Cardiac, ventilatory and behavioural arousal responses evoked by electrical brain stimulation in the goldfish (Carassitis auratus)

Physiology & Behavior, Vol. 43, pp. 715-727. Copyright 0 Pergamon Press plc, 1988. Printed in the U.S.A.

0031-9384/88 $3.00 + .OO

Cardiac, Ventilatory and Behavioural Arousal Responses Evoked by Electrical Brain Stimulation in the Goldfish (Carassius auratus) ILONA Department

A. QUICK AND PETER R. LAMING

of Biology, The Queen’s University Belfast BT7 lNN, Northern Ireland

Received

of Belfast

24 February 1988

QUICK, I. A. AND P. R. LAMING. Cardiac, ventilatory and behavioural urousal responses evoked by electrical brain stimulation in the goldfish (Carassius auratus). PHYSIOL BEHAV 43(6) 715-727, 1988.-Goldfish (Carassius nuratus) were fitted with intracranial stainless steel microelectrodes for electrical evocation of behavioural arousal and its cardiac

and ventilatory correlates. Behaviour was monitored on a videosystem and ECG electrodes and a buccal catheter were implanted to monitor physiological responses. Threholds for responses were described in relation to the current spread likely to excite CNS tissue. Two types of responses were obtained. These were (A) cardiac and ventilatory responses alone, apparently due to stimulation of primary sensory pathways and (B) these responses and behaviourrd arousal responses which were elicited at higher thresholds. These latter, more complete expressions of arousal resulted from stimulation of the Dm/Dc region of the telencephalon, the dorsal diencephalon and the midbrain tegmentum. Response thresholds were higher and physiological response magnitudes lower in the midbrain tegmentum compared to the forebrain regions. Teleosts Arousal Midbrain tegmentum

Brain stimulation Telencephalon

Cardiac responses

Ventilatory responses

response as well as nonspecific arousal or stimulus registration [2]. The nonunitary nature of the response to novelty or orientation reaction (OR) of mammals is associated with inconsistent physiological correlates [2]. Recent research on fish and amphibia (reviewed by Laming [23]) suggests that in phylogenetically older vertebrates arousal is consistently the prevalent component of the OR. Teleost fish, especially, have provided a useful model for studying mechanisms of arousal. In these animals arousal is a nondirectional response to a novel stimulus of any sensory modality and has been shown to be associated with welldefined behavioural and physiological changes [29]. The behavioural components of the arousal response of teleosts consist of fin movements which rarely change the animal’s position in the water. These have been described for a variety of teleost fish; Carassius auratus [29,45], Rutilus rut&s [26], Halichoeres bivittatus [25], Holocentrus rufus [22]. In goldfish, a marked behavioural arousal response is charactermed by a change in the beat of pectoral, pelvic and caudal tins, an erection of the dorsal fin and movements of the eyes. Physiological changes associated with behavioural arousal responses include an increase in amplitude and frequency of the EEG [20], a reduction in visceral venous blood flow [28] and decreases in ventilatoty amplitude and cardiac and ventilatory rate [29].

SINCE the work of Moruzzi and Magoun [36] who produced behavioural arousal and cortical EEG desynchronisation by reticular formation (RF) stimulation in cats, the RF has been implicated in the regulation of levels of consciousness such as sleep, wakefulness, arousal and attention [3,17]. The concept of an ascending activating system [34] derived from the earlier experiments. Electrical stimulation of the reticular formation was also found to cause an activation of diverse sensory structures. Thus, reticular stimulation enhances visual evoked potentials [48] and causes an increase in 14C-2deoxyglucose (2DG) uptake in auditory pathways [12]. The reticular formation, however, is not the only region involved in behavioural arousal in mammals as it can also be evoked by stimulation of various cortical sites [46]. The RF can also exhibit inhibitory influences. For example, Chin et al. [4] obtained a reduction in auditory evoked potential with mesencephalic RF stimulation and medullary areas of the RF have been associated with the inhibition of general motor activity [33]; Gonzalez-Lima and Scheich [13] reported a suppression of 2DG uptake in some cortical areas after RF stimulation. The functional significance of the RF and its interactions with other regions can therefore be described as complex and diverse. Most information on elicitation of arousal has come from studies on mammals where subjects often show a directional

