A visual pathway that mediates fear-conditioned enhancement of acoustic startle

Brain Research, 276 (1983) 55--71 Elsevier

55

A Visual Pathway that Mediates Fear-Conditioned Enhancement of Acoustic Startle MARC D. TISCHLER and MICHAEL DAVIS* Yale Universtiy School of Medicine, Department of Psychiatry, Connecticut Mental Health Center, New Haven, CT (U.S.A.) (Accepted February 8th, 1983) Key words: startle - - conditioning - - lateral lemniscus - - superior colliculus - - visual cortex - - lateral geniculate

Eighty rats received 10 light-shock pairings on two successive days. Seventy-two h after the final training session, subjects received lesions directed at the primary visual areas (deep and superficial layers of the superior colliculus, dorsal lateral geniculate nucleus, pretectal nuclei, visual cortex and thalamic reticular nucleus) and at the nuclei of the lateral lemniscus and reticularis pontis caudalis, proposed components of a primary acoustic startle circuit in the rat. Control animals were sham operated. One day later, all animals were tested for startle by presenting noise bursts of 3 different intensities in the presence or absence of the light conditioned stimulus. Potentiated startle (the difference between light-noise vs noise-alone trials) was significantly attenuated or eliminated by lesions directed at the dorsal nucleus of the lateral geniculate, deep layers of the superior colliculus, visual cortex, and the posteroventral region of the nucleus of the lateral lemniscus. Lesions directed at pretectal nuclei, superficial layers of the superior colliculus, thalamic reticular nucleus, nucleus reticularis pontis caudalis or dorsal nucleus of the lateral lemniscus did not attenuate potentiated startle. The results suggest that the visual pathway that mediates potentiated startle goes from the retina to the dorsal lateral geniculate nucleus to visual cortex to deep layers of superior colliculus and down to the postero-ventral region of the lateral lemniscus where acoustic startle is modulated.

INTRODUCTION T h e way in which neural systems mediate associative learning represents an area of increasing interest to physiologists, pharmacologists and psychologists. W h e t h e r working in simple invertebrate systems or in the intact v e r t e b r a t e , a necessary first step in this e n d e a v o r is to isolate the neural circuit where conditioning occurs. O n c e this is done it should be possible to d e t e r m i n e how conditioning is brought about at a cellular level. O n e way to a p p r o a c h this p r o b l e m is to begin with a simple reflex that can be modified by prior associative learning. T h e p o t e n t i a t e d startle effect in the rat4. 7 fulfils these requirements. In this p a r a d i g m , startle magnitude is increased when the startle-eliciting stimulus is p r e s e n t e d in the presence of a cue (CS) that has previously been p a i r e d with a shock. H e n c e , with potentiated startle, conditioning is expressed through some neural circuit that is activated by the light and ultimately impinges on the startle circuit. The advantage this p a r a d i g m has over o t h e r associative learn-

ing p a r a d i g m s is that conditioning is measured by the interaction of two p r e s u m a b l y isolatable neural systems: a p a t h w a y that transmits information about the ' c o n d i t i o n e d stimulus' (e.g. the conditioned stimulus circuit) and a simple reflex circuit (e.g. the acoustic startle circuit). Recently, a primary acoustic startle circuit in the rat has been delineatedS. This consists of the ventral cochlear nucleus (VCN), the dorsal and ventral nuclei of the lateral lemniscus ( D L L and V L L ) , the nucleus reticularis pontis caudalis (RPC) and inter- and m o t o n e u r o n s in the spinal cord. Having d e l i n e a t e d the reflex circuit involved in the potentiated startle effect, the next task is to determine (a) the point within the startle circuit where the light m o d u l a t e s transmission following conditioning and (b) to delineate the 'visual circuit' that allows the cond i t i o n e d stimulus to alter startle. To d e t e r m i n e where CS presentation modulates transmission, we have been eliciting startle-like responses electrically from various points along the startle p a t h w a y before and after CS presentation in trained rats o r in controls in which light and shocks

* To whom correspondence should be addressed at: 34 Park Street, New Haven, CT 06508, U.S.A. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.

