Consequences of amygdala kindling and repeated withdrawal from ethanol on amphetamine-induced behaviours

It has been shown previously that chronic ethanol treatment in mice leads to accelerated behavioural sensitization to psychomotor stimulants [Manley & Little (1997) J. Pharmacol. Exp. Ther., 281, 1330-1339], whilst repeated experience of ethanol withdrawal sensitizes pathways underlying seizure activity (Becker & Hale (1993) Alcohol Clin. Exp. Res., 17, 94-98]. The aim of the current experiment was to investigate the consequences of repeated withdrawal from ethanol on amphetamine-induced behaviours in the rat and compare this with animals with electrical kindling of the amygdala, a procedure that has been shown to enhance alcohol withdrawal seizures [Pinel et al. (1975) Can. J. Neurol. Sci., 2, 467-475]. For the kindling experiments, electrodes were surgically implanted in the left basolateral amygdala and were stimulated daily at the afterdischarge threshold until a criterion of three consecutive stage 5 seizures was reached. Fully kindled rats showed a marginally significant reduction in sensitivity to the locomotor stimulant effects of acute amphetamine compared with sham and partially kindled rats which had experienced subthreshold stimulation of the amygdala. Sham and partially kindled rats sensitized readily to the locomotor activating effects of amphetamine (0.125 mg/kg) following repeated treatments, but the fully kindled rats did not. Fully kindled rats also failed to show place preference conditioning to amphetamine (0.5 mg/kg). Rats, withdrawn three times from chronic ethanol (liquid-diet), kindled more quickly to PTZ (30 mg/kg, i.p.) than rats with the same overall exposure to ethanol (24 days) followed by a single withdrawal or control animals. However, there was no difference in the locomotor stimulating effects of acute amphetamine (0.25-1 mg/kg, i.p.), the rate of sensitization to amphetamine (0.125 mg/kg, i.p.) or amphetamine induced conditioned place preference (1 mg/kg, i.p.). These observations suggest that, in rats, repeated withdrawal from a relatively mild chronic ethanol treatment modulates neuronal systems that may also be involved in PTZ-induced kindling but not those involved in either the acute stimulant effects of amphetamine or behavioural sensitization or appetitive conditioning following repeated amphetamine administration. Behavioural changes following amygdala kindling differed from those following repeated ethanol withdrawal, suggesting that withdrawal kindling from a mild ethanol treatment differs in its effects from amygdala kindling.     

Impaired fear conditioning but enhanced seizure sensitivity in rats given repeated experience of withdrawal from alcohol

Repeated experience of withdrawal from chronic alcohol treatment increases sensitivity to seizures. It has been argued by analogy that negative affective consequences of withdrawal also sensitize, but repeated experience of withdrawal from another sedative-hypnotic drug, diazepam, results in amelioration of withdrawal anxiety and aversiveness. We tested whether giving rats repeated experience of withdrawal from alcohol altered their ability to acquire a conditioned emotional response (CER). Male Hooded Lister rats were fed a nutritionally complete liquid diet as their only food source. Different groups received control diet, or diet containing 7% ethanol. Rats receiving ethanol diet were fed for either 24 days (Single withdrawal, SWD), or 30 days, with two periods of 3 days, starting at day 11, and 21, in which they received control diet (Repeated withdrawal, RWD). All rats were fed lab chow at the end of their liquid diet feeding period. Starting 12 days after the final withdrawal, groups of Control, SWD and RWD rats were given pentylenetetrazole (PTZ; 30 mg/kg, i.p.) three times a week, and scored for seizures. The occurrence of two successive Stage 5 seizures was taken as the criterion for full PTZ kindling. Other groups of control, SWD and RWD rats were trained to operate levers to obtain food, and were then exposed, in a fully counterbalanced design, to light and tone stimuli which predicted unavoidable footshock (CS+), or which had no consequences (CS-). Rats consumed approximately 17.5 g/kg/day of ethanol, resulting in blood alcohol levels of approximately 100 mg/dL. Repeated administration of PTZ resulted in increasing seizure scores. RWD rats achieved kindling criterion faster than either Control or SWD rats. No differences were seen in the groups in flinch threshold to footshock (0.3 mA). At a shock intensity of 0.35 mA, Control, but not RWD or SWD rats showed significant suppression to the CS+ CS- presentation did not affect response rates. The three groups differed in their response to pairing the CS+ with increasing shock levels, the Controls remaining more sensitive to the CS+. SWD rats showed significant suppression of lever pressing during CS+ presentations only at 0.45 and 0.5 mA, and RWD rats only at 0.5 mA. Giving rats repeated experience of withdrawal from chronic ethanol results in increased sensitivity to PTZ kindling, but reduces their ability to acquire a CER. Withdrawal kindling of sensitivity to anxiogenic events does not seem to occur under circumstances which give rise to kindling of seizure sensitivity.     

