Exp Brain Res (1993) 93:231 241

Experimental Brain Research �9 Springer-Verlag 1993

Enhancement of the acoustic startle response by stimulation of an excitatory pathway from the central amygdala[basal nucleus of Meynert to the pontine reticular formation Michael Koch, Ulrich Ebert

Tierphysiologie, Universitfit Tfibingen, Auf der Morgenstelle 28, W-7400 Tfibingen, Germany

Received: 24 July 1992 / Accepted: 26 October 1992

Abstract. The acoustic startle response (ASR) is a simple motor reaction to intense and sudden acoustic stimuli. The neural pathway underlying the ASR in rats is al- ready fairly well understood. As the ASR is subject to a variety of modulations, this reaction can serve as a model for vertebrate neuroethologists to investigate the neural mechanisms mediating sensorimotor transfer and their extrinsic modulation. We report here on experiments in rats which were undertaken in order to investigate the neural mechanisms underlying the enhancement of the ASR. An increased amplitude of the ASR can be ob- served during states of conditioned and unconditioned fear. By employing neuroanatomical tract-tracing meth- ods, we describe a pathway from neurons of the medial division of the central amygdaloid nucleus (cA) and the basal nucleus of Meynert (B) to the caudal pontine retic- ular nucleus (PnC), an important relay station in the acoustic startle pathway. Extracellular recordings from acoustically responsive neurons in the PnC showed that electrical stimulation of the cA/B facilitates the tone- evoked response of these neurons. Behavioural tests fol- lowing chemical stimulation of the cA/B with NMDA (N-methyl-d-aspartate) in awake rats indicated that acti- vation of this pathway increases the ASR. The lack of sufficient spatial resolution of our stimulation techniques did not allow us to differentiate the relative contributions of the cA and the B to this effect. As the amygdaloid complex has been implicated in emotional behaviour, particularly in the mediation of fear, these findings sub- stantiate the concept that the amygdaloid complex plays a key role for the enhancement of the ASR by condi- tioned and unconditioned fear.

Key words: Acoustic startle response- Amygdala Basal nucleus of Meynert - Pontine reticular formation Rat

Correspondence to: M. Koch

Introduction

The acoustic startle response (ASR) is elicited by sudden and intense stimuli, and is mediated by a relatively simple neural circuit including the cochlear nuclei, neurons in the pontine reticular formation and spinal motoneurons (Davis et al. 1982a; Lingenh6hl 1992). As the ASR is subject to a variety of modulations, it is considered a promising model for vertebrate neuroethologists to in- vestigate the mechanisms of sensorimotor transfer and their extrinsic modulation (Davis and File 1984). Exam- ples of modulations of the ASR are two types of response inhibition which occur after presentation of a non- startling stimulus some 50 ms before the startle stimulus (pre-pulse inhibition) and after repeated presentation of a startle stimulus (habituation), respectively. Another ex- ample for the plasticity of the ASR is an increase in the response amplitude. The enhancement of the ASR can be observed during conditioned and unconditioned states of fear, both in rats (Davis et al. 1991) and humans (Grillon et al. 1991). In order to understand the different mecha- nisms of modulation of the ASR, it is necessary to char- acterise the input to neurons of the primary startle path- way in relation to its possible effects on sensorimotor transfer. Here we address the question of how the fear-en- hanced ASR can be explained.

The caudal pontine reticular nucleus (PnC) has long been implicated in the mediation of the ASR in rats (Sz- ab6 and Hazafi 1965; Davis et al. 1982a), and more re- cently, the subpopulation of acoustically responsive, gi- ant reticulospinal neurons of the PnC was identified as a sensorimotor interface of the ASR (Wu et al. 1988; Ebert and Koch 1992; Koch et al. 1992; Lingenh6hl and Friauf 1992). Therefore, these neurons would be ideal targets for relaying extrinsic modulatory input impinging upon the startle pathway. In fact, a pathway from the central amygdaloid nucleus to the pontine reticular brainstem, which probably mediates the effects of conditioned fear on the ASR, has recently been described by Rosen et al. (1991) and Hitchcock and Davis (1991).

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The objective of the present s tudy was to describe in more detail the ana tomica l connec t ion between the amygdalo id complex and the PnC and to investigate the physiological effects of amygdalo id s t imula t ion on acous- tically responsive PnC neurons and on the A S R in rats. We therefore employed retrograde and anterograde t ract- t racing techniques in order to demons t ra te projec- t ions from the amygdalo id complex to the pon t ine bra in- stem, with special a t t en t ion to the giant ret iculospinal neurons of the PnC. In a second set of experiments, we used extracellular electrophysiology in order to charac- terise the effects of electrical s t imula t ion of this project ion on acoustic responses of PnC neurons. Finally, we mea- sured the A S R in awake rats after chemical s t imula t ion of the amygdalo id complex in order to proof the be- havioura l relevance of the pa thway from the amygdalo id complex to the pon t ine reticular formation.

