Functional activity mapping of the rat brainstem during formalin-induced noxious stimulation

Neuroscience Vol. 41, No. 2/3, pp. 667-680, Printed in Great Britain

0306-4522/91 $3.00 + 0.00 Pergamon Press plc c 1991 IBRO

1991

FUNCTIONAL ACTIVITY MAPPING OF THE RAT BRAINSTEM DURING FORMALIN-INDUCED NOXIOUS STIMULATION* C.

A.

PoRRo,~~.

M. CAVAZZUTI,$

A. GALETTI~

and L.

SASSATELLI~

SIstituto

di Fisiologia Umana, Via Campi 287, 41100 Modena, Italy §Clinica Neurologica, Via de1 Pozzo 71, 41100 Modena, Italy

Abstract-Functional

activity

changes

in 35 selected

structures

of the rat brainstem

elicited

by subcu-

in a forepaw were investigated by the [r4C]2-deoxyglucose method in unanesthetized, freely moving animals. Experiments were initiated 2 min (“early” group) or 60 min (“late” group) after the injection. Treatment induced a significant increase of [‘4C]2-deoxyglucose uptake relative taneous

formalin

injection

to controls in 17 structures of the “early” group, including portions of the bulbar, pontine and mesencephalic reticular formation, nucleus raphe magnus, median and dorsal raphe nuclei, the ventrolatera1 and dorsal subdivisions of the periaqueductal gray matter, deep layers of the superior colliculus and the anterior pretectal nucleus. Most changes were bilateral, with the exception of the increases observed in the nucleus reticularis paragigantocellularis and the lateral parabrachial area, which were contralateral, and the one in the mesencephalic reticular formation, which was ipsilateral to the injected paw. In pentobarbital-anesthetized rats a significant difference in metabolic activity values between formalin- and saline-injected animals was only detected at the medullary level. In the “late” unanesthetized formalin group functional activity levels were higher than controls in four structures, including the lateral reticular and paragigantocellular nuclei, contralaterally, and nucleus cuneiformis and ventrolateral periaqueductal gray matter, bilaterally. No between-groups difference was observed in visual or auditory structures. These results provide evidence for activation of several brainstem regions, which are conceivably involved in different sensory, motivational or motor circuits, during the initial phase of formalin-evoked noxious stimulation in unanesthetized animals. Functional changes blunted over time as did pain-related behavior integrated at the supraspinal level, but they persisted in some brainstem regions for which involvement in endogenous antinociceptive systems have been suggested. The mechanisms underlying these time-related changes need to be clarified.

Brainstem centers are the recipients of ascending pathways carrying nociceptive information and are thought to play an essential role in pain mechanisms. Indeed, nociceptive neurons have been recorded by electrophysiological experiments in several brainstem nuclei, which are conceivably involved in different functions, such as arousal, motor adjustments and vegetative reactions, in addition to the rostra1 transmission of noxious information.“J5~93~9s Additionally, behavioral analgesia or inhibition of spinal neurons responding to noxious stimuli may be elicited by electrical stimulation or opiate microinjection from a number of brainstem sites.94,9* The actual involvement of the individual brainstem structures in pain states is, however, still largely unknown. The problem is further complicated by the fact that most physiological studies have so far been performed in anesthetized animals. Under these con-

ditions the excitatory and inhibitory drives, and therefore neuronal responses, may be differently affected according to drug and dosage.16.32,36 Although electrophysiological recordings may be performed in awake, freely moving animals,3s’4,64 this technique allows a limited sampling in terms of number of cells and more so of structures in the same animal. Other techniques such as the 2-deoxyglucoseE4 (2-DG) and c-fos methods37.79 may provide a more comprehensive map of pre- and postsynaptic metabolic changes induced by noxious stimuli in CNS structures. In the present study the extent of functional activation of 35 selected brainstem structures has been studied by the 2-DG method in unanesthetized and pentobarbital-anesthetized rats after formalin injection in a forepaw.23

EXPERIMENTAL

*Communicated in part at the XIIth Annual Meeting of the European Neuroscience Association, Turin, 1989. tTo whom correspondence should be addressed. Abbreviations: APT, anterior oretectal nucleus: 2-DO. 2-deoxyglucose; LGU, local’ glucose utilization;- LI&: lateral reticular nucleus; MI, metabolic indexes; PAG, periaqueductal gray; PAGD, dorsal periaqueductal gray.