attentional

715

Ventilatory and heart rate responses have been the most consistently used quantitative measures of the arousal response in teleosts and it has been shown that the magnitude of the response is directly related to the strength of the stimulus [44]. Electrical stimulation studies on arousal in fish have been restricted to the work of Savage [45] and Laming [ 211. These experiments were primarily concentrated on telencephalic and superficial tectal stimulation sites. They also used either behavioural [45] or physiological [21] responses alone to define arousal and may therefore have interpreted results as ‘arousal’ which were more specific in their nature. This study, therefore, describes the effects of stimulation of more diverse and deeper brain regions, using cardiac. ventilatory and behavioural responses together as criteria of arousal. Further emphasis has been laid on a precise location of areas involved in the evoked response. Response thresholds, therefore, were described according to the distance over which current spread might be expected to excite neuronal elements [41]. The present study was performed to more clearly and unequivocably describe brain regions which on electrical stimulation are concerned with the expression of behavioural arousal and its physiological correlates in the teleost, C‘rrrrr.\.si/lsN~{Y(~/~Js. METHOD Suhjrct.5

Goldfish (Curussius a~trcltrrs) of 1 l-15 cm in length were obtained from a regular supplier. They were maintained in 1.5~1.5x0.5 m deep aquaria containing aerated, filtered water at 17~2°C for at least four weeks prior to experiments. Twenty-one animals were randomly selected from stock and underwent the following surgical and experimental procedures. Operations

Fish were anesthetised by immersion in a solution of 1: 10000 MS222 (tricaine methane sulphonate) until opercular movement had just ceased. Animals were then equipped with: (a) a ventilatory catheter consisting of a IO cm length of vinyl-tubing (i.d. 0.63 mm, o.d. 1.4 mm) inserted into the buccal cavity through a hole in the midline between the nares [29]; (b) a pair of ECG electrodes (insect pins, size 0) insulated, with varnish to the tip, to record the electrocardiogram (ECG), implanted as previously described [42]; (c) two stainless steel microelectrodes (tip diameter 5.6 pm) for electrical brain stimulation. Stimulation electrodes were insulated with three coats of insulating varnish (Radiospares) leaving an exposed area of l-l.5 mm at the tip. They were implanted through two burr holes above the desired brain areas using a Narashige micromanipulator and fixed in position with Permabond adhesive and a drop of Howmedica activator followed by a layer of Simplex dental cement. The wires of the two sets of electrodes were held in a dorsal suture just rostra1 to the dorsal fin. A cube of polystyrene foam was attached to the wires to compensate for the negative buoyancy imparted by the assembly. Fish were then revived by perfusion of their gills, via the mouth, with fresh water. When breathing movements recommenced, fish were returned to the stock aquaria. Rrcording

and Stimulatior~

After 24 hr postoperative recovery, fish were transferred to a 22 x 6 x 10 cm deep, 1 cm wire mesh, trough suspended in a 35 x 25 x 25 cm deep perspex tank which was half-filled with

water from the stock-aquaria. After this period ot tune nor ma1 behavioural and physiological responses have returned 1291 and in this situation lish. though restrained from freely swimming. could be observed during behavioural arousal. The tank was situated in a black box and a 80 W fluorescent tube gave a constant background illumination of 250 lux: consequently, fish were visually isolated from any external stimuli. Animals were left undisturbed for 1 hr to adapt to the experimental situation. This set-up provided comparable conditions between stimuli and individual fish. Electrical brain stimulation consisted of 60 Hz monopolar square waves with a pulse duration of 1 msec delivered for 1-2 set by a Grass S44 stimulator and PSI constant current unit. Stimulation currents ranged from 0.1 to 150 PA (in some cases up to 200 PA) applied in increments of 0.5-10 PA and were delivered at intervals of not less than 2 min. Behavioural responses to electrical brain stimulation were observed by an overhead video camera. A Beckman 411 dynograph was used to monitor both ventilatory changes via a pressure transducer and cardiac responses. The magnitudes of cardiac and ventilatory (C + V) arousal responses were determined by the ratio of the longest interbeatiinterbreath interval in the 10 set poststimulus period to that in the 10 set prestimulus period [27]. Threshold stimulation current was considered to have occurred if on 3 consecutive stimulations of the same current the response ratio exceeded the mean + standard error of similar random measures on the unstimulated animal prior to the experiment. Similarly, thresholds for behavioural arousal responses were considered to have been achieved if three stimuli of the same current induced at least two components of the behavioural arousal response as described in the introduction.