5~ were randomly paired. 'Startle' elicited by electrical stimulation at or before the point in the circuit where the CS modulates transmission should show potentiation, whereas 'startle' elicited beyond this point should not. The results suggest that the CS modulates startle in the ventral nucleus of the lateral lemniscus (Berg and Davis, in preparation). Therefore, the task of this paper is to delineate a pathway that might mediate transmission of the CS from the eye to the ventral nucleus of the lateral lemniscus. Deposition of horseradish peroxidase (HRP) in medial regions of the nuclei of the lateral lemniscus and paralemniscal zone reveals a heavy projection from deep layers of the superior colliculus to these nucleilS,30; (Tischler and Davis, in preparation). In turn. the superior colliculus receives direct projections from the retina as well as indirect projections from the retina via the dorsal lateral geniculate and visual cortex or via connections involving pretectal areas. To determine which of these structures might be involved in mediating potentiated startle, each of these areas was systematically lesioned following training to evaluate how this would affect potentiated startle performance. The data suggest that the conditioned stimulus pathway involves the dorsal lateral geniculate nucleus (dLGN), visual cortex, deep layers of the superior colliculus (SC), and that the modulation produced by this circuit is expressed in the ventral nuclei of the lateral lemniscus. MATERIALSAND METHODS

Animals A total of 80 male albino Sprague-Dawley rats weighing between 300 and 400 g were used. The rats were housed in group cages of 5 rats each and maintained on a 12 h:12 h light/dark schedule. Food and water were continuously available.

Potentiated startle training apparatus Five identical boxes (30 x 25 x 25) were used during training. The sides and tops of the boxes were made of aluminum and the fronts and backs of clear Plexiglas. The floors were composed of 4.76 mm stainless steel bars spaced 19 mm apart. The boxes were located on a shelf within a 1 x 1 z 2 m ventilated, sound-attenuated chamber. The conditioned stimulus was produced by a pair of 15 W incandescent

bulbs located 1 m from the cages. Shocks (US) were delivered from 5 LeHigh Valley Shock Generators (SGS-004) located outside the chamber. Shock intensities were measured with an oscilloscope across a I K resistor in series with a 100 K resistor connected between adjacent grid bars in the shock boxes. Current was defined as the rms voltage across the 1 K resistor where mA = 0.707 x 0.5 x peak-peak voltage. A 0.6 mA shock measured this way corresponds to a setting of 3.6 mA on the meter of the SGS-004 shock generator.

Startle testing apparatus The apparatus used to measure startle has been described previously7. Briefly, 5 separate stabilimeters were used to record the amplitude of the startle response. Each stabilimeter consisted of an 8 x 15 x 15-cm Plexiglas and wire mesh cage suspended between compression springs within a steel frame. Cage movement resulted in displacement of an accelerometer where the resultant voltage was proportional to the velocity of displacement. Startle amplitude was defined as the maximum accelerometer voltage that occurred during the first 200 ms after the startle stimulus was delivered and was measured by a PDP-11 computer. The stabilimeters were housed in a dark, ventilated, sound attenuated chamber, each 10 cm from a high frequency speaker (Radio Shack Supertweeter). The startle stimulus was a 50 ms burst of white noise having a rise-decay time of 5 msec that varied in intensity (95, 105, 115 dB). Background white noise, provided by a white noise generator, was 55 dB. Sound level measurements were made within the cages with a General Radio Model 1551-C sound level meter (A-scale).

Pre-operative testing Prior to training, groups of 15 rats were placed in the startle test cages and 5 rain later presented with 30 noise bursts at 20-s intervals. There were 10 noise bursts at each of three different intensities (95, 105 and 115 dB) presented in an irregular, balanced sequence across the session. These data served as a pre-operative baseline to determine how the various lesions or treatments would alter subsequent startle levels.

57

Trainingprocedure

Testingprocedures

Rats were placed in the shock cages and 5 min later presented with 10 light-shock pairings (trials) in which a 0.6 mA-shock was presented during the last 500 ms of the 1000 ms CS. Trials were presented at an average inter-trial interval of 5 min (range 4-6 min). The 10 conditioning trials were presented on 2 successive training days, creating a total of 20 conditioning trials.