Material and methods

Retrograde and anterograde tract tracing

The animals were anaesthetised with 420 mg/kg chloral hydrate (injected intraperitoneally) supplemented by the local anaesthetic lidocaine, which was applied prior to craniotomy.

The retrograde tracer Fluoro-Gold (FG; 2% solution in 0.1 M cacodylate buffer pH 7.5; Fluorochrome Inc) was injected ion- tophoretically (+5 pA, 20 min, 5 s on/off) through glass mi- cropipettes (30 gm tip diameter) into the pontine reticular forma- tion of 15 male Wistar rats. After a survival period of 7 days, the animals were re-anaesthetised and perfused through the aorta with 0.01 M phosphate-buffered saline (PBS), followed by cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The brains were removed and placed in 30% sucrose in PBS until they sank. Coronal sections (40 gin) were then taken on a freezing microtome, mounted, coverslipped with DePeX, and examined under a fluores- cence microscope. Adjacent sections were stained with thionin.

The anterograde tracer Phaseolus vuIgaris-leucoagglutinin (PHA-L, 2.5% solution in 0.01 M phosphate buffer pH 8.0) was injected iontophoretically ( + 6 pA, 20 min, 5 s on/off) through glass micropipettes (20-30 gm tip diameter) into the central amygdaloid nucleus or the caudal part of the basal nucleus of Meynert in ten male Wistar rats. After survival periods of 7-10 days, the animals were re-anaesthetised and perfused through the aorta with 0.01 M PBS followed by 200 ml cold 4% PFA in 0.1 M acetate buffer (pH 6.5) and 300 ml cold 4% PFA in 0.1 M borate buffer (pH 11.0). The brains were removed, postfixed in the last fixative for 3 h and placed in 30% sucrose in PBS overnight. Coronal sections, 40 gm thick, were taken on a freezing microtome and rinsed in Tris-buffered saline (TBS). The sections were incubated for 1 h in a blocking solution containing 10% normal goat serum, 2% bovine serum albumin (BSA) and 0.3% Triton X-100 in 50 mM TBS at pH 7.6. They were then transferred into the solution of primary antibody (rabbit anti-PHA-L from DAKO diluted 1:3000 in a carrier con- taining 1% normal goat serum, 1% BSA, and 0.3% Triton X-100 in TBS) and incubated for 48 h at 4 ~ After several washes in TBS, the sections were transferred into a solution of unlabelled swine anti-rabbit IgG antiserum (DAKO) diluted 1:50 in carrier, and incubated for 1.5 h at room temperature. Thereafter, the sections were thoroughly rinsed in TBS and incubated in rabbit peroxidase anti-peroxidase (PAP) complex (DAKO) diluted 1:150 in carrier. After 1.5 h, the sections were rinsed in TBS. The distribution of PHA-L in the brain was visualised by incubating the sections for 5-15 rain in a solution of 0.05% 3,3-diaminobenzidine hydrochlo- ride and 0.01% hydrogen peroxide in TBS. The incubation was stopped by transferring the sections into TBS. The sections were mounted on gelatine-coated slides, air-dried, dehydrated in graded alcohols, cleared in xylene and coverslipped with entellan. Adjacent sections were stained with thionin. The location of the injection sites

and the distribution of PHA-L labelled fibres in the pontine reticu- lar brainstem were examined using a microscope with brightfield illumination and Nomarski optics.

Electrical brain stimulation and extracellular electrophysiology

Five rats were anaesthetised with chloral hydrate (420 mg/kg i.p.) supplemented by topical application of lidocaine. Anaesthesia was maintained by injecting one-third of the initial dose of chloral hy- drate every 2 h. During the experiment, the animal's electrocardio- gram was continuously monitored, its body temperature kept at 37~ and its electrolytic balance maintained by subcutaneous injec- tions of isotonic saline (1 ml/h). The rat was placed in a stereotaxic frame and bipolar stimulation electrodes (cut and twisted insulated stainless steel wires) were placed bilaterally into the medial part of the central amygdaloid nucleus, using the stereotaxic coordinates from the atlas of Paxinos and Watson (1986). The electrodes were fixed to the skull with stainless steel screws and dental acrylic ce- ment. The brainstem was exposed by aspirating parts of the cerebel- lum located underneath the parietal bone. The animal was then released from the stereotaxic frame and its skull was fixed by a nail embedded in dental acrylic cement for open-field acoustic stimula- tion. Acoustic stimulation was performed using a high-frequency speaker located in a sound-attenuated chamber. Pure tone stimuli of 50 ms duration and 2.5 ms rise/fall times were presented at a rate of 1 Hz. The recording electrode (Teflon-insulated tungsten elec- trodes; impedance: 10 M~) was lowered through the pontine retic- ular formation by a hydraulic motor microdrive at a caudorostral angle of 20 ~ and a mediolateral angle of 10 ~ in order to record from single units in the PnC. Peristimulus time histograms (PSTHs) from dot displays of 20 or 50 consecutive stimuli were produced on-line by a PDP-11 computer. Spikes occurring in a time frame of 50 ms after the neurons' minimal response latency were taken as tone- evoked activity. Spike rates of tone-evoked activity with and with- out amygdaloid stimulation were calculated from the PSTHs. Elec- trical stimulation of the amygdala was performed ipsilaterally to the recording site in the PnC using single square pulses of 50 gs dura- tion and a mean voltage of 38 _+ 10 V. Electrical stimuli to the amyg- dala were delivered 1-5 ms before tone onset. A response of a PnC neuron to amygdaloid stimulation was defined as a change in the unit's activity exceeding _+ 10% of the control activity. The differ- ence in activity between tone alone and tone plus amygdaloid stim- ulation was tested by the Wilcoxon signed rank test.