Methods

PROCEDURES

were described in detail in Ref. 69a and will only be summarized here. Male Sprague-Dawley rats weighing 230-280 g were injected with a small amount of dilute formaldehyde solution (O.OW.08 ml, 5%) into one forepaw, and immediately returned to their home cages. Experiments were started 2-3 min (n = 6) or 60min (n = 6) later by intravenous 667

668

C. A. I%RR~ el ~1.

administration of a bolus of [‘4C]2-deoxyglucose ([14C]2DG; 100 pCi/kg). Control rats were either similarly handled but not injected (n = 6) or injected with an equivalent volume of saline into one forepaw (n = 4). During the experiments animals were freely moving in their cages, An additional group of eight rats was anesthetized with sodium pentobarbital (40 mg/kg, i.p.) 10min before the subcutaneous injection of dilute formalin (n = 4) or saline (n = 4) in one forepaw; experiments were initiated 2 min later as previously described. Body temperature was maintained at 36.5537”C by a heating lamp. The level of anesthesia was assessed by testing the cornea1 reflex, which was abolished in all rats. In another group of animals the same anesthetic regimen induced a moderate increase of PCO, and decrease of pH values in arterial blood samples. Mean arterial blood pressure values were slightly depressed, but increased relative to control unanesthetized rats after formalin injection (see Ref. 69a). Forty-five minutes after the [14C]2-DG injection all rats were killed by an overdose of pentobarbital, i.v. Immediately after death animals were decapitated, the brain and spinal cord rapidly removed and frozen in Freon chilled to -70°C. Serial coronal 20pm sections were later cut by a Reichert Frigocut 3700 cryostat at -2O”C, mounted on conventional glass slides and immediately dried on a plate at 60°C. Autoradiograms were obtained by contact exposure of sections to high resolution films (Hyperfilm Betamax, Amersham) for 3 days in X-ray cassettes. Calibrated plastic standards (Amersham) with known concentrations of 14C were exposed along with brain and spinal cord sections in order to allow quantification of the isotope uptake. One out of every five brain sections was routinely saved and exposed. In most brains the same sections were later stained with Cresyl Violet or Toluidine Blue for histological identification. Analysis of autoradiograms was performed using programs developed on a computerized image processing system.‘” Mean 14C concentration values were calculated from 35 brainstem regions (following Paxinos and Watson’s atlas of the rat brain@‘). Metabolic activity indexes (MI) for each region (I) were obtained according to the formula: Mlr = ([‘“C]r/[‘“C]

vestibular

nuclei) x 100

MI values from each region

were submitted to separate ANOVA, with one between-subjects (treatment) and one within-subjects (side) factors. Post hoc comparisons were run by the Student’s t-test. A value of P less than 0.05 was assumed as the level of statistical significance. Since data from the two unanesthetized control groups were not significantly different from each other, they were pooled together in subsequent analyses. Brains from five additional unanesthetized rats (two controls, two “early” and one “late” formalin-injected animals) were cut in the sagittal plane in order to better appreciate the rostrocaudal distribution of [“‘Cl2-DG uptake by means of computer-generated maps. Data from these animals were not included in the statistical analysis. RESULTS

Animal

behaviour

has

been

and therefore only the results iments are presented here.

in Ref. 69a of the 2-DC exper-

described

Ununesthetized animals Early group. Treatment induced significant changes in comparison to controls (ANOVA, P < 0.05) in 17 out of 35 brainstem structures studied (Table 1 and Fig.