About 24 h after the lesions, rats were placed into the startle test cages and were then presented with 60 noise bursts at 30-s intervals. The intensities of the noise bursts were either 95, 105 or 115 dB. Half of each of these were presented in darkness (NoiseAlone) and half 0.5 s after the onset of the CS (LightNoise). The 10 occurrences of each of the 6 different trial types were presented in a balanced, irregular order across the 30-min test session. Potentiated startle was defined as the within-subject change in startle on the Light-Noise vs Noise-Alone trials, using the median startle amplitude across the 10 occurrences of each trial type as scores.

Lesion Procedure About 72 h after the final training session, animals were anesthetized with chloral hydrate (400 mg/kg). Number 00 insect pins (0.25 mm in diameter) insulated to within 0.5 mm of the tip were used to make DC anodal lesions (0.1 mA, 60 s in duration). Lesions were placed using the following coordinates: dorsal lateral geniculate nucleus (dLGN): 4.0 anterior to lambda (A), 4.0 lateral to midline (L), 4.5 below the top of the skull (D); ventral lateral geniculate nucleus (vLGN) 4.0 A, 4.0 L, 5.5 D; deep layers of superior colliculus (deep SC): 1.4 and 2.4 A, 1 and 2 L, 5.1 D; superficial layers of SC: 1.4 and 2.4 A, 1 and 2 L, 4.1 D; pretectal nuclei: 3.0 A, 3 L, 4.8 D; dorsal nucleus of the lateral lemniscus (DLL): 0.7 A, 2.3 L, 7.5 D; ventral nucleus of the lateral lemniscus (VLL): 0.6 A, 2.2 L, 8.7 D; nucleus reticularis pontis caudalis (RPC): 1.5 posterior to lambda (P), 1.5 L, 9.0 D; thalamic reticular nucleus (RNT): 5.6 A, 2.8 L, 5 and 6 D and 6.4 A, 2.2 L, 5 and 6 D. The visual cortex was removed by aspirating the tissue through a hand-held Pasteur pipette attached to a suction pump. This was done under visual control in chloral hydrate anesthetized rats after removing the occipital bone at the back of the skull. Enucleation was carried out in chloral hydrate anesthetized rats by clamping the eyeball with a forceps, retracting the eyeball and severing the optic tract with a no. 11 scalpel blade. Sham operations were performed on all control rats using identical procedures as for normal lesioned animals. However, in the sham rats no current was passed through the implanted electrode. Within each group of 15 rats, 2-4 rats were saved as sham operated animals to be sure that the lack of potentiated startle in a given lesion group could not be attributed to the particular batch of rats used. Across the entire study, therefore, the total number of sham rats (n = 24) was larger than any one group of lesioned animals.

Histology After testing, animals were sacrificed by chloral hydrate overdose and peffused intracardially with 0.9% saline followed by 10% formalin. The brains were stored for at least 1 day in 10% formalin and subsequently, 50 # m frozen coronal sections were cut through the areas containing the lesions. Brain sections were mounted and stained with cresyl violet. Lesion locations were transcribed onto modified atlas sections 18,24 using Prado universal microscopic slide projector.

Data analysis Degree of attenuation was evaluated using 3-way analyses of variance with lesion type a between-subject variable and noise intensity and trial type (noise alone (NA) vs light-noise (LN)) as within-subject variables. All sham operated groups were compared against each other. No interaction was found between trial type and 'lesion type' for this comparison. Therefore, all shams were pooled into a single group against which all actual lesion groups were compared. A significant trial type x lesion interaction indicates that the lesion attenuated potentiated startle. RESULTS Enucleation abolished potentiated startle (see Table I), indicating that potentiated startle is indeed a visual phenomenon. Lesions directed at the dLGN, a primary recipient of direct retinal ganglion cell projections, also eliminated potentiated startle (Table I, Figs. 1 and 2). Lesions of at least 50% of each dLGN

58 TABLE I Effects o f various lesions on potentiated startle Lesion

Tone intensity 105 dB

95 dB NA

LN

NA

Sham n = 24 DLL n=5 RPC n=4 Superfic. SC n=7 Pretectal n. n=4 Ant. Cortex n=2 Unilat. LGNd n=6 RNT n=4

Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post

35 39 50 1 24 1 26 27 45 50 18 24 36 26 25 31

--~ 92 -9 -4 -76 -105 -60 -55 -65

70 70 77 3 44 3 50 53 74 54 39 24 75 42 41 64

Enucleate n=3 Bilat. LGNd n=4 Vis. Cortex n=3 Deep SC n=5 Deep SC + LGNd n=4 VLL-Posterior n= 5

Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post

35 35 22 16 64 4 22 68 54 19 47 2

-27 -23 -5 -77 -23 -1

77 47 52 33 92 12 38 93 74 47 63 2

resulted

in significant a t t e n u a t i o n

of p o t e n t i a t e d

Mean % potentiation

115 dB LN

NA

--

77

128 -22 -10 --

58

101 --

103

--

59

85

--

95

--

87

32 62 80

92

--

106

n.s.