The electrode tracks were marked by electrolytic lesions (15 gA, 15 s) in order to localise recording sites. Upon termination of the experiments, the rats were decapitated, the brains removed and immersion-fixed in formaldehyde. Recording and stimulation sites were localised on thionin-stained sections.

Behavioural experiments

Chemical stimulation of the amygdaloid complex during the ASR was performed in 22 adult male Wistar rats (200-300 g). They were housed in groups of four to six animals per cage before and after surgery. The rats had free access to food and water and were kept under a continuous light-dar-k cycle (on 06.00 h, off 18.00 h).

The animals were anaesthetised with chloral hydrate (420 mg/ kg) supplemented by topical application of lidocaine and placed in a stereotaxic frame. Twenty-three-gauge, stainless steel guide can- nulae were bilaterally implanted into the brain aiming at the medial division of the central amygdaloid nucleus (according the coordi- nates of Paxinos and Watson 1986) and fixed to the skull with dental acrylic cement and two anchoring screws. The wound was sutured and stylets were inserted into each of the guide cannulae to main- tain patency. The rats were allowed to recover from surgery for one week during which time they were handled daily. The measurement of the ASR was accomplished after placing the rat in a wire mesh cage (19.5 x 9 x 8 cm) mounted on a digital balance (Sartorius L2200 S) inside a sound-attenuated chamber (100 x 80 x 60 cm). The deflections of the balance caused by the rats' movements were

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digitised and fed into a computer for further analysis. Acoustic stimuli which elicit the ASR (10 kHz tone bursts, 100 dB SPL, 20 ms duration, 0.4 ms rise and fall times, 30 s interstimulus interval) were delivered through a loudspeaker at a distance of 40 cm from the test cage. In addition, continuous white background noise of 40 dB SPL was presented. The whole-body startle amplitude was calculated from the difference between the peak-to-peak amplitudes of the output of the balance within time-windows of 80 ms after and 80 ms before the onset of the acoustic stimulus. In order to avoid a possi- ble ceiling effect of the startle response amplitude on the day of drug treatment, the animals were exposed to 40 startle stimuli the day before testing began. This procedure reduces both the amplitude and the variance of the ASR during the tests due to a long-term habituation process.

We performed a chemical stimulation of the cA/B in order to make sure that the changes we might observe were due to the excitation of local neurons rather than fibres-of-passage. For testing the effects of NMDA in the amygdala on the ASR, the rats received bilateral microinjection cannulae (30-gauge stainless steel) and were placed in the wire mesh cage. The cannulae were connected to two 1.0 gl Hamilton syringes outside the test chamber by a length of flexible PVC tubing (inner diameter 0.015 in; Reichelt Chemie Tech- nik). Immediately after the rats were placed in their test cage they received ten startle stimuli, which reduced the response amplitude and variance to the subsequent stimuli due to short-term habitua- tion. The mean response amplitude to the next ten stimuli was taken as the pre-injection value. The animals then received bilateral injections of either 0.3 gl of artificial cerebrospinal fluid (ACSF, pH 7.2) or 0.3 gl of a 0.013 M solution of N-methyl-d-aspartate (NM- DA; Sigma; pH 7.2) in ACSF into the amygdaloid complex. This dosage of NMDA was found to be effective in preliminary tests and is based on the recommendations of Schmidt and Bury (1988). The injections were given at a rate of 0.1 gl/s (Routtenberg 1972). The injection cannulae were left in the brain during the test. The mean response amplitude to the next ten stimuli was taken as the post-in- jection value. All rats received both treatments (drug and vehicle) on two separate days in a randomised serial order. The percent difference between pre- and postinjection mean startle amplitudes was calculated for each animal for both treatments. As the ampli- tude of the ASR shows a considerable degree of variance, an in- crease of the startle amplitude of more than 100% compared to vehicle injection was considered as an effect. According to this crite- rion, the animals were divided into two groups, one containing the rats with an increased startle amplitude and the other where NM- DA was without effect. Statistical analysis of the difference in re- sponse amplitudes between injections of ACSF and NMDA was performed using the t-test for both groups. Animals with unilateral injections, due to occasional block of one of the cannulae, were excluded from the analysis.

