flattened and/or pleomorphic clear synaptic vesi- cles (TJ. All the terminals make synaptic contacts over the cell soma (short arrows; S, cell soma). Scale bars: a = 1 pm; b, c = 0.25 pm.

ketlike structures which surround nonimmunoreactive cell Thus, there were terminals which contain small, clear, somata and proximal dendrites. round vesicles and terminals which contain flattened or

At least two different morphological types of terminals pleomorphic vesicles. The former type also contain dense- could be distinguished on the basis of their vesicle content. cored vesicles, but these vesicles do not appear in all serial

10 I. BLASCO ET AL.

Fig. 8. Light (a) and electron (b) micrographs of a cell body sur- rounded by GAL-IR terminals and located in the SPFpc. A blood vessel used to correlate the light and electron micrographs is marked with an asterisk. Some of the terminals observed with light microscopy were identified at the ultrastructural level (arrows in a and c). One of the ter-

minals (T in b and c) establishes a symmetric synaptic contact over the cell soma (b, short arrow). Next to it, an unstained terminal also pre- sents a symmetric synaptic contact (double arrowhead). Both terminals contain clear, round and some dense-cored synaptic vesicles. Scale bars: a = 10 pm; b = 0.25 pm; c = 1 pm.

sections taken through a profile, indicating that they are seggregated in clusters. The classification of a distinct mor- phological fiber type based on the presence or absence of dense-cored vesicles is therefore extremely difficult and may be misleading.

Some, but not all, of the terminals containing clear, round, agranular, synaptic vesicles show GAL immunoreac- tivity in ultrathin sections obtained from superficial layers of vibratome sections. This observation suggests that this terminal type may be further classified attending to the neurochemicals contained within it. In this investigation we have focused our ultrastructural study on galaninergic ter-

minals contacting cell somas of the SPFpc. The presence of galanin suggests that this class of terminal may have a spi- nothalamic origin.

Only symmetric synaptic contacts were observed between GAL-IR terminals and cell somas. Strikingly, some of the larger profiles surrounding the cell soma make asymmetric synaptic contacts over dendrites and dendritic spines. This observation raises the possibility that GAL-IR terminals contacting cell somas may also establish synaptic contacts with neighbouring dendrites.

GAL-IR fibers and terminals appear to contain a particu- larly large number of dense-cored vesicles. This fact and the

GALANIN-ir IN THE RAT POSTERIOR THALAMUS 11

they may represent a distinct projection from the spinal cord which is different from the classical lateral spinotha- lamic fibers which end in the ventrobasal complex. In addi- tion spinothalamic endings in the ventrobasal complex were found very rarely adjacent to cell bodies and only ocasion- ally made axosomatic synapses (Ma e t al., '87). However, we found that GAL-IR spinothalamic fibers ending in the me- dial posterior thalamus contact mainly cell somas and proxi- mal dendrites. All these data suggest that spinothalamic GAL-IR terminals may constitute a special spinothalamic subsystem with different morphology and distribution than lateral spinothalamic fibers ending in the ventrobasal com- plex of the thalamus.

Fig. 9. A GAL-IR terminal surrounding a somatic spine (ss). S, cell soma. Scale bar = 0.25 urn.

classical consideration that dense-cored vesicles may have a peptidergic content (Hokfelt et al., '80) fit well with previ- ous studies indicating that several neuropeptides may exist in these basketlike nerve terminals (Fallon and Leslie, '86; J u et al., '87).

To our knowledge, this is the first ultrastructural study on the rat SPFpc area. However, there are several reports on the ventrobasal complex of cat, rat, and monkey thalamus, an area which also receives spinothalamic inputs (MacAllis- ter and Wells, '81; Ralston et al., '85; Ralston, '85; Pe- schanski et al., '85; Ma e t al., '87). In these studies, three morphological types of terminals have been described in the rat and four in the cat and monkey. Comparing our results with these classifications, we found that the F terminals of Ralston et al. are similar to our small terminals containing flattened or pleomorphic vesicles. F terminals have been shown to he GABAergic (Ralston et al., '85), and their origin was found to be the reticular nucleus of the thalamus (Pe- schanski et al., '83; Ralston, '85). In addition, Ottersen and Storm-Mathisen ('84) have described some GABAergic fi- bers which surround cell bodies in the rat thalamus. Many of these fibers are located in an area dorsal to the medial lemniscus, and this area may correspond to the SPFpc. Therefore, some of the terminals which make symmetric contacts over cell bodies in the SPFpc and which contain flattened or pleomorphic synaptic vesicles may be GABAer- gic.