1). In the caudal

[“‘Cl2-DG nucleus

uptake and

lateral

medulla

was detected reticular

a bilateral

increase

in the dorsal nucleus

(LRt).

of

reticular In this

latter region, as well as in the more rostrally situated nucleus reticularis gigantocellularis, pars ventralis (and pars alpha) and parvocellular reticular nucleus. there appeared to be a slight side difference, the effect being more conspicuous on the side contralateral to the injected paw. The increase in nucleus reticularis paragigantocellularis was present only on the contralateral side. At more rostra1 levels bilateral functional activity changes were evident both in the caudal and oral portion of the pontine reticular formation and nucleus cuneiformis. In the lateral parabrachial area only values from the contralateral side were higher than controls, although a tendency towards bilateral increase was observed. All three raphe regions investigated, namely raphe magnus, median and dorsal nuclei, displayed a variable degree of metabolic activation. At the mesencephalic level, an MI increase was found both in the ventrolateral and dorsal subdivisions of the periaqueductal gray matter. In the former region highest values were usually observed at the level of the caudal half of the superior colliculus, at the border with nucleus intercollicularis and mesencephalic reticular formation (Fig. 2). In deep mesencephalic gray the increase in the [14C]2-DG uptake was limited to the ipsilateral side. The intermediate and deep layers of the superior colliculus, but not the superficial ones, also exhibited a bilateral enhancement of metabolic activity. At the mesodiencephalic junction, MI values from the anterior pretectal nucleus were bilaterally increased with respect to controls over the whole rostrocaudal extent of the nucleus. The highest values, however, were consistently found in the rostra1 half (Fig. 3). The maximum percentage increase over controls (ventrolateral central gray) was of the order of 24%. Late group. Metabolic activity indexes from this group were different from controls in four structures (Fig. 4). At the medullary level, an increase of [14C]2-DG uptake was detected in the lateral reticular nucleus and nucleus reticularis paragigantocellularis on the side contralateral to the injected paw. More rostrally, a bilateral metabolic increase was found in nucleus cuneiformis and the ventrolateral periaqueductal gray. In most of these areas, values from the late group fell between those of the early group and controls (Table 1). Values from auditory (superior olivary complex, inferior colliculus) and visual (superior colliculus, superficial layers) structures were not different in the three groups. Pentoharbital-anesthetized

rats

from saline-injected and formalin-injected rats are shown in Table 2. A significant effect of treatment (ANOVA, P < 0.05) was found in four medullary regions, namely dorsal and ventral reticular nuclei, the spinal trigeminal subnucleus caudalis Values

669

Functional activity mapping of the rat brainstem Table 1. Metabolic activity indexes of unanesthetized rats Formalin-injected Controls L Cuneate n. Gracilis n. N. caudalis V MdV MdD N. solitary tract Lateral ret. n. Inferior olivary complex N. ret. gig., dors. N. ret. gig., vent. N. ret. paragig. N. raphe magnus Parvocellular ret. n. Facial n. Cerebellar med. int. lat. Main V Superior olivary c. N. ret. pont. caud. N. ret. pont. oralis Lat. parabr. n. Median raphe n. Dorsal raphe n. Inferior colliculus N. cuneiformis Periaq. gray, ventrolateral dorsal Deep mesenc. gray Sup. colliculus, superficial layers deep layers Substantia nigra, pars compacta pars reticulata Red nucleus Ant. pretectal n.

(late)

(early) C

I

C

I

70 * 3 56 + 1 78 k4 67 k 3 70 * 3* 62 + 2 55 + 2+

70 + 3 55 + 1 75 *4 66& 3 69 + 3* 62 k 3 53 * 2+

62 + 2 55 * 2 67 &-3 61+2 64*2 58 f 2 53+ 1*

64+2 54 * 2 67 f 3 59 * 2 63 5 2 58+ 1 51 k 1

R

65 & 2 55*2 66 * 3 59 * 2 62 i 2 57 f 1 48 + 1

64+2 54 * 2 67 + 3 59 + 2 61 +2 56 k 2 47 * 1

71*2 58 k 2 53+ 1 54& 1 48 f 62 & 1 64+ 1 78 k 1 84k 1 84 + 2 67 + 2 102+3 54 + 2 57+ 1 46+ 1 79 * 61 k 122+4 48 & 2

70+ 1 58 k 2 53 * 1 54 + 1 1 62* 1 64+2 79 * 1 85 + 2 86 + 2 67 k 2 105k2 54+2 58k 1 46 k 1 3 1 124k4 48 k 1