Abbreviations. ACSF: artificial cerebrospinal fluid; ASR: acoustic startle response; B: basal nucleus of Meynert; BSA: bovine serum albumin; cA: central amygdaloid nucleus; CGPn: central gray pons; coA: cortical amygdaloid nucleus; CPu: caudate putamen; En: endopiriform nucleus; EP: entopeduncular nucleus; FG: fluo- ro-gold; GP: globus pallidus; I: intercalated amygdaloid nucleus; ic: internal capsule; 1A: lateral amygdaloid nucleus; LH: lateral hypothalamic area; mA: medial amygdaloid nucleus; Mo5: motor trigeminal nucleus; AL: nucleus ansa lenticularis; NMDA: N- methyl-D-aspartate; ot: optic tract; PAP: peroxidase anti-peroxi- dase; PB: parabrachial nucleus; PBS: phosphate buffered saline; PCRtA: parvocellular reticular nucleus (pars alpha); PFA: para formaldehyde; PHA-L: Phaseolus vulgaris leucoagglutinin; Pir: pir- iform cortex; PnC: caudal pontine reticular nucleus; Pr5: principal sensory trigeminal nucleus; PSTH: peristimulus time histogram; py: pyramidal tract; RtTg: reticulotegmental pontine nucleus; Rt: reticular thalamic nucleus; scp: superior cerebellar peduncle; s5: sensory root of the trigeminal nerve; SOC: superior olivary com- plex; SubCV: subceruleus nucleus, ventral; TBS: Tris buffered sa- line; Tz: trapezoid body nucleus; VPL: ventral posterolateral thala- mic nucleus; ZI: zona incerta; 6: abducens nucleus; 7n: facial nerve.

After completion of the behavioural tests the animals were anaesthetised with chloral hydrate and 0.5 gl of alcian blue (5% solution, Sigma) was injected into the brain in order to visualise cannulae placements. The animals were decapitated under anaes- thesia and their brains immersion-fixed with formaldehyde. Fifty micrometer sections were taken on a freezing microtome and stained with neutral red. The injection sites were drawn onto plates from the atlas of Paxinos and Watson (1986).

Results

Retrograde and anterograde tracing experiments

After F l u o r o - G o l d (FG) in ject ions in to the cauda l pon- t ine re t icu la r fo rmat ion , we found r e t rog ra de ly labe l led neurons in va r ious par t s of the bra in , inc luding par t s of the m e d u l l a r y and mesencepha l ic re t icu lar fo rmat ion , the coch lear nucleus, the super io r o l ivary complex, the cen- t ra l grey, the p e d u n c u l o p o n t i n e and l a t e r o d o r s a l t egmen- tal nuclei, the deep mesencepha l ic nucleus, the super io r coll iculus, the subs t an t i a nigra, the zona incerta , the lat- eral h y p o t h a l a m u s , as well as in the cent ra l a m y g d a l o i d nucleus (cA), and in the basa l nucleus of M e yne r t (B).

We focus on the p ro jec t ions f rom the cA and the B in the present s tudy. These p ro jec t ions are largely ips i la te ra l and show a me d io l a t e r a l t o p o g r a p h y (Fig. la ,b) : injec- t ions of F G into the cauda l pon t ine re t icu la r nucleus (PnC) labe l led cells in the med ia l pa r t of the cA and in a n a r r o w b a n d of cells media l f rom the cA tha t be longs to the cauda l pa r t of the B (Fig. la). On ly very few re t ro- g rade ly label led cells were found in the an te r io r pa r t of the B. The d i s t inc t ion be tween the media l cA and the B can also be based on m o r p h o l o g i c a l differences be tween the cells: neurons of the cA are m e d i u m to smal l sized and mul t ipo la r , whi ls t the cells of the B are b ipo la r , spin- d l e - shaped neurons . F igure 2 shows r e t rog ra de ly labe l led cells p ro jec t ing f rom the media l d iv is ion of the cA and the cauda l pa r t of the B to the PnC.

In jec t ions of F G into a reg ion u n d e r n e a t h the m o t o r t r igemina l nucleus (Mo5) and dorsa l to the super io r oli- va ry complex (SOC), the vent ra l pa r t of the subcoeru leus nucleus (SubCV) after Pax inos and W a t s o n (1986), re t ro- g rade ly labe l led neurons in the centra l pa r t of the cA and to a m i n o r extent in the ven t r a lmos t pa r t of the B (Fig. lb) which merges wi th the nucleus of the ansa lent icular is (AL). Since we were unab le to d is t inguish the nuc lear b o u n d a r i e s be tween the B and the AL, it is poss ib le tha t some of the r e t rog rade ly labe l led cells be longed to the AL.

The results of the r e t r og ra de t rac ing exper iments , showing a t o p o g r a p h y in the p ro jec t ion f rom the cA/B to the PnC, were conf i rmed and ex tended by the an te ro - g rade t rac ing exper iments .