There are no ultrastructural similarities between GAL-IR terminals and the spinothalamic terminals of the ven- trobasal complex described by Ralston et al. ('85) and Mac- Allister and Wells ('81). Spinothalamic terminals in the ventrobasal complex are large and characterized by the presence of numerous mitochondria, scattered round, clear vesicles, and a few dense-cored vesicles. In contrast, the GAL-IR terminals observed in this study were smaller and contained densely packed, clear, round, synaptic vesicles and numerous dense-cored vesicles.

The special characteristics of the medial GAL-IR spino- thalamic nerve fibers (peptide content, axosomatic basket- like structures, and small terminal diameter) suggest that

Physiological considerations

The existence of GAL-IR terminals surrounding cell so- mas and proximal dendrites implies the existence of a strong spinothalamic input to SPFpc and PF nuclei. Among affer- ent fibers from several sources, spinothalamic afferents to the P F have been previously described (Lund and Webster, '67; Faull and Mehler, '85). In contrast, the SPFpc afferent connections are ill defined (Faull and Mehler, '85). The presence of a spinothalamic input to PF and SPFpc is in agreement with electrophysiological studies performed in cat, which have demonstrated the existence of numerous neurons activated by noxious stimuli in the PF and SPFpc (Dong et al., '78). In fact, stimulation of similar areas of the posteromedial thalamus produces analgesia (Mayer et al., '71; Mayer and Liebeskind, '74; Rhodes and Liebeskind, '78). It has been shown that cell groups located in the P F and related areas in the mesodiencephalic junction project to nuclei involved in descending pain suppression systems such as the periaqueductal gray and nucleus raphe magnus (Semba et al., '81; Peschanski and Mantyh, '83). Immedi- ately ventral to the fasciculus retroflexus and extending into the prerubral field are located cells which may project as far caudally as the spinal cord (Peschanski and Mantyh, '83). This area has been called the subfascicular area. These con- nections suggest that cell groups related to the PF nucleus and other regions surrounding the fasciculus retroflexus and extending into the mesodiencephalic junction may be in- volved in some aspects of the descending pain suppression systems. Accordingly, i t has recently been shown that stim- ulation of the rat nucleus parafascicularis activates pain suppression systems involving neurons in the periaqueduc- tal gray and nucleus raphe magnus (Sakata et al., '88). The presence of GAL-IR fibers with a spinothalamic origin sur- rounding cell bodies in these nuclei indicates that some pain suppression inputs arising from the parafascicular nucleus or related areas may be directly controlled by direct spino- thalamic fibers.

In contrast to the PF, the cells located in the SPFpc pro- ject mainly to the amygdala. In fact, the nucleus in the rat has been defined as an amygdalopetal cell group located in the posteromedial thalamus (Faull and Mehler, '85; Le'Doux e t al., '85) since it is not clearly defined in Nissl- stained sections of the rat thalamus. This cell group is better defined in cat and monkey, where it has been shown that also projects mainly to the amygdala (Mehler, '80; Russchen, '82). Therefore the SPFpc may be a relay nucleus sending spinothalamic information, including nociception, to the amygdala and linking somatosensory systems with the limbic system.

12 I. BLASCO ET AL.

ACKNOWLEDGMENTS upon single thalamic neurons. Neuroscience 22925-934. Mayer, D.J., T.L. Wolfe, H. Akil, B. Carder, and J.C. Liebeskind (1971) Anal-

gesia from electrical stimulation in the brainstem of the rat. Science 174:1351-1354.

Mayer, D.J., and J.C. Liebeskind (1974) Pain reduction by focal electrical stimulation of the brain: An anatomical and behavioral analysis. Brain Res. 68:73-93.

Mehler, W.R. (1980) Subcortical afferent connections of the amygdala in the monkey. J. Comp. Neurol. 190:733-762.

Melander, T., T. Hokfelt, and A. Rokaeus (1986) Distribution of galanin immunoreactivity in the rat central nervous system. J. Comp. Neurol.

The authors wish to thank Dr* J.M. Polak for the galanin antisera used in this research and Dr. F. Rei- noso, Dr. c. Avendafio, and Dr. J.V. Priestley for their criti- cal reading and helpful comments. We also express our gra- titude to Mrs. M. Lbpez, Mrs. M. Campos, and Mr. J. Lozano for their technical assistance.

This work was supported by *Ondo de Investigaciones Sanitarias de la Seguridad Social (grants 88-1679) and CICYT (grant C.S.I.C. 88EA036). 248:475-517.

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