54 + 2 52& 1 58 k 2

52 + 2 52+ 1 59& 1

65 k 3** 6Oi3’ 62 + 2

66 * 3*** 61 *4* 66 * 1**

59 * 2* 54* 1 62 f 2

60&2* 54 + 1 63k2

64&l 69 + 2

67 & 1 71 + 1

69 k 3 76 + 3*

71 k3 78 & 3*

66k4 66 f 3

68 f 4 68 * 3

65 + 56 k 67 k 82 k

67 k 57 + 68 + 84 +

64+2 57* 1 70 + 3 97 f 2***

63 k 3 56+ 1 71*2 99 + 2***

70 f 2 59 & 2 67 k 3 87 k 2

70 + 2 60 k 2 66 & 2 87 k 2

2 2 1 2

74 f 3 76 k 3 64*3 64*3 57 * 2+ 59 * 2+ 60 k 3 62 k l** 54* 1** 67 + 3* 69 f 2** 68 +4 69 & 3 83 + 1 82 k 1 90 + 1 89k 1 90 * 2 88 + 2 72+ 3 71*3 102 + 3 102 + 3 63 + 2** 62 f 2* 67 k 2*+ 66 f 2** 49 + 1 50&l* 91 * 3* 73 * 2*** 123 + 5 122&3 55 f 1** 53 * 2*

2 2 2 3

71*3 3 59 * 2 2 55+ 1 1 1* 55* 1 49+ 1 66 2 2 65 & 2 65 f 2 64+2 81 &2 80 + 2 86k2 86 + 3 89 f 2 87 k 3 73 +2 73 * 2 105 * 5 108+3 54* 1 53& 1 58& 1 54 + 2 50 5 2 48 + 1 76k2 63 + 1 124k4 118k4 57 * 2*+ 56 k 2’+ 70 f 59 * 56k 59*

Values are means k S.E.M. (n = 6 in fonnalin groups, n = 10 in control group) of metabolic activity indexes, calculated as described in Experimental Procedures. L, left; R, right; C, contralateral; I, ipsilateral to the injected paw. MdV, medullary reticular field, ventral; MdD, medullary reticular field, dorsal. *P < 0.05, **P < 0.01 and ***P < 0.001, significantly higher than controls. and parvocellular

reticular

nucleus.

No change

with

respect to controls was detected in any pontine or mesencephalic structure. Mean percentage increases over the corresponding control groups in unanesthetized vs anesthetized

Abbreviations

APT CA1 CNF DpMe DR DSC GIA LPB LPGI

anterior pretectal nucleus CA1 field of the hippocampus cuneiform nucleus deep mesencephalic gray dorsal raphe nucleus superior colliculus, intermediate and deep layers gigantocellular reticular nucleus, alpha lateral parabrachial area lateral paragigantocellular nucleus

formalin-injected rats are reported in Fig. 5, which shows that the metabolic increase induced by treatment was somehow preserved at more caudal levels, while it was abolished in rostra1 structures.

used in the figures

LRt MdD MnR PAGD PAGVL PCRt PnC PnO RMg RSC

lateral reticular nucleus medullary reticular field, dorsal median raphe nucleus periaqueductal gray, dorsal periaqueductal gray, ventro-lateral parvocellular reticular nucleus pontine reticular nucleus, caudal pontine reticular nucleus, oral raphe magnus nucleus retrosplenial cortex

670

C. A. PORROel al DISCUSSION

Methodological issues To our knowledge, this is the first study which shows, by the [14C]2-DC method, functional correlates of prolonged noxious stimulation, as induced by formalin injection, in brainstem structures of unanesthetized free-moving animals. Local glucose utilization (LGU) changes represent the integrated metabolic activity of a whole region over a relatively long period of time. This may explain why no effect was found in some sensorimotor structures despite the between-groups differences in phasic motor behavior (licking, grooming).

Moreover, due to the limited spatial resolution of the technique, it is likely that the metabolic correlates of sensory stimulation in a given structure may not be detected unless they are related to activation of a large number of neural elements in that area. It should be further underlined that the adopted semiquantitative 2-DC technique is less sensitive to changes in local metabolic rate than the fully quantitative method. For instance, MIS were unaffected by treatment in dorsal column nuclei, where a relatively small percentage of neurons respond to noxious stimuli in the monkey.25 In this respect it should be noted, however, that cells at the origin of the postsynaptic dorsal column pathway,” which is

-11

Fig. la.