94%

n.s,

95%

n.s.

167%

n.s.

134%

n.s.

1%

84

--

76

68

--

71

--

--1%

45 90 13 48 117

43 -14 -148

--

88

--

47

65

67

--

78

--

3

467%

80%

64

3

n.s.

143

--

29 -17 -113

492%

123

50

141

--

126 --

72

--

101%

156 -52 -14 --

76

121

LN --

78 86 10 71 2

Trial × lesion interaction

3

n.s. P

<

0.001

P

<

0.001

24%

P < 0.005

22%

P < 0.005

5%

P < 0.001

0%

P < 0.001

sions of the visual c o r t e x d e p r e s s e d b a s e l i n e startle

startle (Fig. 3, T a b l e II). I n contrast, lesions that

a m p l i t u d e s on the n o i s e - a l o n e trials, p e r h a p s be-

w e r e f o u n d u p o n histological e x a m i n a t i o n to h a v e

cause o f the short lesion-test i n t e r v a l e m p l o y e d . In

c a u s e d o n l y u n i l a t e r a l d a m a g e to the d L G N did not

fact, of all t h e lesions p e r f o r m e d in this w o r k , o n l y

b l o c k p o t e n t i a t e d startle ( T a b l e I). Similarly, lesions

the cortically a b l a t e d rats a p p e a r e d d e b i l i t a t e d d u r -

f o u n d to be d o r s a l to t h e d L G N , in the o v e r l y i n g hip-

ing testing, p r o b a b l y because of the c o n s i d e r a b l e

p o c a m p u s o r c o r t e x , also failed to a t t e n u a t e p o t e n -

b l e e d i n g during the lesion p r o c e d u r e and s u b s e q u e n t

t i a t e d startle, despite c o m p a r a b l e tissue d a m a g e (Fig. 1). L e s i o n s of t h e d L G N t e n d e d to depress

edema

b a s e l i n e levels of startle, a l t h o u g h this did n o t o c c u r

during testing.

However,

comparable

de-

creases in baseline startle p r o d u c e d by a b l a t i o n of ant e r i o r cortical regions (e.g. Fig. 4) w e r e not asso-

in e v e r y rat. T h e visual c o r t e x r e c e i v e s a f f e r e n t input f r o m the d L G N and lesions o f the visual c o r t e x m a r k e d l y at-

ciated with a d e c r e a s e in p o t e n t i a t e d startle.

t e n u a t e d p o t e n t i a t e d startle ( T a b l e I, Fig. 4). In con-

6). In cases w h e r e the lesions w e r e s y m m e t r i c a l l y bi-

trast, c o m p a r a b l e tissue d a m a g e to m o r e a n t e r i o r

lateral and i n c l u d e d roughly the p o s t e r i o r two thirds

cortical regions did n o t significantly affect p o t e n -

of t h e d e e p layers of the s u p e r i o r colliculus, p o t e n -

tiated startle ( T a b l e I, Fig. 4). In several cases, le-

tiated startle was d e c r e a s e d (Fig. 7, T a b l e II). T h e s e

Lesions o f the d e e p layers of the s u p e r i o r colliculus also a t t e n u a t e d p o t e n t i a t e d startle ( T a b l e I, Figs. 5,

59

TABLE II

Median startle amplitudes at each of 3 noise intensities (95, 105 and 115 dB) prior to lesioning (pre-test) and after lesioning on the noisealone (NA) or light-noise (LN) trials Group

Pre-test (dB)

Test-NA (dB) 95

Test-LN (dB)