Selective deficits in appetitive conditioning as a consequence of ethanol withdrawal

The acquisition of a conditioned response to a cue associated with a fearful event has been shown to be impaired in animals that had been repeatedly withdrawn from ethanol, but not in animals with the same chronic ethanol treatment but only a single withdrawal episode [D. N. Stephens et al. (2001) Eur. J. Neurosci., 14, 2023-2031]. Lesion studies have shown that the amygdala plays a vital role in this type of conditioning process. Here we investigate aspects of conditioning for appetitive reinforcers in operant tasks, also shown to rely on amygdala processing, in rats following repeated withdrawal from ethanol. Rats were chronically treated with either an ethanol-containing liquid diet for 24 days continuously (single withdrawal) or interspersed with 2 x 3-day withdrawal periods (repeated withdrawal), or with a control diet (control). Two weeks after the final withdrawal, operant training began. In tasks that are impaired by lesions of the basolateral amygdala, conditioned reinforcement and reinforcer devaluation, there was no effect of chronic ethanol treatment or withdrawal on acquisition or performance. However, in a task that is dependent upon functioning of the central nucleus of the amygdala, Pavlovian-to-instrumental transfer, the single and repeated withdrawal groups were significantly impaired. Therefore, chronic ethanol treatment and withdrawal resulted in deficits in behavioural tasks that are sensitive to central but not to basolateral amygdala lesions, and may reflect different sensitivities of these areas to ethanol.     

Repeated withdrawal from ethanol impairs acquisition but not expression of conditioned fear

Repeated withdrawal from ethanol impairs acquisition of conditioned fear [Stephens, D.N., Brown, G., Duka, T. & Ripley, T.L. (2001) Eur. J. Neurosci., 14, 2023-2031]. This study further examined the effect of repeated withdrawal from ethanol on the expression and acquisition of fear conditioning. Following training, presentation of a cue associated with footshock (CS+) resulted in a suppression of operant responding for food reinforcement. In different groups, shock thresholds were manipulated to give weak or severe behavioural suppression. Rats were subsequently chronically treated with ethanol-containing liquid diet either continuously (single withdrawal) or with three withdrawal periods (repeated withdrawal). Ethanol treatment and withdrawal had no effect on conditioned suppression of responding tested 2 weeks after the final withdrawal, at either shock intensity. Nevertheless, extinction of conditioned fear was impaired in the repeated withdrawal group exposed to the higher shock intensity. In the high intensity group, the stimulus-shock association was then reversed, so that the previously neutral conditioned stimulus (CS-) became the CS+. Acquisition of suppression to the new CS+ was significantly less in the animals previously given repeated experience of withdrawal, confirming our previous finding. Thus, repeated withdrawal from ethanol lead to disruption in the acquisition of fear conditioning but had no effect on retrieval of an association formed prior to the ethanol-withdrawal experiences.     

Development of long-term subsensitivity to GABA in dorsal raphe neurons of amygdala-kindled rats.

Previously we reported a long-term change in neuronal sensitivity to GABA following amygdala kindling. Dorsal raphe neurons of amygdala-kindled rats exhibited significant subsensitivity to GABA 4 weeks after the last fully generalized (Stage 5) seizure. We hypothesized that this alteration in GABA sensitivity might reflect neuronal changes corresponding to kindled seizure susceptibility and subsequent experiments have investigated this hypothesis. The progression towards neuronal subsensitivity to GABA during amygdala kindling can be correlated with the Stage to which an animal has been kindled. That is, when measured 4 weeks after the last kindled seizure, dorsal raphe neurons are supersensitive to GABA following a Stage 2 seizure, not different from controls following a Stage 3 seizure and subsensitive to GABA following a Stage 5 seizure. In addition, subsensitivity to GABA appears to be permanent in that it is still measurable 3 months after the last Stage 5 seizure. Thus, amygdala kindling produces long-term, perhaps permanent, changes in neuronal sensitivity to GABA and these changes reflect the Stage to which an animal has been kindled.     