Functional activity mapping of the rat brainstem

671

-4.52mm Fig. lb. Fig. 1. Schematic coronal sections of the rat brainstem, modified from the Paxinos and Watson atlas of the rat brain,@ showing the location of structures in which a significant increase of [14C]2-DG uptake was detected in the “early” unanesthetized, formalin-injected group. The size of stippling is related to increase in functional activity (mean values from the whole structure); see also Table 1 and Fig. 5. Values from the side contralateral to the injected paw are represented on the left. Numbers represent A-P coordinates with respect to bregma.

a major candidate for supplying noxious input to dorsal column nuclei, do not display clear nociceptive responses in the rat.3’ On the other hand, false negative (or positive) effects might result if undetected absolute LGU changes in the reference structure do occur in

the different experimental groups. Values from regions presumably not directly affected by treatment (such as visual or auditory structures) were not different in the three groups, suggesting that this possible source of error had a minor effect.

672

C. A.

PORRO

et al.

Fig. 2. Representative autoradiogram from coronal section of the rat brain at the level of the caudal third of the superior colliculus in a formalin-injected rat of the “early” unanesthetized group. Formalin injection was performed in the left forepaw. The original image was digitized, filtered and gray-coded according to metabolic values. Arrows point to the location of the highest MI in the lateral portion of the periaqueductal gray matter, near the border with surrounding structures. Note the bilateral uptake of [14C]2-DG. The right side of the figure corresponds to the right side of the brain.

Despite all these caveats, it seems reasonable to assume that regions displaying significant effects are at least the most heavily involved in processing of prolonged noxious stimuli. Functional changes in the early group

creased by formalin injection (see Ref. 69a). A minority of spinal projections are collaterals of fibers directed to the medial and ventrobasal thalamus.34~40.UHowever, the observed effects are also conceivably related to the highly complex pattern of interconnections between the various brainstem nuclei, as well as to descending pathways from higher brain levels.6,8,27,67.82.69.90

In the unanesthetized, formalin-injected group metabolic activity was increased in a number of regions from the caudal medulla to the mesoOn the basis of available electrophysiological, bediencephalic junction, for which a role in nocihavioral and anatomical knowledge it is likely that ceptive or antinociceptive mechanisms has been the activated regions play distinct roles. For instance, suggested.4~32~93~94 In all of these structures the extent LRt is part of the corticocerebellar 100~s~~but also of the functional changes was considerably less than receives a strong spinal projection,53,59,‘02which origthe one observed at the spinal level, the maximum inates in part from nociceptive cells.‘* Since most LRt nociceptive neurons project to the cerebellum,85 both percentage increase over control values being 24% in structures are likely to participate in adaptive motor the ventrolateral periaqueductal gray (PAG), as compared to 62% in the ipsilateral superficial dorsal responses to noxious stimuli. A number of reticular and raphe regions were horn. activated in the early group but not in the late one, In most instances metabolic activation induced and at least some of them (namely pontine and by the noxious stimulus was bilateral, which can mesencephalic) may be related to the arousal reaction be predicted on the basis of afferent spinal pathelicited by the noxious stimu1us.63.*2 ways.53B1o2Indeed, the majority of the affected The increase observed in nucleus reticularis regions receive a direct input from spinal projection dorsalis of the medulla confirms recent electrophysiocells, including nociceptive neurons.‘“,93,9s Both logical observations describing a population of cells spinal neurons projecting to and brainstem nociceptive neurons usually have large receptive fields.33~56~‘Wresponding to noxious stimuli from the whole body (or large portions of it) in the halothane-anesthetized The cells of origin of spinoreticular and spinomesencephalic tracts are located in different laminae of rat.Y’ This region projects to the spinal cord and brainstem and limbic structures9a9’ Evidence for a the spinal cord gray matter, as well as in the interstitial nucleus of the dorsolateral funicu1us,‘5,57,59 role of most rostra1 aspects of the medullary reticular formation, such as nucleus gigantocellularis and all regions where metabolic activity was highly in-