95

105

115

105

115

11 33 28 16

33 60 91 25

49 77 139 2

0

9

22

61 0 2

107 2 12

115 22 21

30 7 34 27 10

71 18 48 31 22

55 13 106 39 25

156 0 121 45 16

178 9 171 71 35

197 20 205 105 59

5 69 8 5 49 35 11

14 154 28 13 65 49 27

33 160 27 40 64 67 15

0 43 30 0 28 0 91

7 93 56 1 63 0 148

43 62 42 41 27

53 95 52 51 39

71 123 68 51 35

1 6 1 1 17

30 43 94 21 63

92 88 100 20 83

104 89 91 37 111

27 10 8 52

36 25 26 87

68 34 40 38

96 43 104 54

95

%Potentiation 105

115

0

4

81 0 12

92 1 11

12 118 19 21

--48 3 --17 26

170 0 157 48 9

193 19 202 83 68

197 30 297 115 103

5 68 32 11 63

22 141 102 3 68 8 187

21 93 88 2 67 43 224

56 133 117 8 139 26 225

84 167 180 22 142 65 225

455 42 104 700 119 1575 58

1 4 1 1 21

1 7 1 1 28

1 2 1 1 41

1 8 1 1 55

1 5 1

0 --11 0

1

0

108

209

1 0 0 0 1

3 2 0 1 11

2 1 26 4 19

1 19 2 11 14

4 27 28 9 40

6 93 74 45 41

83 4533 300 1200 206

113 36 24 110

1 0 1 0

2 0 1 3

3 2 6 0

6 3 1 7

8 8 8 15

10 10 9 26

300 950 125 1500

109 80 158 63

95 81 22 0

107 54 56 12

132 69 64 24

153 145 97 26

212 124 117 31

213 132 90 58

73 97 114 219

dLGN 1

2 3 4 SC-DEEP 5 6 7 8 9 SC-Superficial 10 11 12 13 14 15 16 VLL 17 18 19 20 21 DLL 22 23 24 25 26 RPC 27 28 29 30 PRE-TECTAL 31 32 33 34

lesions also resulted in increased baseline levels of

deep layers of the SC and bilateral lesions of the

startle relative to the pre-lesion baselines, t (4) = 2.31, P < 0.05. In contrast, lesions of the superfi-

d L G N eliminated potentiated startle, but had no

cial layers of the superior colliculus did not significantly attenuate potentiated startle (Table I, T a b l e

ble I). The V L L receives heavy projections from the d e e p

II, Figs. 5, 8, 9), nor did they alter baseline levels o f

layers of the SC and represents one o f the primary

startle. In other animals, c o m b i n e d lesions of both the

nuclei in the acoustic startle pathway. As expected,

consistent effect on baseline startle amplitudes (Ta-

lesions of the V L L essentially eliminated acoustic

60 D C.R S~., N~CLE US

LATERAL

GEN~CULAI E CON ~R'- [

j

. •

~

J

o J,'.

~

S

~ -

J

startle, t (4) = 4.19, P < 0.01 (Table I, Table II, Figs. 10, 11), provided the lesions were placed bilaterally in posterior regions of the VLL (Fig. 12). In such animals the light failed to increase startle on the light-noise trials (Table I, Table II, Figs. 11, 12). In fact, the lesions have to be quite posterior, since the one rat in the V L L group that still showed potentiated startle did have sparing of posterior VLL regions (rat 21). However, the fact that lesions in this area markedly attenuated acoustic startle makes the interpretation of a blockade of potentiated startle problematic. It is particularly noteworthy, therefore, that lesions of the D L L also markedly depressed baseline startle levels on the noise-alone trials, t (4) = 6.14, P < 0.01 (Table I, Table II, Figs. 10, 13, 14). Despite this, every D L L rat startled consistently, and frequently at very high levels, on the lightnoise trials, indicating that this lesion did not prevent potentiated startle. Similarly, lesions of the RPC, another part of the acoustic startle pathway, also depressed acoustic startle, t (3) = 2.92, P < 0.05, yet

~

/,

[ [ [ ] ~IGH T-

NOISE

BO" N O I S E ALONE

,5o u~

FI

120~

95

IO5

115

95

NOISF BURST [NTENSIT Y (dB)

IO5

j

o

__

Fig. 1. Top: comparison of histological comparison of reconstructions taken from brain sections of rats with lesions (black) directed at dLGN vs lesions found to be more dorsal in the overlying hippocampus and cortex• Bottom: corresponding plot of median startle amplitude vs noise intensity for noisealone trials (black) and light-noise trials (white)•

11

~15

Fig. 2. Photomicrograph showing bilateral lesions (arrows) of the dorsal lateral geniculate nucleus (dLGN).