In jec t ions of P H A - L into the c A p r o p e r labe l led fibres in the la te ra l pa r t of the ips i la te ra l pon t ine bra ins tem, i.e. in the S u b C V and in the p a r a b r a c h i a l nucleus (PB), as well as in the pa rvoce l lu l a r re t icu lar nucleus (PCRtA, Fig. 3 ra t =~ 1). In jec t ions in to the med ia l d iv is ion of the cA and the B label led fibres in the ips i la te ra l PnC p r o p e r in a d d i t i o n to the PB, SubCV, and P C R t A (Fig. 3 ra t 4# 10; Fig. 4a). Very few fibres were found on the con- t r a l a t e ra l side in this case. Labe l l ed axons coursed media l

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Fig. 1. a,b. Retrograde tracing with Fluoro-Gold (FG). The upper part of the figure shows two injection sites of FG into the pontine reticular formation on coronal sections. The black area corresponds to the gliosis observed in the centre of the injection, the stippled area corresponds to the fluorescent halo. The bottom part of the figure

depicts retrogradely labelled cells in the basal forebrain, a FG injec- tion into the PnC resulted in retrogradely labelled cells in the medi- al part of the ipsilateral cA and in the caudal part of the ipsilateral B. b FG injection into the SubCV resulted in retrogradely labelled cells in the central part of the cA and in the B

neuropil (Fig. 4b,c) or close to small and medium-sized neurons of the PnC (Fig. 4d) and the SubCV. Occasional- ly, presumed terminal varicosities were observed near the somata of the giant PnC neurons. Injections of PHA-L into the caudal part of the B labelled fibres in the ipsilat- eral PnC, in the SubCV, and the PCRtA (Fig. 3; rat ~ 9). Beaded fibres were only occasionally seen in close prox- imity to the somata of giant PnC neurons. No pre- sumable axosomatic contacts were observed on the giant PnC neurons, rather it can be assumed, on the basis of our material, that the cA/B afferents contact the proxi- mal and distal parts of the dendrites (Fig. 4e). Hence, it can be concluded that the projections from the B and cA form axodendritic and/or axoaxonic rather than axoso- matic synapses on giant PnC neurons.

Fig. 2. Photomicrograph of retrogradely labelled cells in the medial division of the cA and the B after an injection of FG into the PnC. Notice that neurons in the cA are medium-sized and multipolar, whilst neurons in the B are spindle-shaped and bipolar. Scale bar 100 gm

to and through the Mo5, and formed presumed terminal arborizations and boutons-en-passant in the ventrolater- al part of the PnC and in the SubCV (Fig. 3 rat :H: 10; Fig. 4a,b). These fibres appeared to terminate mainly in the

EIectrophysiological experiments

Extracellular recordings were obtained from 15 units which were all located in the PnC. These PnC neurons were driven by acoustic stimuli with latencies of 2-10 ms (median: 4 ms) and had their best frequencies around 10 kHz with minimal threshold intensities of about 80 dB SPL. Electrical stimulation of the amygdaloid complex induced spikes in nine (60%) of the PnC neurons with latencies in the range of 3-8 ms (median 6 ms). The tone-

235

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Fig. 3. Anterograde tracing with PHA-L. Line drawings of coronal sections of three representative rats (1, 9 and 10). The upper row shows the injection sites, the middle and bottom rows show PHA-L labeling in the pontine reticular formation at the level of the Mo5 and the 7n, respectively. Rat 1 was injected into the central part of cA and exhibits labelled fibres and terminals ipsilaterally, mainly in the PB, SubCV and PCRtA. Rat 10 was injected into the medial division of the cA and into the B. Labelled fibres and terminal

arborizations are found in the PB, SubC~ PnC, and PCRtA ipsilat- erally. A few fibres cross the midline to the contralateral PnC. Rat 9 was injected into the B, and labelled fibres with presumable termi- nals were found in the ipsi- and contralateral PnC as well as in PCRtA. These findings indicate that the projection from the cA and B to the pontine reticular formation is organized in a mediolateral topography. Scale bars 1 mm

evoked discharge rate was enhanced in 13 (87%) neurons after stimulation of the amygdaloid complex.

Recordings from two representative units are shown in Fig. 5a and b. The peristimulus time histograms (PSTHs) show the units' responses to acoustic stimula- tion alone and to electrical stimulation of the amygdaloid complex alone (upper two panels of 5a and b). The effect of a conjunctive stimulation is shown in the third panel: the numbers of spikes recorded from PnC neurons during simultaneous acoustic and amygdalobasal stimulation clearly indicate a facilitation of the acoustic responsive- ness of the neurons by stimulation of the amygdaloid complex. After termination of the amygdalobasal stimu- lation, the tone-evoked response declined to the control level (recovery). An immediate recovery of the discharge rate to control levels after terminating the stimulation of the amygdaloid complex was observed in eight units. In some cases, the facilitating effect of amygdalobasal stimu- lation lasted for more than 10 min but the maximal dura-

tion of the facilitation was not investigated. The increase of the tone-evoked activity of PnC neurons after stimula- tion of the amygdaloid complex was statistically signifi- cant (Wilcoxon signed rank test: a - -5 ; n = 1 5 ; P < 0.001). The increase was in the range of 41-507% (mean 158 _ 40%). All stimulation electrodes were localised in the medial division of the cA and in the B.