Functional activity mapping of the rat brainstem

673

Fig. 3. Representative autoradiograms from coronal sections of the rat brainstem at the level of the rostra1 pate of the anterior pretectal nucleus from a control animal (left) and a fo~alin-inj~~d animal of the “early” unanesthetized group (right; see anatomical scheme below). Note the increase induced by treatment, most evident in this section on the side ipsilateral to the injection (arrow). The maximum [‘4C]2-DG uptake is distributed in the rostra1 third of the nucleus in both groups.

paragigantocellula~s, in nociceptive and antinociceptive mechanisms and spinosupraspinal loops is compelling.4*32*93.94

Raphe neurons respond to noxious stimulation26*64*80and project to brainstem and forebrain regions, each nucleus having a specific pattern of

connections (see Refs 67 and 89). A role of nucleus raphe magnus neurons in adaptive processes such as stress and alertness, in addition to supraspinal control of somatosensory input,4J0,62 has been proposed on the basis of electrophysiological studies in unanesthetized animals.‘*@ The lateral portion of the parabrachial area is part of the pain pathways;z9~4’~s*~s3 moreover, electrical stimulation of the parabrachial area induces potent analgesia.19 Nucleus cuneiformis receives input from the spinal cord, PAG, and deep layers of the superior collicu1~s’~” and sends fibers to the nucleus raphe magnus and surrounding reticular formation, as well as to the

nucleus reticular-is parv~ellula~s.~~‘oi Stimulation of nucleus cuneiformis determines inhibition of responses to noxious stimuli of spinal units,13 behavioral analgesiaa’~‘O’and defense-like reactions.60 The metabolic increase observed in the deeper layers of the superior colliculus is of particular interest since this region receives a direct projection from the spinal cord and several somatosensory structures.2~s3~74~‘oz Nociceptive-specific and multireceptive neurons with receptive fields on the face and forelimbs have recently been described in the rat; they are candidates for the mediation of withdrawal behavior elicited by painful stimuli.” The ~~aqueductal gray matter is linked to a variety of functional circuits (see references in Ref. 54), including endogenous antinociceptive systems4sq4 In the ventrolateral PAG, where the spatial distribution of metabolic activity was heterogeneous, higher values were observed in the lateral part, at the border with surrounding reticular formation. The

C. A. PORRO Ed al.

674

- 14.30mm

- 11.96mm

Fig. 4. Schematic coronal sections of the rat brainstem, modified from the Paxinos and Watson atlas of the rat brain,@ illustrating the location of structures in which significant changes of metabolic activity were found in the “late” unanesthetized, formalin-injected group. Values from the side contralateral to the injected paw are shown on the left.

majority

of direct spinal afferents end in this realthough they constitute only a relatively small fraction of PAG connections.6,48.49.54 This subregion also displays the highest levels of cytochrome oxidase reactivity, which is thought to be related to the presence of synaptic terminals.17 gion,43.53.99.102

To our knowledge the response properties of APT neurons have not been systematically explored by electrophysiological techniques, although units responding to noxious stimuli have been described.‘4,7’ In the rat, the anterior pretectal region almost exclusively receives input from somatosensory structures

675

Functional activity mapping of the rat brainstem Table 2. Metabolic activity indexes of pentobarbital-anesthetized Saline-injected I

C Caudate n. Gracilis n. N. caudalis V MdV MdD N. solitary tract Lateral ret. n. Inferior olivary complex N. ret. gig., dors N. ret. gig., vent. N. ret. paragig. N. raphe magnus Parvocellular ret. n. Facial n. Deep cerebellar n. medial interposed lateral Main V Superior olivary c. N. ret. pont. caud. N. ret. pont. oralis Lat. parabr. n. Median raphe n. Dorsal raphe n. Inferior colliculus N. cuneiformis Periaq. gray, ventrolateral dorsal Deep mesenc. gray Sup. colliculus, superficial layers deep layers Substantia nigra, pars compacta pars reticulata Red nucleus Ant. pretectal n.

rats

Formalin-injected C

I

56+2 54& 1 60&l* 56f l* 61 & I** 56+2 52 * 2

60+2 53+ 1 62 + 2* 56k 1* 60& l* 56 k 2 52 + 2

68 & 2 65 k 3 52+ 1 50* 1 50 & 2 50 f 2 51* 1 50* 1 48k 1 53 * 1 53 + 1 54 * 1 54 * 2