Amygdala and pyriform cortex kindling in vasopressin deficient rats (Brattleboro strain)

Homozygous and heterozygous Brattleboro rats and Long--Evans control rats were subjected to repeated electrical stimulation of the amygdala or pyriform cortex in a kindling paradigm. The homozygous Brattleboro group stimulated in the amygdala was retarded in its kindling rate relative to heterozygous Brattleboros and Long--Evans controls. The retarded kindling rate of the homozygous Brattleboros stimulated in the amygdala is attributed to a delay in seizure development at stages 1 and 2 which suggests that vasopressin may be necessary for normal kindling from the amygdala to take place.     

Amygdaloid kindling with bicuculline methiodide in rats

The effect of repeated intraamygdaloid injection of bicuculline methiodide (BM) was studied in rats. Chemitrodes for both microinjection and electrographic recording were implanted into the left basolateral amygdala. Two weeks after the surgery, a subconvulsive dose of BM (0.2 or 0.4 nmol) was administered through the chemitrodes every fourth day. Repeated injections caused a progressive seizure development which was comparable to that seen with electrical kindling. The kindling effect persisted after a 6-month interruption of the stimulation. When a mixture of BM and GABA agonist (GABA 20 nmol, muscimol 2 nmol, or baclofen 5 nmol) was injected into the amygdala of kindled rats, seizure activity was markedly suppressed to stage 0 or 1. On the other hand, an intraamygdaloid injection of picrotoxin (0.8 nmol) brought about the same seizure as induced by BM, whereas no seizure was observed with strychnine (4 nmol). No histological change specific to this kindling was detected. The present results indicate that chemical kindling can be induced by repeated local injection of BM into the amygdala, and that the mechanism underlying this kindling is closely associated with local postsynaptic GABA receptors in the amygdala. This GABAergic system may also be important in other types of kindling.     

Repeated ethanol exposure and withdrawal impairs human fear conditioning and depresses long-term potentiation in rat amygdala and hippocampus.

BACKGROUND: In rats, repeated episodes of alcohol consumption and withdrawal (RWD) impair fear conditioning to discrete cues. METHODS: Fear conditioning was measured in human binge drinkers as the increased startle response in the presence of a CS+ conditioned to aversive white noise. Secondly, the ability of tone CSs, paired with footshock, to induce c-fos expression, a marker of neuronal activity, in limbic structures subserving emotion was studied in rats. Additionally, consequences of RWD on subsequent induction of long term potentiation (LTP) in external capsule/lateral amygdala and Schaffer collateral/hippocampus CA1 pathways were studied in rat brain slices. RESULTS: Fear conditioning was impaired in young human binge drinkers. The ability of fear-conditioned CSs to increase c-fos expression in limbic brain areas was reduced following RWD, as was LTP induction. Rats conditioned prior to RWD, following RWD showed generalization of conditioned fear from the tone CS+ to a neutral control stimulus, and a novel tone. CONCLUSIONS: Binge-like drinking impairs fear conditioning, reduces LTP, and results in inappropriate generalization of learned fear responses. We propose a mechanism whereby RWD-induced synaptic plasticity reduces capacity for future learning, while allowing unconditioned stimuli access to neuronal pathways underlying conditioned fear.     

Can a generalized kindling seizure induce a reward state?