After determination of thresholds for cardiac, ventilatory and behavioural responses up to a maximum current of 150 PA for each electrode position, fish were anesthetized and a 300 FA DC current was applied for 30 set to form electrolytic lesions at the electrode site. Fish were then killed in 1:5000 MS222, the brains removed, fixed in 5% formalin, wax-embedded, sectioned at 15 pm and stained with Haematoxylin and Eosin. Electrode positions were determined from lesion damage. A topographical map of transverse sections of the goldfish brain was constructed. Datu

Treutnwrlt

Response thresholds for electrode positions were grouped into 5 threshold ranges: (1) below 10 PA; (2) 10-20 PA; (3) 20-40 WA; (4) 40-80 PA; (5) over 80 PA for cardiac and ventilatory responses; and for behavioural responses: (1) below 20 PA: (2) 20-40 PA; (3) 40-50 PA; (4) 55-80 PA; (5) over 80 PA. In order to determine which areas of the brain were involved in evoking the obtained responses at the determined thresholds, results were described by the distance over which a given current can be expected to excite CNS tissue. Ranck’s review [41] of 10 different studies in which current distance relations for neuronal thresholds in mammalian CNS tissue were examined, was used for the calculations. Ranck normalized the individual results to allow direct comparability and graphically summarized them. For the purposes of the present work, values from Ranck’s [41] summary were corrected for 1 msec pulse duration. Minimal and maximal distances over which neuronal elements would be expected to become excited at a given current were calcu-

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FIG. 1. Graph of minimal (m) and maximal (0) current spread with distance to achieve threshold for excitation of CNS tissue: derived from calculations based on the review of ten studies by Ranck [41] and corrected for 1 msec pulses.

lated and are shown in Fig. 1. Minima1 and maximal current spreads for the various response threshold ranges were derived from this graph (Table 1) and each electrode position was then shown with its particular response threshold current density bands (RTCDB) for the three types of responses on line drawings of the representative serial sections (Fig. 3A, B, C: Sl-14). The outer RTCDB for any given threshold range represents the limit of current spread which will be sufficient to excite neurons, the inner band the minimum distance at which neurons may be excited. As the outer RTCDB for the next lower threshold range always exceeded the inner RTCDB of the threshold range under consideration, the space between bands reflects the volume of CNS tissue in which neurons are most likely to be activated by the electrical stimulus to evoke the response. A series of two-way ANOVAs, followed by Tukey-tests for multiple comparisons, were carried out to test for the relationships between response thresholds and areas of the brain stimulated. Each electrode position was described in two ways: (1) Longitudinal distance (LD): This was the distance of the electrode position from the anterior telencephalic border as a proportion of the total distance from the anterior telencephalon to the posterior cerebellar border. (2) Radial distance (RD): This was the distance of the electrode position from the central longitudinal axis of the brain as a proportion of the radius of the brain passing through that position in that transverse section. After estimation of RD and LD, electrode sites were split into 4 equal groupings for each RD and LD. In the case of LD these groups represented (a) most of the telencephalon: (b) posterior telencephalon, diencephalon and anterior half

TABLE

1

MINIMUM AND MAXIMUM DISTANCES EXPECTED FOR ADEQUATE CURRENT SPREAD FOR NEURONAL EXCITATION DERIVED FROM FIG. 1 Current

Minimum

Maximum

Cardiac and Ventilatory Response Threshold Ranges Below IO PA IO-20 PA 2(&40 FA 40-80 /.LA

180 pm 300 pm 410 I*rn 620 pm

380 pm 550 pm 720 Frn 1090 pm

Behavioural Response Threshold Ranges 20 PA 3&40 PA 4&55 PA 55-80 PA

300 pm 410 pm 490 wrn 620 Frn

550 pm 720 pm 850 pm 1090 pm

of the midbrain; (c) posterior midbrain and (d) the cerebellum. In addition to the estimation of thresholds for cardiac, ventilatory and behavioural responses, quantitative measures of response magnitude were determined for cardiac and ventilatory responses [27] at their thresholds and at that of the behavioural response. A two-way ANOVA was used to examine the relationships between electrode position, response threshold and the magnitude of the response. In addition, paired t-tests were carried out to compare response magnitudes at physiological and behavioural thresholds.