61 f 2 54 * 2 55 f 1 55&2

68 + 54 + 55+ 56 k

74 & 2 76 k 3 77 + 2 78 k 2 80 & 1 80 + 1 62 + 2 61 k3 124+7 125k4 48 + 1 48 k 1 49 + 2 50+ 1 47 & 2 47* 1 59 + 3 48 + 2 113*4 114&3 47 + 2 48 & 2

74&4 78+2 80+ 1 62 f 3 12929 50 + 1 525 1 48 + 1

56 + 2 52 & 3 54+ 1 51&2 51 * 1 52 f 3 48 k 1

55 + 1 51*2 55 &2 50 * 2 54 * 2 52 f 3 41* 2

4 2 1 2

48 f 2 60&2’ 58 k 2+ 58 +2 58 + 2 73 *4 78 k 3 79 f 2 64+3 128+8 50 k 2 53& 1 48k 1

64+3 48 k 3 ll8+4 ll8&6 49 + 2 49 * 2

48 k 2 48 & 2 50 * 2

49 * 2 47 * 3 49 k 2

49 + 3 50 & 2 50 * 2

50 f 2 5Ok 3 49* 1

66 * 3 56 k 1

67 & 5 56 + 1

65 f 3 58 + 2

65 + 3 58 + 2

58 + 50 + 53 * 60 f

59*2 52 + 2 52 k 2 59 + 2

60 + 2 51*2 54 * 3 60 + 3

60 If: 2 52 k 2 54 f 2 60 + 4

2 2 3 2

Values are means f S.E.M. (n = 4 per group) of Mls, calculated as described in Experimental Procedures. C, contralateral; I, ipsilateral to the injected paw. *P < 0.05 and **P < 0.01, significantly higher than controls.

such

as the spinal cord and dorsal column nuclei,45,46,50 brainstem,67~89~90~‘0’and cortica15.96 re-

Effects of anesthesia

gions. Similarly, anterior pretectal nucleus (APT) receives different somatic inputs in the cat.‘s41 In anesthetized rats electrical stimulation of APT provokes behavioral analgesia70~75~77 and selectively inhibits the response of multireceptive neurons of the deep laminae of the dorsal horn to noxious stimuli.‘* Effective sites are located within the anterorostral portion of the nucleus, where the highest metabolic values were found in the present study; this subregion has been proposed as a separate entity on anatomical grounds.*’ Although the activation of motor outputs may conceivably contribute to the observed effect, the finding of a substantial LGU increase after formalin injection supports the hypothesis that APT is part of a negative feedback loop inhibiting the rostra1 transmission of noxious information.

In pentobarbital-anesthetized rats metabolic increases in the formalin group were only detected at the medullary level. Previous 2-DG studies in barbiturate-anesthetized rats employing electrical stimulation of the saphenous nerve” or of the dental pulps3 failed to reveal any LGU increase in brainstem structures, with the exception of the ipsilateral trigeminal complex in the latter study. Barbiturate effects on nociception are complex and dose-dependent. Intrathecal pentobarbital depresses nociceptive reflexes in spinal and intact rats (e.g., increases tail-flick latencies’2.2’J6), while intraperitoneal administration of low doses of the drug yields opposite effects’2l81and diminishes the antinociceptive potency of morphine injected into the rat periaqueductal gray.6s The facilitatory effects of low

676

C . A . PORRO et al.

caudal medulla, while it was abolished at higher levels. The bilateral increase in the subnucleus caudalis of the trigeminal complex was unexpected. A possible explanation is that this enhancement of metabolic activity is related to inhibitory heterotopic effects (Diffuse Noxious Inhibitory Control) induced by the noxious stimulus, as described in electrophysiological experiments.2° A trend towards LGU increase was also observed in unanesthetized animals (Table 1). The increase in dorsal medullary reticular field may be due to changes in the respiratory rate. 9j

Metabolic activity indexes Mean % differences from controls

Med. ret. n., dorsal Med. ret. n., ventr. Spinal n. V, caud. Lateral ret. n.