The postictal behavioral depression (PBD), characterized by behavioral immobility and unresponsiveness to environmental stimuli, observed after a stage 5 kindling seizure is opioid dependent. Morphine injection prolongs while naloxone and naltrexone (opioid antagonists) reduce or eliminate PBD. Opioids have clear rewarding actions that can be easily detected by place preference conditioning (PPC). In the present study, we evaluated if the opioid release after a stage 5 kindling seizure that produces PBD could induce PPC. Male rats were kindled in the medial preoptic area (MPOA), the amygdala (AMG) or insular cortex (IC). After kindling was established their initial preference in a three-compartment chamber was determined. During conditioning, subjects received a standard kindling stimuli that evoked a stage 5 seizure. At the end of the after discharge and during the PBD the animals were placed in the non-preferred chamber for 30 min. On alternate days they were placed without stimulation in the preferred chamber. At the end of conditioning the kindled groups showed a clear change of preference. This change of preference was completely blocked by injection of naloxone. These results suggest that opioid release after a stage 5 kindling seizure can induce a positive affect of sufficient intensity and duration to induce conditioning.     

The glutamate release inhibitor riluzole attenuates the formation of conditioned place aversion induced by naloxone in rats undergoing a single morphine exposure

Acute morphine exposure has been hypothesized to produce long-lasting central changes that contribute to the withdrawal aversion. We have most recently demonstrated that those changes may involve the glutamatergic system, including multiple classes of receptors. The present study was undertaken to further determine the involvement of the glutamatergic system by examining the effect of riluzole, a glutamate release inhibitor, on the motivational component of withdrawal from acute morphine dependence. The role of the amygdala in the action of riluzole was also assessed. We investigated the effects of riluzole on the conditioned place aversion (CPA) induced by naloxone-precipitated withdrawal from a single morphine exposure 24 h before, and on c-Fos expression within the amygdala during the withdrawal period in rats. Riluzole (2, 4, 8 mg/kg) dose-dependently attenuated the CPA without producing place conditioning itself. This result provided further evidence that glutamatergic mechanisms may be recruited in adaptational changes following acute morphine exposure and play a role in withdrawal aversion. In addition, riluzole appeared to produce nonspecific effects on c-Fos expression by itself, without specifically modifying c-Fos expression following naloxone-precipitated withdrawal in any region of the amygdala examined, suggesting that the amygdala may not serve as a specific site of action for riluzole.     

Amygdala kindling induces muscarinic cholinergic receptor declines in a highly specific distribution within the limbic system

The term kindling refers to the phenomenon whereby repeated administration of an initially subconvulsive electrical stimulus results in progressive intensification of stimulus-induced epileptic activity, culminating in a generalized seizure. Once established, this enhanced sensitivity to electrical stimulation (i.e., kindling effect) is permanent. Pharmacologic studies indicate that the interaction of acetylcholine with brain muscarinic receptors may contribute to two important events in kindling: (i) the development of the permanent enhanced sensitivity to electrical stimulation by which kindling is defined; and (ii) suppression of spontaneous interictal spiking. The present report confirmed and extended our previous findings by demonstrating that the muscarinic cholinergic receptor declines involved not only the stimulated amygdala, but also both hippocampal formations of kindled rats. The decline of muscarinic receptors within the hippocampal formations involved mainly the dentate gyri and spares the CA1 regions. Correlative electrophysiologic and biochemical studies did not confirm a relationship between spontaneous interictal spiking and the receptor declines in kindling. The spiking and seizures induced by the convulsant agent, pentylenetetrazol, failed to induce a receptor decline. The results imply that the receptor declines of the kindled animals are related to some property of the kindling per se. In light of the propensity of hippocampal circuitry to develop long-term potentiation, the biochemical evidence of hippocampal involvement in amygdala kindling is intriguing.     

Bidirectional transfer between kindling induced by excitatory amino acids and electrical stimulation

Chemical (C) kindling by means of repeated spaced injections into the amygdala (AM) of a subconvulsive dose of L-glutamate and L-aspartate combined in a molar ratio of 1:3. (Glu/Asp) produced progressive seizure development culminating in generalized convulsion strikingly similar to electrically kindled Stage 5 seizure in rats. These C-kindled AM sites responded readily to electrical stimulation with very rapid development of kindled seizure. When a separate group of rats electrically kindled at the AM were subjected to identical C-kindling at the kindled AM site, a similar positive transfer effect was observed. In addition, 30 days following the last injection, kindled Stage 5 seizure was triggered with a single injection of Glu/Asp, one-half of the dose used for C-kindling. These results suggest that the glutamate and/or aspartate systems participate in the development and persistence of increased seizure susceptibility induced by AM kindling.     