QUICK

71x

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__-._._ ._

AND LAMING

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ABBREVIATIONS CbG CbM cc CE CM CoCb DC Dd DF DI Dm DP EG FR G H HbCo Hd Hv LH LIH LL LV MED

granular layer of cerebellum molecular layer of cerebellum cerebellar crest nucleus centralis of the inferior lobe corpus mammillare corpus cerebellum area dorsalis pars centralis area dorsalis pars dorsahs nucleus diffusus of the inferior lobe area dorsalis pars lateralis area dorsalis pars medialis area dorsalis pars posterior eminentia granularis of the cerebellum fasciculus retroflexus nucleus glomerulosus habenula habenular commissure dorsal zone of periventricular hypothalamus ventral zone of periventricular hypothalamus nucleus lateralis hypothalami inferior lobe of hypothalamus lateral lemniscus nucleus lateralis vafvulae medulla

MLF nMLF OT PC0 PGa PGI PM PoA PTA T TA TB TCo TL TLa TLo TS VCL VCM Vd VI VS vv

RESULTS The major topographical features of the goldfish brain are described from representative serial sections at 450 Al.intervals (Fig. 2), Section numbers are denoted by the prefix ‘S’. The experimental data from 17 successfully tested animals were used for the study of responses to electrical stimulation. Four other fish had to be excluded due to inadequate histological verification of electrode positions. Results from one electrode (Fig. 3, SlO) were not included in the statistical analysis as stimulation with currents up to 200 PA had no effect, probably due to faulty connection of the electrode leads during stimulation. Behavioural

Responses

The variability of elicited behavioural responses was small, partly due to the nature of the experimental set-up. In some cases, where stimulation evoked behavioural arousal responses, further increase in current resulted in “swimming’ ’ movements. These were characterized by similar fin-movements, but unlike arousal responses, caused a significant displacement of the animal in the water. The intensity of this response seemed to be correlated with current strength; physiologically it was associated with either a bradycardia or tachycatdia or both. Other infrequently elicited behavioural responses were muscular spasms and twitches of the body, which occurred at currents above 100 PA. Only in one case was this type of motor response evoked at the low current of 40 PA (Fig. 3C, Sll). Muscular spasms were associated with a reduction in cardiac and ventilatory rate, which could exceed the 10 set poststimulus period; subsequently, there was a tachycardia in these cases.

medial longitudinal fasciculus nucleus of the medial longitudinal fasciculus optic tectum posterior commissure nucleus preglomerulosus anterior nucleus preglomerulosus lateralis posterior mesencephaiocerebellar tract preoptic area prectectal area telencephalon nucleus tuber-is anterior tectobulbar tract tectal commissure tectal laminae nucleus of the torus lateralis torus longitudinalis torus semicircularis lateral valvula cerebellum medial valvula cerebellum area ventralis pars dorsalis area ventralis pars lateralis area ventralis pars supracommissuralis area ventralis pars ventralis

Behavioural arousal, itself, consistently involved a movement of pectoral and pelvic fins and a slight undulation of the caudal fin. Pectoral fin movements were found to occur first, i.e., at lower currents; a further increase in current then resulted in movement of other fins. Only on three occasions was pectoral fin movement not a part of the arousal response. Response

Threshold Current Density Bands (RTCDB)

In Fig. 3 each electrode position is shown with its RTCDBs, the size of which was calculated for the threshold current at which arousal was evoked. The RTCDBs give an estimate of the area within which neurons were most likely to be involved in producing the evoked response from stimulation of a single electrode. The most corroborative evidence for an area’s involvement, therefore, is represented by the zones of greatest overlap (darkest hatching of several RTCDBs derived from different electrodes). Cardiac and ventilatory response types (Fig. 3, A and B) were evoked by stimulation of most electrodes. In the diencephalon and mesencephalic tegmentum, areas of greatest overlap for C and V response types and behavioural response type were found in midline regions (Fig. 3, A, B, C, S6, S8 and SlO). In telencephalic areas, however, C + V RTCDBs were relatively small and behavioural responses were evoked less consistently which complicated the determination of an involved area, therefore, a closer statistical analysis was carried out in order to classify relationships between electrode site, threshold and type of responses. Effects of electrode position on response threshold. Initial analyses were performed to determine whether differences

FACING PAGE FIG. 2. Topographical map of the goldfish brain. Photographs of 15 pm, transverse, Haematoxylin-Eosin-stained sections shown at distances of 450 pm apart, are on the left; major cell groups or fibre tracts are indicated in the corresponding line drawings on the right.