B e h a v i o u r a l e x p e r i m e n t s

Bilateral microinjections of N M D A and ACSF were pos- sible in 12 rats and the behavioural data from these ani- mals were taken for final analysis.

Figure 6 shows the injection sites of N M D A and ACSF with triangles corresponding to injections where N M D A increased the startle response. Bilateral microin- jection of 3.9 nmol N M D A led to a mean increase of the ASR amplitude of 170 • 16% compared to the injection

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Fig. 4a-e. Photomicrographs of the caudal pontine brainstem after anterograde tracing with PHA-L. Dorsal is up and medial is left. a Labelled fibres and presumed terminals ventromedial to the Mo5 in the PnC and SubCV of rat 10. b Presumed terminal varicosities and boutons-en-passant in the PnC in rat 10. Notice that no presumed axosomatic contacts can be seen. e Descending fibre coursing

through the PnC in rat 10 forming boutons-en-passant (arrows). d Presumed terminal varicosities in close apposition to the somata of medium-sized (arrowheads) and small (arrows) PnC neurons in rat 10. e Fibre coursing through the PnC (arrows) of rat 9 forming presumed terminals on the apical dendrite of a giant PnC neuron (arrowheads). Scale bars 250 gin, b-e (Nomarski optics) 25 gm.

of ACSF (Fig. 7a) if the injections were made in the vicin- ity of the cA and B (n = 6; triangles in Fig. 6). In this case, the difference between the effect of ACSF and N M D A was statistically significant (t=6.4, df=5, P < 0.001). N M D A injections into the medial amygdaloid nucleus and thalamic nuclei (n = 6; see filled circles in Fig. 6) were without effect on the ASR, i.e. no significant difference between N M D A and ACSF was observed ( t=-0.42, df= 5, P=0.65) (Fig. 7b). Apar t from the effect on the ASR, the N M D A injections into the cA/B were without much effect on the rats' behaviour: Immediately after drug injection, a slight trembling of the masseter muscles was observed and the locomotor activity was reduced. The increased amplitude of the ASR was evaluated for only 5 min following the injection. In a few animals, a long-lasting ( > 30 min) and continuous increase in the startle amplitude was observed, but this effect was not investigated in detail.

Discussion

Retrograde and anterograde tracing

Our data demonstrate a direct projection to the pontine reticular formation from two immediately adjacent, yet distinguishable nuclei of the basal forebrain; the central nucleus of the amygdala and the basal nucleus of Meynert. The retrograde tracing experiments revealed a multitude of afferent projections to the PnC. We focused on the projections from the cA and B in the present study, because this projection was found to be relevant for the enhancement of the ASR (Davis et al. 1991). The anterograde tracing experiments show that the descend- ing amygdaloreticular fibres course through and appear to terminate in the lateral part of the pontine brainstem, in the PB, PCRtA, SubCV, and PnC. It can be assumed that varicosities of PHA-L labelled axons represent

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J , . i i . r r }s I l l i l I

40 60 80 100 120

consecutive stimuli is given by n in each PSTH. The upper panel shows the tone-evoked response of the units [tone (control)]. The middle panels show that in both units, stimulation of the cA/B evoked spikes in the PnC (amygdala), and that the tone-evoked response of the PnC neurons was facilitated by amygdalobasal stim- ulation (tone and amygdala). The bottom panel shows that the tone- evoked activity of the neurons reaches the control level after stop- ping the amygdalobasal stimulation ["tone (recovery)"]

238

-~ 140-

~. 120 E m 100 _r

80-

"~ 60- 8,

4o

2o E

o

c -20

- 4 0 a b

ACSF NMDA ACSF NMDA

Fig. 7a,b. Bar diagram showing the effect of microinjections of ACSF and NMDA on the ASR. The mean percent change (+ sem) of the ASR amplitude after injection compared to the pre-injection amplitude is given for injections into the cA/B (a: n=6) and for misplaced injections (b: n = 6)

Fig. 6. Injection sites of NMDA/ACSF traced onto drawings of serial coronal sections of the rat brain at 0.5 mm intervals, based on Paxinos and Watson (1986). Triangles show injection sites where NMDA increased the amplitude of the ASR by more than 100% compared to the injection of ACSF into the same locus (n=6). Filled circles represent ineffective injections of NMDA/ACSF with respect to the above criterion (n = 6)

presynaptic boutons (Wouterlood and Groenewegen 1985), although some care should be exercised with this interpretation because electron microscopic validation has shown that these boutons need not necessarily repre- sent synaptic contacts (Meredith and Wouterlood 1990). The findings from the anterograde tracing experiments are in accordance with the results of previous anatomical studies on amygdaloreticular or amygdalotegmental pro- jections in rats and cats, most of which being based on different methods (Hopkins and Holstege 1978; Krettek and Price 1978; Inagaki et al. 1983; Veening et al. 1984; Milner and Pickel 1986; Sakanaka et al. 1986; Takeuchi et al. 1988; Rosen et al. 1991; Wallace et al. 1992). It