N. ret. paragig. N. ret. gig., alpha Parvocell. ret. n.

N. raphe magnus N. ret. pontis caud. N. ret. pontis or. Lat. parabrachial n. Median raphe n. Dorsal raphe n. N. cuneiformis Periaq. gray, V-L Periaq. gray, Dors. Deep sup. oollioulus Ant. pretectal n.

Functional changes in the late group

-5

0

5

1Unanesth.

10

15

20

25

30

[~Anesth.

• ANOVA, p ~ 0.05 Fig. 5. Histograms depict mean percentage differences relative to controls of metabolic activity values of unanesthetized or pentobarbital-anesthetized formalin-injectedrats ("early" groups), in structures in which treatment exerted significanteffects. Data refer to the side contralateral to the injected paw. pentobarbital doses on the tail-flick reflex and on the noxious-evoked responses of spinal neurons 16'36may be due to blockade of tonic descending inhibitory systemsJ 2'28'8~ On the other hand, responses of nociceptive neurons of the reticular formation and thalamus are readily abolished. 38'93The mechanisms for these effects are not known, although they are likely to be related to the drug-induced disruption of the synaptic circuitry76 of different neural structures. It has long been demonstrated that pentobarbital potentiates presynaptic inhibition,24and this was later shown to be due to the drug-induced enhancement of GABA mechanisms.39'76'86At the spinal level, GABAimmunoreactive neurons are likely to be a major fraction of inhibitory interneurons, which may act pre- and postsynaptically to reduce the transmission of nociceptive information.4'88'97 On the other hand, recent results suggest that GABAergic neurons in the periaqueductal gray matter tonically inhibit descending inhibitory pathways to the spinal cord (see Ref. 73). Barbiturates may independently depress the strength of excitatory synaptic transmission at different CNS sites. 76 Therefore, the net effect after systemic administration of pentobarbital is conceivably the result of the balance between the suppression of noxious-evoked activity at spinal and supraspinal sites and the removal of descending inhibition. At the dosage employed in the present study the metabolic increase induced by formalin injection was at least in part preserved at the spinal cord and in the

Metabohc actwlty changes with respect to controls were observed in few structures. Moreover, with the exception of nucleus cuneiformis, the extent of the increase was less than the one found in the early group. Similar results were obtained at the spinal cord level (see Ref. 69a). This time-related decrease in formalin-evoked LGU enhancement is likely to be due to peripheral factors, namely to parallel changes in the discharge of nociceptors from the injured area, which in turn result in a lesser activation of spinal ascending systems and spinosupraspinal loops (see discussion of Ref. 69a). It is also possible, however, that endogenous antinociceptive systems are triggered by the noxious input and this eventually leads to inhibition of the transmission of nociceptive information at one or more CNS sites. Biochemical studies have provided evidence for enhanced [Met]enkephalin release from nucleus reticularis gigantocellularis42and increased beta-endorphin-like immunoreactivity levels in the ventral periaqueductal gray matter 69 after formalin injection. These changes require time to develop, being evident 30~50 min following treatment. These biochemical effects could be accompanied by parallel activation of at least some brainstem antinociceptive circuits, of which opioid peptides are considered to be an essential link.4'35'87 In fact electrical or chemical stimulation in or near structures whose metabolic activity was elevated in the late group may elicit analgesia and antinociception (see Refs 22 and 94). Although most studies have so far been directed to descending control at the spinal cord level, neurons in the periaqueductal gray matter have been reported to affect noxious-evoked responses at supraspinal sitesJ '61 Further studies are needed to test this hypothesis. CONCLUSION

The present study gives evidence for dynamic changes of functional activity occurring over time in brainstem structures after formalin injection in one paw. Since the biological significance and the consequent adaptive responses are conceivably diverse

Functional activity mapping of the rat brainstem according to the nature, site and intensity of noxious stimuli, it would be helpful to investigate whether different populations or structures are activated under different painful situations.

611

Acknowledgements-The authors wish to thank Prof. R. Corazza and Prof. G. Carli for helpful comments on the manuscript. This work was supported by funds of the Minister0 della Pubblica Istruzione and Consiglio Nazionale delle Ricerche, Italy.

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