6

FIG. 3A FIG. 3. Response threshold current density bands (RTCDB) for (A) cardiac, (B) ventilatory and (C) behavioural responses in goldfish (Carussius aururus). Dots: electrode positions: outer concentric ring: maximal limit of current spread to achieve CNS threshold excitation values; inner concentric circle: minimal limit of current spread to achieve excitation. The region between the RTCDBs is therefore the area in which CNS tissue would be most likely to be activated to produce the response (see text for further details). Solid circles: RTCDBs with electrodes at that level in the neuraxis. Dashed circles: RTCDBs extending from more anterior or posterior sites in the neuraxis. Squares: sites for which no response was obtained at currents up to 80 PA. Asterisk: site for which muscular spasm was obtained (3C; Sll).

in response thresholds existed either due to a longitudinal (LD) or radial (RD) position of the electrodes. Longitudinul distance (LD). Significant differences in response threshold were found between the LD groups, F(3,84)= 3.26, ~~0.05. The Tukey-test revealed that thresholds for all three types of responses were higher when electrodes were placed in the posterior midbrain area (LD3) (Table 2). Radial distance (RD). No significant differences were found when considering all 3 types of responses in four RD groups, F(3,84)=0.064, p=NS. Figure 3C, however, suggests that stimulation of laterally placed electrodes was less successful

in evoking a behavioural response. Beyond a RD of 35% there was only one position from which a behavioural arousal response was elicited. Therefore, a one-way ANOVA was performed on behavioural responses comparing the combined two inner RD groups (RD O-W%, MeantSEM threshold=96.84+- 12.08) with the two outer groups (RD W-100%, Mean*SEM threshold= 136.15+14.87), F(1,30)=4.23, p
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6

FIG. 3R

the specified ranges (Table 1) and the corresponding RTCDBs (Fig. 3) already reflected differences in current necessary to evoke either physiological or behavioural responses. This relationship was therefore further examined in terms of (1) differences in thresholds for evoking the three kinds of responses and (2) overlapping RTCDBs. Since stimulation of one electrode could evoke up to three responses it was necessary to examine whether these RTCDBs overlapped in order to establish those brain regions which were involved in elicitation of all three responses. In this context, overlapping RTCDBs decribed an interresponse type relationship rather than the interelectrode relationship mentioned earlier. Threshold. The two-way ANOVA revealed (Table 2) that there were differences in thresholds for the different types of responses: those for cardiac and ventilatory responses being significantly lower than those for behavioural responses re-

gardless of the topographical orientation. Thus, significant differences were obtained when comparing response types: for LD groups, F(2,84).=25.59, ~~0.01; and for RD groups, F(2,84)=29.57, ~~0.01. Thresholds for C + V responses usually fell into the same current ranges (Fig. 3A, B); only in four cases were ventilatory responses grouped into the next higher range (Fig. 3B, S4, S7, and S9). Overlapping RTCDBs. As a result of the similarities in thresholds, RTCDBs for C + V responses often overlapped (Fig. 3A, B). In the cases where C + V responses but no behaviour or very high threshold behavioural responses were evoked, the area described by the C + V RTCDBs was not overlapped by a behavioural RTCDB. These were electrodes placed in the dorsal lateral area (Dv + Dl) of the mid-to posterior telencephalon (Fig. 3C, S3 and S4), the optic tectum (Fig. 3C, SS and S7), torus semicircularis (Fig. 3C, S7, S8 and SlO) and electrodes situated in the torus

722

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1

C ,

Q lmm

6

FIG. 3C longitudinalis (Fig. 3C, S6 and SlO) orjust ventral to it in the lateral valvula (Fig. 3C, S7). Stimulation of 12 sites, however, caused all three types of arousal responses with the RTCDBs for each response type showing some overlap. These were 3 electrode positions in the Vd and bordering Dm/Dc regions of the telencephalon (Fig. 3, S2-4), one in the deep anterior tectum (Fig. 3, S5), 2 located near the fasciculus retroflexus (Fig. 3, S6 and S7), 4 in the posterior tegmentum (Fig. 3, S9 and Sll) and 2 situated in the hindbrain (Fig. 3, S12 and S13). Relationship between response threshold and response magnitude. In the 12 cases in which stimulation of a single electrode evoked all three kinds of responses, behavioural arousal was consistently associated with cardiac and ventilatory responses. This allowed an examination of response magnitudes at physiological and behavioural thresholds and the relationship of these to electrode position.