61

DORSAL LATERAL GENICULATE LESIONS

I

2

3

4

Fig. 3. Histological reconstructions of lesions (black) of the dLGN in rats 1-4. The data for each rat are in Table II. CORTEX VISUAL

LU150"

8 F-

~

failed to abolish potentiated startle (Table I, Table II, Figs. 15, 16). Finally, lesions directed at the pretectal nuclei (Table I, Table II, Figs. 17, 18) and thalamic reticular nuclei (Table I) did not measureably alter either baseline startle or potentiated startle. However, because of the extensive and complicated structure of the thalamic reticular nucleus, it is not certain if this nucleus was destroyed completely and further work on this point is clearly necessary. Nevertheless, considerable damage to this area still spared potentiation. Similarly, it was not possible to isolate the effects of lesions directed at the v L G N on potentiated startle due to the close proximity of the v L G N to the optic tract.

ANTERIOR

[]

LIGHT-NOISE

•

NOISE ALONE

1

120-

90-

60-

30-

95

105

115

95

NOISE BURST INTENSITY (dB)

105

I

JI5

Fig. 4. Top: comparison of histological reconstructions taken from brains of rats with lesions (black) directed at visual vs frontal-parietal cortex. Bottom: corresponding plot of median startle amplitude vs noise intensity for noise-alone trials (black) and light-noise trials (white).

62 DISCUSSION According to the present results, the potentiated startle effect observed when an acoustic stimulus oc-

SUPERIOR COLLICULUS DEEP LAYERS

'iit

SUPERFICIAL LAYERS

[ ] LIGHT-NOISF

,

11

!i 95

I05

115

95

I05

115

NOISE BURST INTENSITY (dB)

Fig. 5. Top: comparison of histological reconstructions taken from brain sections of rats with lesions (black) directed at the deep or superficial layers of the superior colliculus. Bottom: corresponding plot of median startle amplitude vs noise intensity for noise-alone trials (black) and light-noise trials (white).

curs in the presence of light previously paired with a shock is mediated by a visual circuit involving the retina, d L G N , visual cortex and deep layers of SC which projects upon the nuclei of the lateral lemniscus. Systematic exploration of visual pathways begins, out of necessity, with the eye. Although it is generally acknowledged that potentiated startle reflects a visual phenomenon that modulates an acoustic response, it was necessary to rule out other possible contributing circuits. The light bulbs used in the present investigation emit a very faint onset click that is barely detectable under silent background conditions. To demonstrate that this auditory component was not a significant factor contributing to potentiation, rats were enucleated and tested 24 h later. As expected, enucleation blocked potentiation. Substantial work on the projection patterns of retinal ganglion cells suggest that the primary recipients of retinal projections are the superficial layers of the S C 6,16.17,25.32,33 and d L G N 5.11,1a,27. Lesions directed at superficial layers of SC did not attenuate potentiated startle. Bilateral lesions of the d L G N , however, completely blocked potentiation. Histology performed on sections taken from these animals revealed that only discrete dorsal lesions were required and that the optic tract and v L G N were spared. Unilateral lesions of dLGN did not block potentiation. The dLGN projects primarily to the areas 17, 18 and 19 of the visual cortex 2,~j,13,24.33-34,35. The possible importance of visual cortex to the potentiated startle effect is further stressed by the observation that cooling the visual cortex abolishes all visual responsiveness in the deep layers of the SC (see ref. 10). Accordingly, aspiration lesions were made throughout the visual cortex. These successfully blocked the potentiated startle effect in all rats. The visual cortex projects to several sites within the visual pathway including vLGN, d L G N , thalamic reticular nucleus, pretectal nuclei and superior colliculus1,12,15,20,28.29,30. The SC was singled out due to its close proximity to the primary acoustic startle circuit, the fact that cooling visual cortex abolishes visual responsiveness in deep SC, and the observation that the deep layers innervate many brainstem structures 10. In particular, Henkel l~ in cat, and Tischler and Davis (in preparation) in rat report a significant projection from the deep layers of SC to the ipsilateral para-lemniscal region. Accordingly, lesions

Fig. 6. Photomicrograph

showing

bilateral

SUPERIOR

Fig. 7. Histological reconstruc for each rat are in Table II.

lesions (arrows)

COLLICULUS

6 :tions of lesions (black)

of intermediate

and deep layers of the superior

LESIONS

-DEEP

colliculus.