should be noted here that there exist some differences in the nomenclature of the pontine reticular formation. For example, Newman (1985) subdivided the PnC into a large medial part (pars beta) and a smaller ventrolateral part (pars alpha). Andrezik and Beitz (1985) characterised the PnC proper by the presence of giant neurons and there- fore used the term PnC only for the central part of the caudal pontine reticular formation, which would corre- spond to Newman's pars beta. The region underneath the motor trigeminal nucleus (Mo5) is termed subcoeruleus nucleus, ventral part (SubCV) and corresponds roughly to Newman's caudal pontine reticular nucleus pars al- pha. Newman's nomenclature is based on hodological and morphological data indicating that the medial part of the PnC (pars beta) is characterised by the presence of giant neurons projecting into the ipsilateral spinal cord, whereas the ventrolateral part of the PnC (pars alpha) consists of smaller cells, larger in number, projecting mainly to the contralateral spinal cord. Since the stan- dard rat atlas of Paxinos and Watson (1986) follows the nomenclature of Andrezik and Beitz (1985), we used their nomenclature and nuclear boundaries in the present study.

The medial part of the PnC (PnC proper, according to Andrezik and Beitz 1985) is reached mainly by fibres from the B and medialmost part of the cA. The presump- tive terminal fibres are not seen approaching the somata of giant reticulospinal cells of the PnC, which are likely to mediate the ASR (Koch et al. 1992), in contrast to projec- tions from the cochlear nucleus, which are frequently found to terminate on the somata of giant PnC neurons (Kandler and Herbert 1991). Nevertheless, it seems rea- sonable to assume that the amygdalobasal projections directly influence giant PnC neurons via axodendritic contacts, because these giant PnC neurons have extreme- ly large dendritic trees (more than 1 mm in diameter) covering the whole area where the amygdalobasal projec- tions are found (Lingenh6hl and Friauf 1992). Both the

239

low intensity of the retrograde labeling of cells in the cA and B, when compared to the intensity of other retro- gradely labelled cells (e.g. in the superior colliculus), and the relatively low density of anterogradely labelled fibres, indicate that the projections from the cA and B to the PnC are comparatively weak.

Our data also demonstrate an amygdalobasal projec- tion to the SubCV and PCRtA but the contribution of these nuclei to the ASR is, at present, unknown. Previous studies in our laboratory have shown that chemical le- sions confined to the giant PnC neurons significantly re- duce the ASR amplitude, but that a virtually complete block of the ASR occurred only after large lesions which also affected the SubCV and PCRtA (Koch et al. 1992). Therefore, it might be possible that part of the amyg- dalobasal input influences the ASR indirectly via other nuclei of the pontine brainstem.

Injections of PHA-L which were confined to the cau- dal part of the B revealed a direct projection from the B to the PnC proper. The fact that the B is a prominent part of the basal forebrain cholinergic system (Woolf 1991) raises the question as to whether this projection is cholin- ergic. Preliminary data from our laboratory show, how- ever, that the subset of neurons of the B projecting to the PnC are not ChAT-immunopositive (Koch, unpublished observations). The transmitters used by neurons project- ing from the B and the cA to the PnC are not known. These neurons probably use glutamate as a transmitter, and it is likely that the neurons of the cA use neuropep- tides as co-transmitters (Price et al. 1987).

Electrophysiological experiments

The extracellular single unit recordings in the PnC demonstrated that the amygdalobasal afferents can en- hance the acoustic responsiveness of PnC neurons. The response latencies of 3 8 ms fit very well to the response latencies found in identified giant PnC neurons during intracellular recordings after stimulation of the cA (Lin- genhShl 1992) and are in accordance with the work of Rosen and Davis (1988a; 1990), who demonstrated that the latency for the effects of electrical stimulation of the amygdala to reach the brainstem startle circuit is 3 5 ms.

It is very likely that we recorded from the giant reticu- lospinal PnC neurons which are known to be particularly important for the mediation of the ASR (Koch et al. 1992) because the electrophysiological data (e.g. response latency, characteristic frequency and threshold to acous- tic stimulation, as well as the response latency to amyg- dalobasal stimulation) of the present study closely corre- spond to the findings of intracellular recordings from identified giant PnC neurons (Lingenh6hl 1992) and to the data from our previous study (Ebert and Koch 1992). Interestingly, we did not find a marked habituation of the tone-evoked response in single PnC units during 1 Hz- stimulation, although the ASR is found to rapidly habit- uate after stimulation at a 2-s interstimulus interval (Davis 1970), which might result from a change at the PnC (Davis et al. 1982b). Our observation could be due to the anaesthesia used for the unit recording. The magni-

tude of enhancement of the acoustic responses of PnC neurons following amygdalobasal stimulation found in the present study, indicates that this projection is of be- havioural relevance. Therefore, our data suggest that the giant PnC neurons not only function as sensorimotor relay neurons within the primary circuit of the ASR, but additionally serve as the recipients of important modula- tory influences.