Ejfects of LD on response magnitude. Since the previous analysis had revealed that response thresholds were higher in posterior midbrain areas (LD3) it was interesting to find out whether response magnitudes were also different in relation to the longitudinal position of the electrodes. Owing to the small number of electrode positions from which all three responses were obtained on stimulation, the two-way ANOVAS were carried out using combined LD groups 1 and 2 (Fig. 3, Sl-S7) compared with groups 3 and 4 (Fig. 3, SS-S14). The magnitudes of cardiac and ventilatory responses were significantly higher in the combined anterior groups which included the telencephalon and diencephalon. This was demonstrated by plotting response magnitude against LD (Fig. 4) and by ANOVA results for cardiac and ventilatory magnitudes at their physiological threshold (cardiac magnitudes Mean+SEM LD 1 + 2=1.62+-O&, LD 3 + 4= 1.47~0.1; ventilatory magnitudes Mean+SEM LD 1 +2=

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FIG. 4. Relationship of longitudinal distance to cardiac and ventilatory response magnitudes at physiological and behavioural thresholds. A=cardiac responses at physiological threshold; B=cardiac responses at behavioural threshold; C=ventilatory responses at physiological threshold; D=ventilatory responses at behavioural threshold. The horizontal solid and dashed lines represent the Mean+SE of the response ratio in unstimulated fish. Note that the largest responses were obtained from the telencephalon (LD O-30) and the diencephalon (LD 35).

TABLE CARDIAC, VENTILATORY AND

Cardiac LD Group

Mean ? SEM

THRESHOLDS

(MEAN + SEM) IN

Ventilatory Tukey

Mean ? SEM

Behaviour Tukey

Mean ? SEM

Tukey

* 14.8

30

? 14.8

84 + 18.08

(C + V)

2 Fig. 3 s4,ss, S6,S7

30.5 + 10.1

34

2 11.04

95.5 2 11.16

(C + V)

3 Fig. 3 S8,S9,SlO

51.3 ? 10.46

4 Fig. 3 Sll,Sl2, s13,si4

39

1

30

2

BEHAVIOURAL RESPONSE FOUR LD GROUPS

Fig. 3 Sl,S2,S3

2 10.04

(~2~4)

57.7 2 10.9

39

2 10.04

(1,2,4)

121.8 2

76

t

9.98

(C + V) (132.4)

9.79

(C + V)

Tukey=Tukey-test for multiple comparisons. Significant differences @<0.05) denoted by C, V or B for types of responses and 1, 2, 3, or 4 for LD groups.

TABLE 3 CARDIAC, THRESHOLDS

LD Group LD I+

2

VENTlLATORY AND BEHAVIOURAL RESPONSE (MEAN 5 SE, n= 121IN COMBINED LONGITUDINAL. GROUPS I -I- 2 and 3 - 4

Cardiac (Mean t SEM)

Ventilatory (Mean + SEM)

22.08 k 9.01

14.8

t

9.85

38.33 ~?r9.09

33.33 2 10.21

Behaviour (Mean k SEM) 50

2

8.06

61.66 t

12.49

Fig. 3 Sl-s7 LD3+4 Fig. 3 S%Sl4

2.16kO.35, LD 3 + 4= 1.49-~0.09), F(1,20)=4.49,p<0.05, and at their behavioural threshold (cardiac magnitudes Mean? SEM LD 1 + 2= 1.7820.12, LD 3 + 4=1.45&O. 11; ventilatory magnitudes Mean+SEM LD 1 + 2=2.19?0.37, LD 3 + 4=1.46?0.11). F(1,20)=6.14, p
Arousal, monitored by behaviour and its physiological correlates of cardiac and ventilatory rate reductions, was evoked by electrical stimulation of diverse regions of the goldfish brain. Thresholds were used to describe the involvement of an area in facilitating arousal responses. Additionally, the interactions of areas involved was estimated by overlapping of a calculation of spread of effective current. The description of results in relation to current spread presumes a possible relationship between strength of current and the likelihood of an arousal response occurring. Such a relationship, however, was not definitively obtained, as currents of 150 PA did not evoke responses though the estimated spread overlapped regions which evoked responses at low thresholds. Lack of a distinct relationship may have been due to the high currents and large volume spread involving many regions, some of which may have had an in-

hibitory effect on arousal. Alternatively, the estimate\ of current effective in eliciting neuronal activity derived I’~om mammalian studies 1411 may have given an overestimate ot effective current spread in teleosts. Cardiac and ventilatory arousal responses were elicited at similar thresholds. A high correlation between C - c’ responses was also reported by Laming and Savage 1291 who examined the physiological changes associated with behavioural arousal and fright evoked by a variety of environmental stimuli. In the present work, thresholds for eliciting behavioural arousal responses were consistently higher than those for physiological responses. Only in 36% of the stimulated sites was the physiological response succeeded by a behavioural response so that all three RTCDBs showed some overlap. It was shown that stimulation of more peripherally and laterally placed electrodes was less successful in eliciting a behavioural response. It therefore appears that two types or degrees of arousal were evoked, characterised by (Aj physiological responses alone and (B) by behavioural, cardiac and ventilatory correlates of arousal. Type A: Physiological