Insular cortex lesions and taste aversion learning: effects of conditioning method and timing of lesion

The specific role of insular cortex in acquisition and expression of a conditioned taste aversion was assessed using two different conditioning methods, which vary mode of taste delivery. Involvement of insular cortex in the induction of c-Fos-immunoreactivity in the nucleus of the solitary tract, a cellular correlate of the behavioral expression of a conditioned taste aversion, was also assessed. Electrolytic lesions of insular cortex blocked behavioral expression of a conditioned taste aversion and this was evident not only when lesions were placed prior to conditioning, but also when they were made after conditioning but before testing. In contrast to the effects on behavior, lesions did not completely block the c-Fos-immunoreactivity which accompanies re-exposure to the aversive taste. In addition, the blocking of behavioral evidence of aversion conditioning by cortical lesions was seen both in animals trained under an intraoral acquisition procedure and those trained with bottle-conditioning. This contrasts with previous work with amygdala lesions which showed that amygdala was absolutely necessary for taste aversions conditioned with the intraoral method but not for those conditioned using bottle presentation of the taste. Overall, these findings imply that the details of the neural circuitry involved in taste aversion learning, including its anatomical distribution, complexity and degree of redundancy, vary with the type of conditioning method employed.     

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor autoradiography in mouse brain after single and repeated withdrawal from diazepam

Withdrawal from chronic treatment with benzodiazepines is associated with increased neuronal excitability leading to anxiety, aversive effects and increased seizure sensitivity. After repeated withdrawal experiences, seizure sensitivity increases while withdrawal-induced anxiety and aversion decrease. We used autoradiographical methods employing [(3)H]Ro48 8587, a selective ligand for glutamatergic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, to study withdrawal-induced changes in AMPA receptor binding in areas of the mouse brain postulated to be involved in these responses. Mice were given 21 days treatment with diazepam (15 mg/kg, s.c. in sesame oil) followed by withdrawal (single withdrawal) or three blocks of 7 days treatment interspersed with 3-day periods to allow washout of drug (repeated withdrawal). In keeping with heightened excitability in withdrawal from chronic diazepam treatment, the single withdrawal group showed, 72 h after their final dose of diazepam, increased [(3)H]Ro48 8587 binding in several brain areas associated with emotional responses or seizure activity, including hippocampal subfields, amygdalar and thalamic nuclei and motor cortex. In contrast, the repeated withdrawal group showed no changes in [(3)H]Ro48 8587 binding in any brain area studied. These observations are consistent with up-regulation of AMPA receptor-mediated transmission being important in withdrawal-induced anxiety and aversion but not in increased seizure sensitivity associated with repeated withdrawal. As changes in AMPA receptor subunit expression alter the functionality of the receptor, future studies will address this possibility.     

Lateral geniculate kindling and long-lasting photosensitivity in cats

The kindling response of the lateral geniculate body (GL) was compared with that of the amygdala, using cats. Daily electrical stimulation in the GL group led to the generalized tonic-clonic convulsion in most subjects and the resulting state of seizure susceptibility was long-lasting, as in the amygdala group. The kindling response of the GL differed from that of the amygdala in some respects, i.e., rapid kindling, short latency for seizure generalization, a different pattern of behavioral seizure development, and seizure regression during the course of kindling. The effects of photic stimulation with pentylenetetrazol administration were also examined before and after kindling in both groups. This study revealed that the photically induced myoclonus, at times proceeding to the generalized tonic-clonic convulsion, was provoked repeatedly as a result of GL kindling, whereas none of the amygdala-kindled cats showed such marked photosensitivity. These photically induced seizures were invariably observed for at least 4 weeks after GL kindling. Our results suggest that a neural mechanism participating in GL kindling is different from that in amygdala kindling, and that there might be cross-sensitization between seizure susceptibility resulting from GL kindling and photosensitivity.     