LAYERS

of the deep and intermec Sate layers of the superio sr colliculus

in rats 5-9.

64

Fig. 8. Photomicrographic showing bilateral lesions (arrows) of superficial layers of the superior colliculus.

SUPERIOR

~7-

! ,~_ ~

', :-

~2

COLLICULUS

LESIONS-SUPERFICIAL LAYERS

)

lo II 12 13 14 15 t6 Fig. 9. Histological reconstructions of lesions (black) of the superficial layers of the superior eolliculus in rats 10-16. The data for each rat are in Table II.

65

LATERAL LE MNISCUS

DORSAL

VENTRAL

@@ ,:, ":~:

I00-

n ~E

[]

LIGHT- NOISE

•

NOISE ALONE

80.

project directly to the startle circuit, did not block potentiated startle. Additional evidence suggesting a contribution of the deep layers of the SC to the potentiated startle effect can be found in the fact that lesions directed medial and anterior to the dorsal nucleus of the lateral lemniscus appear to attenuate potentiation but do not measurably reduce baseline startle. Analysis of the histology reveals that these lesions extend into the cuneiform nucleus. Edwards 10 observed that the descending crossed projections of the deep layers of SC and the cuneiform nucleus are actually fused into a single bundle with fibers of the cuneiform nucleus occupying a more ventral position. Lesions just posterior to the cuneiform nucleus presumably sever this bundle, effectively blocking the visual circuit. Lesions of the deep layers of the SC usually resulted in substantial increases in baseline levels of startle on noise-alone trials. It is possible that this resuited ~,om damage to the SC itself, since local infusion of picrotoxin into deep layers of the SC have been reported to produce hyperreactivity 21, Hence physical removal of the SC might be similar to chemical removal of a tonic inhibitory G A B A system by picrotoxin. On the other hand, increased startle after lesions of the deep layers of the SC might have been caused by damage to the underlying central grey matter.

~60Fm ~40,

20-

105

115

95

105

NOISE BURST iNTENSITY (dB)

Fig. 10. Top: comparison of histological reconstructions taken from brain sections of rats with lesions (black) directed at VLL vs DLL. Bottom: corresponding plot of median startle amplitude vs noise intensity for noise-alone trials (black) and lightnoise trials (white).

were placed throughout the deep layers. These were found to block potentiated startle in all rats where histology revealed complete lesioning of at least the posterior two-thirds of the structure. In contrast, lesions of the superficial layers of the SC, which do not

Fig. 11. Photomicrograph showing bilateral lesions (arrows) of the VLL and paralemniscal regions.

~6

VENTRAL LATERAL LEMNISCUS LESIONS /

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'~

21

Fig. 12. Histological reconstruction of lesions (black) of the VLL in rats 17-21. The data for each rat are in Table II. One might argue, therefore, that the apparent blockage of potentiated startle by lesions of the deep layers of the SC resulted because these lesions increased baseline startle levels, leading to a 'ceiling effect'. However, this was not the case, since these rats clearly were not at any ceiling when tested with the weaker noise intensities, yet they still failed to show potentiated startle. In addition, combined lesions of the deep layers of the SC and d L G N did not consistently alter baseline startle, yet they markedly attenuated potentiated startle. Lesions of the pretectal and thalamic reticular nuclei did not measurably attenuate potentiated startle. It appears, therefore, that these structures do not contribute to the visual circuit involved in potentiated startle. On the other hand, exploratory studies indicated that lesions of the vLGN generally did depress potentiation. However, interpretation of these data is complicated by the fact that lesions of the v L G N also damaged the overlying d L G N as well as the optic tract. Hence, it is not possible at this time to determine the role of the vLGN in potentiated

startle. This leaves a pathway consisting of the retina, d L G N , visual cortex and deep layers of SC. H R P histochemistry 17.3° (Tischler and Davis, in preparation)

Fig. 13. Photomicrograph of bilateral lesions (arrows) of the DLL.