It is noteworthy that in a few cases, the enhanced re- sponsiveness of PnC neurons to acoustic stimulation out- lasted the stimulation of the amygdaloid complex for a few minutes. However, whether this effect corresponds to some kind of long-term potentiation in the amygdaloid complex (Clugnet and LeDoux 1990) remains unknown.

Behavioural experiments

The objective of our behavioural experiments was to demonstrate that excitation of the amygdalobasoreticu- lar pathway is relevant for the ASR. Our data show that the ASR can be enhanced by stimulation of the amyg- dalobasal complex with NMDA. This finding is in accor- dance with the results of Rosen and Davis (1988b) which were obtained using electrical stimulation. Their data in- dicate that the most sensitive site for the electrical facili- tation of the ASR is located just medial to the cA, and these authors conclude that this effect might result from stimulation of the fibres of the amygdalofugal pathway. As we have found that cells within this region belonging to the B also project to the PnC, it could be conjectured that electrical stimulation of these B neurons was to some extent responsible for the enhanced ASR. Although the chemical stimulation technique offers the possibility of distinguishing between the effects of axonal and neuronal stimulation (Gelsema et al. 1987), our present data do not allow for a decision on the issue of whether neurons of the B are more relevant for the enhancement of the ASR than neurons of the cA, because the spatial resolution of the microinjection technique is limited due to the possi- ble spread of NMDA from the B to cA [we estimate from Myers' (1966) experiments that a volume of 0.3 gl NM- DA has an average spread of 0.5 mm over 5 mini.

It cannot be excluded that the chemical stimulation of the cA/B increased the ASR secondarily through the amygdala's well-known influence on cardiovascular re- sponses (Iwata et al. 1987), or through other neuroen- docrine or autonomic changes. However, since our elec- trophysiological data have unequivocally shown a direct effect of amygdalobasal stimulation on acoustically driv- en PnC neurons, we are confident that the results of the chemical stimulation of the cA/B represent a specific ef- fect on the ASR.

It is likely that the increased ASR after infusion of NMDA into the cA/B reflects a sensitization process (Hitchcock et al. 1989). NMDA is a potent glutamate agonist and is therefore expected to stimulate neurons in the cA/B expressing ionotropic glutamate receptors of the NMDA subtype (Monaghan and Cotman 1985). It cannot be excluded, however, that fear-conditioning con- tributes to the increased ASR amplitude as well. This

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assumption is based on the findings of Leaton and Cran- ney (1990), who showed that acoustic startle stimuli act as aversive stimuli for a fear-conditioning to contextual cues during startle experiments. Since the activation of N M D A receptors in the amygdala can be implicated in the acquisition of conditioned fear (Clugnet and LeDoux 1990; Miserendino et al. 1990), the direct application of N M D A to the amygdaloid complex might support the fear-conditioning to contextual cues by the aversive startle stimuli.

General discussion

Taken together, the present data demonstrate a direct excitatory projection from the cA and B to the PnC. Fur- thermore, we show that stimulation of this pathway in- creases the ASR. The relative contribution of these two neuroanatomically distinct nuclei to the increase of the ASR cannot be differentiated from our data, because both electrical and chemical stimulation techniques do not have a sufficiently fine spatial resolution. Further studies should address the problem to disclose the effects of the B on the ASR.

A large body of evidence indicates that the B plays an important role in learning and memory, although a more general role in attentional processes might be a better description of the function of the B (Robbins et al. 1989). It was believed for some time that the cholinergic neu- rons of the B are functionally its most important element, but recent studies have shown that the functions of the B in attentional processes are not only fulfilled by its cholinergic subpopulation of neurons, because be- havioural deficits observed after nonspecific lesions of the B could not be replicated with more specific lesions to the cholinergic subpopulation of cells in the B (Dekker et al. 1991 ; Dunnett et al. 1991). However, nothing is currently known about the functional significance of the non- cholinergic B neurons which project to the brainstem.

A fundamental question in behavioural neuroscience is how the motivational impact of integrative brain cen- tres ultimately affects the sensorimotor systems in order to change the animal's responsiveness according to its internal state. Since the ASR very quickly interrupts on- going motor activities and increases muscle tone, it has been regarded as a preparatory act for a flight response (Pilz 1989). Likewise, the amygdaloid nuclei have been implicated in the processing and integration of sensory information in the context of emotions (Turner and Herkenham 1991; LeDoux 1992), particularly in flight, fight, fear and anxiety (Davis 1992). Hence, our present data, together with the recent work of Hitchcock and Davis (1991) and Rosen et al. (1991), demonstrate that integrative brain centres can directly influence a sensori- motor interface and suggest how states of fear and atten- tion could increase the probability of a flight response in a strikingly adaptive way.

Acknowledgements. We thank Drs. E. Friauf, H. Herbert, K. Lin- genh6hl, J. Ostwald, and H.-U. Schnitzler, as well as Mrs. H. Zillus, for various kinds of help with this study. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 307).

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