Arousal

An initial consideration was the possibility of a direct stimulation of cardiac and ventilatory motor centres. The nuclei of the trigeminal, facial and vagus nerves, innervating respiratory and heart musculature, are located in the medulla oblongata [32,39]. However, all electrode sites, except one, were situated in more rostra1 or dorsal brain regions. In the telencephalon C + V responses alone were often obtained as a result of electrical stimulation at low thresholds. Laming [21], using only C + V responses as criteria of arousal also found telencephalic regions with low thresholds for arousal. These were superficial sites near the anterior commissure and in some lateral parts of the telencephalon. In the present work C + V reponses resulted from stimulation of electrodes situated in the area dorsalis lateralis and area dorsalis posterior. The latter region is known to be a prime area of terminating olfactory inputs [37]. In the dorsal midbrain C + V responses at low thresholds were obtained when the optic tectum was stimulated, where the outer layers [stratum opticum (SO) and stratum fibrosum et griseum superficiale (SFGS)] are primarily concerned with the processing of visual information since they receive direct retinal input [3 1,491. In deeper midbrain areas effective sites for stimulation were more lateral, in the vicinity of the torus semicircularis which represents the midbrain centre for acoustic and lateral line information processing in teleosts [6,19]. It therefore appears that C + V responses alone were obtained from areas which are involved in sensory information processing. Stimulation of the torus logitudinalis, however, resulted in the same response pattern [low C + V thresholds but no behaviour even at high (150 PA) current values]. This structure has connections with the tectum and valvula [16] and according to Nieuwenhuys [38] “forms a functional link between tectum and cerebellum” but does not seem to be associated with a specific sensory modality. In most cases, however, it may be that stimulation resulting in C + V responses alone was a result of activation of specific sensory inputs. The evoked C + V responses therefore, may be most appropriately classified as arousal responses to a restricted sensory input. This seems to be a contradiction since a full arousal response, i.e., including behavioural responses, can be evoked by presentation of a

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stimulus of a single modality (e.g., a light-on stimulus) [29]. In this context the differences between an environmental and electrical stimulus have to be considered. For example, a diffuse light-on stimulus would not only excite neurons in peripheral layers of the tectum but the information would also be passed to deeper tectal layers and various relay and integration centers in diencephalic, telencephalic and mesencephalic brain regions. The electrical stimulus applied to SFGS and SO probably stimulated only a small component of that evoked by a general change in visual input. The electrically stimulated regions may not therefore have the broad connectivity necessary to be responsible for the elicitation of a full arousal response. This does not, however, imply that it is impossible to evoke behaviour from, e.g., the tectum. Meyer [35] evoked coordinated “food search” responses by electrical stimulation of the tectum. These responses built up with some latency and continued after cessation of the electrical stimulus; this may have been due to recruitment of various tegmental regions [49]. Indeed, specific behaviours can occur after stimulation of diverse sites of the fish brain (see Demski [5] for review) but most have been categorized as directed responses related to motivational states such as feeding.

Stimulation of electrodes placed in the Dm/Dc region of the telencephalon, the dorsal diencephalon in the vicinity of the fasciculus retroflexus, and medial tegmental regions, resulted in C + V responses and at higher currents also in behavioural arousal responses, with all three RTCDBs overlapping. Previous stimulation studies have also reported behavioural arousal responses and responses in which some components of arousal have been noted. Savage [45], stimulating the telencephalon of goldfish, also described low threshold sites for behavioural arousal in a medial area just posterior to the anterior commissure. Fiebig et al. [9] who evoked eye movements from central and medial portions of the telencephalon in the piranha reported that these were often associated with accelerations or decelerations of gill movements and movements of pectoral fins. In relation to the midbrain, Kashin et al. [ 181 and Peter [40] described tegmental regions involved in ‘locomotion’ similar to those in which arousal was evoked in this study. Kashin et al. [ 181evoked caudal and pectoral fin movements in carp and found that pectoral fin movements were elicited from a larger region of the tegmentum at weaker stimulation currents. The latter result is similar to that in the present data when pectoral fin movements occurred at currents less than those which evoked full behavioural arousal. These results also agree with those on habituation of arousal when pectoral fin movements have been shown to be more resistant to decline than other behavioural components of the response 12.51. Peter [40] lesioned the midbrain tegmentum in goldfish and found an area extending 300 pm caudally from the posterior commissure to be most effective in causing deficits in body orientation. The area described here, however, which on stimulation evokes arousal movements extends caudally to the level of the anterior corpus cerebellum. A direct stimulation of motor nuclei would be unlikely to be responsible for the occurrence of these behaviours since motor nuclei are situated in more caudal brainstem regions 1391. Additionally, stimulation of caudal regions did not resuit in the coordinated movements found by Kashin et al. [ 181.In the work reported here, stimulation of one electrode