67

DORSAL LATERAL LEMNISCUS LESIONS

22

23

24

25

Fig. 14. Histological reconstructions of lesions (black) of the DLL in rats 22-26. The data for each rat are in Table II.

Fig. 15. Photomicrograph of bilateral lesions (arrows) of the RPC.

26

68

PONTINE RETICULAR FORMATION LESIONS

{;

27

"

28

/

29

:30

Fig. 16. Histological reconstructions of lesions (black) of the RPC in rats 27-30. The data for each rat are in Table II.

Fig. 17. Photomicrograph of bilateral lesions (arrows) of the pre-tectal nuclei.

69

PRETECTAL LESIONS

34 32 33 31 Fig. 18. Histological reconstructions of lesions (black) of the.pre-tectal nuclei in rats 31-34. The data for each rat are in Table II.

suggests that the visual circuit is inserting into the primary acoustic startle circuit at the paralemniscal region just medial to the ventral nucleus of the lateral lemniscus. This is corroborated by electrical stimulation studies. In trained animals, electrical elicitation of startle at or before the V L L (e.g. VCN) results in potentiation whereas elicitation of startle beyond the V L L (e.g. RPC, spinal cord) does not (Berg and Davis, in preparation). Finally, lesions directed at V L L block both acoustic startle and its potentiation. Conversely, lesions of RPC and D L L attenuate acoustic startle but do not block potentiated startle. These combined approaches all suggest that the visual circuit is modulating transmission at the paralemniscal region or V L L to mediate the potentiated startle effect. Previous data indicated that lesions of the D L L markedly attenuated startle when testing took place 24 h after surgery with a gradual recovery of startle over the following week 8. Based on this evidence, it was suggested that the D L L formed an integral part of a primary startle circuit but was not essential for startle when a sufficient period of recovery was al-

lowed. In the present study, lesions of the D L L were also found to eliminate startle when testing occurred 24 h after the lesion. However, the most striking finding was that in these animals vigorous startle reflexes could still be elicited in the presence of the light. In several instances we turned up the gains on the accelerometer amplifiers to very high levels to be sure these rats were not moving at the onset of the light. In all cases light onset did not elicit any measurable movement, yet when the startle eliciting noise burst came on, large startle reflexes with appropriately short latencies did occur. Taken together, these data indicate that the D L L is not necessary for startle and suggest that it plays a modulatory rather than a mediating role in acoustic startle. In the present study, we have looked at conditioning as a change in a reflex behavior (startle elicited by a tone) in the presence of a CS. We know that light presentation does not increase startle in rats that have not had light paired with shock 7. Potentiated startle represents, therefore, a change in a visual circuit that begins in the eye and appears to end in the VLL. Having delineated this basic visual circuit, it

70 should n o w be possible to e v a l u a t e exactly w h e r e

ifies transmission within this visual circuit.

within this circuit the CS is altering n e u r o n a l transmission. F o r e x a m p l e , if such an effect w e r e occur-

ACKNOWLEDGEMENTS

ring in the SC, it is c o n c e i v a b l e that electrical stimulation in the SC of t r a i n e d animals p r e s e n t e d with

This r e s e a r c h was s u p p o r t e d by N S F G r a n t B N S -

acoustic stimuli m i g h t elicit p o t e n t i a t i o n . In contrast,

78-01470, N I M H G r a n t M H - 2 5 6 4 2 , N I N C D S G r a n t

a similar effect m i g h t n o t be s e e n in u n t r a i n e d sub-

NS18033, R e s e a r c h Scientist D e v e l o p m e n t A w a r d

jects.

M H - 0 0 0 0 4 to M . D . and the State of C o n n e c t i c u t . W e

By p r o c e e d i n g

systematically f r o m

dLGN,

t h r o u g h o u t the visual c o r t e x and SC, it m i g h t be pos-

t h a n k L e e Schlesinger for technical assistance, Leslie

sible to p i n p o i n t the actual n e u r a l locus w h e r e 'learn-

Fields for t y p i n g the m a n u s c r i p t and J o h n K e h n e for

ing' is taking place, or at least w h e r e learning m o d -

his m a n y h e l p f u l c o m m e n t s on this manuscript.

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