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resulted in a muscular twitch response at a low threshold which might have been due to a direct stimulation of descending motor pathways. The mesencephalic tegmentum is understood to function as a motor coordinating system [391 with possible involvement of the corpus cerebellum [l]. In the present study behavioural arousal was consistently associated with C + V responses whether it was evoked from fore- or midbrain regions. This agrees with data on arousal induced by environmental stimuli [29]. Laming and Savage [29] also showed that, during habituation, behavioural responses disappeared prior to physiological responses which concurs with differences in response thresholds found in this work. The RTCDBs for these responses also indicate that during behavioural arousal a bigger area is involved and it suggests that several regions activated in consort may be required to elicit the full arousal response. However, all three RTCDBs still show some overlap which was the main criterion for inclusion in this group. This was to determine those brain regions which have the integrative capacity to evoke the full arousal response. The similarities between Type B arousal and arousal induced by a variety of environmental stimuli suggest that posterior-medial telencephalic, diencephalic and tegmental regions are involved in producing generalized arousal responses. Cardiac and ventilatory responses may be a more sensitive measure of arousal since they occur at lower thresholds than behavioural responses. They can be associated with generalized arousal (Type B) or can probably also be part of more directed behavioural responses (Type A). It is interesting to note that in the cases of generalized arousal, response thresholds were lower in the forebrain and the magnitudes of the physiological responses were higher. Rooney and Laming [44] found a linear correlation between the intensity of an environmental stimulus and the magnitude of C + V responses. If this relationship is transferred to the present data, it suggests that arousal is more effectively evoked from these forebrain regions. Indeed, there is anatomical evidence that the DC region receives inputs from most surrounding telencephalic regions [37] and has connections with the tectum [S, 14, 16, 311, diencephalic and reticular elements [7,37]. Electrophysiological evidence also suggests a projection from Dc via diencephalic and tegmental relay stations to the corpus cerebellum [30]. The connections of DC are thus manifold and this region therefore meets one of the requirements expected from an area which would be involved in general arousal. However, arousal still occurs after lesions in the posterior DciDm regions and diencephalic areas [26,43] and even after telencephalic ablation [27], although its habituation is impaired. The tegmental regions from which generalized arousal was evoked harbours the mesencephalic reticular formation [39] which in mammals has been shown to be involved in arousal [36,46]. Evidence for such a role from lower vertebrates is scarce. Segura and Kacelnik [47] stimulated the mesencephalic tegmentum of toads, lizards and rats and evoked tachycardias in all three species, desynchronization of the EEG in rats and synchronization in lizards. Desynchronization of the EEG in mammals [36] and a tachycardia in toads [24] have been shown to be associated with arousal. Finkenstaedt [l l] found that the W-2deoxyglucose uptake of the telencephalon after stimulation of the tegmental RF resembled that obtained during active prey-catching and predator avoidance behavior in toads. The present data, for the first time, give evidence that, in fish, general arousal can be elicited from the mesencephalic

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tegmentum, in an area where the involvement of reticular elements may be assumed. It appears that the tegmentum is not solely involved in coordinating locomotor activity but can also be attributed a role in general arousal. However, physiological response magnitudes were lower than those obtained from forebrain regions which indicates that threshold stimulation of the mesencephalic tegmentum constituted a weaker stimulus than the electrical stimulus applied to the forebrain regions. This suggests that in the midbrain inhibitory mechanisms act on the arousal system. This can be supported by findings in mammals where caudal elements of the reticular formation exhibit inhibitory influences [33]. Alternatively, as was shown, the area for which

stimulation was required in the midbrain to elicit arousal is larger than that in the forebrain. This is very interesting since it suggests that regions higher in the neuraxis involved in arousal are more discrete, possibly as a result of neuronal convergence. Inhibitory mechanisms must counteract the facilitatory influences of these forebrain regions since arousal still occurs even after removal of the telencephalon. In summary, in fish, as well as in mammals, the mesencephalic tegmentum is involved in the elicitation of general arousal responses. Additionally, it can also be evoked from dorsal diencephalic areas and from the Dm/Dc regions of the telencephalon. This adds to the evidence that the telencephalon plays an important modulatory role in arousal in fish [I ,211.

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