Altered synapsin I immunoreactivity and fear behavior in male and female rats subjected to long-term amygdala kindling

Long-term amygdala kindling dramatically increases fearful behavior in both male and female rats. In this experiment, we studied the relation between sex, kindled fear behavior, and synapsin I immunoreactivity in various brain regions. Male and female adult Long-Evans rats received either 99 left amygdala kindling stimulations or sham stimulations. One day after the final stimulation, fear behavior was assessed in each rat by measuring exploration and thigmotaxia in an unfamiliar open field, as well as resistance to capture from the open field. Four hours after the behavioral testing, the rats were sacrificed and their brains were processed for immunohistochemical determination of synapsin I expression. As expected, kindling significantly increased fear behavior in both male and female rats. It also increased synapsin I immunoreactivity bilaterally in most hippocampal subfields, but not in the caudate nucleus, sensorimotor cortices, or piriform cortex. Interestingly, kindling decreased synapsin I immunoreactivity bilaterally in the central and basolateral amygdala of male rats but not female rats. Correlational analyses revealed that in male rats, fearful behavior was positively correlated with synapsin I immunoreactivity in hippocampal brain regions located ipsilateral to the site of stimulation (i.e., the CA1 and CA3 subfields of the hippocampus, the dentate gyrus and the hilus) and negatively correlated with synapsin I immunoreactivity bilaterally in the basolateral and central amygdala. In female rats, fear behavior was positively correlated with synapsin I immunoreactivity in the ipsilateral CA1 and CA3 subfields only. These results suggest that altered synaptic plasticity in specific brain regions might be involved in the exaggerated fearfulness produced by long-term amygdala kindling, especially in male rats.     

Effects of postnatal electroshock convulsions on epileptogenesis of the amygdala and hippocampus in adult rats

Adult rats which received repeated electroshock convulsions (ESCs) during postnatal development underwent electrical kindling of the amygdala (AM) and hippocampus (HIPP). Completion of kindling, especially AM kindling, was significantly faster in the groups with ESCs from the neonatal to late infantile age than in the control group. It is discussed that especially the late infantile age in the postnatal development was supposed to play an important role in the faster kindling brought about by postnatal convulsions and that the faster kindling was due in part to abbreviation of the partial seizure process. However, the neonatal ESC group showed significantly delayed completion of HIPP kindling.     

Kindling of the massa intermedia of the thalamus in rats

The kindling response of the massa intermedia (MI) was assessed in rats. Clinical manifestation of the MI kindling was generally similar to that of limbic kindling, and positive transfer to the amygdala (AM) was obtained following MI kindling. However, MI kindling showed (1) a relatively high afterdischarge threshold which sometimes increased during the course of kindling. (2) a seizure stage instability with frequent regression to earlier stages, (3) a failure to establish a generalized seizure triggering threshold with an ‘all-or-none’ property, and (4) a generalized tonic-clonic seizure, which was quite different from a kindledlimbic seizure, during early phase of kindling. Furthermore, the MI stimulation caused violent beating movement of the forelimbs, jumping, or running regardless of presence/absence of afterdischarge. The findings suggest that mechanisms other than a simple activation of limbic structures are involved in the process of MI kindling.     

新型抗癫痫药唑尼沙胺对点燃的影响

目的研究唑尼沙胺对大鼠杏仁核点燃及戊四唑(PTZ)化学性点燃模型的抗癫痫作用。方法建立大鼠杏仁核电刺激点燃、化学性点燃模型,探讨唑尼沙胺的抗点燃作用;测定唑尼沙胺对小鼠最大电休克的影响。结果唑尼沙胺50~200mg·kg-1ig可抑制大鼠杏仁核点燃发作,降低Racine's分级唑尼沙胺50,100mg·kg-1ig抑制PTZ化学性点燃发作,降低Ono分级(P<0.01)与OnoⅣ级发作百分率(P<0.01)。ZNS10.0mg·kg-1ig降低小鼠最大电休克诱发的,惊厥发生率(P<0.01)。结论唑尼沙胺对点燃发作具有拮抗作用。     

Antiepileptic and prophylactic effects of tetrahydrocannabinols in amygdaloid kindled rats

The antiepileptic and prophylactic effects of delta9-tetrahydrocannabinol (delta9-THC) and delta8-THC were examined in rats that developed generalized seizures in response to intermittent electrical stimulation of the amygdala (kindling). Both isomers of the THC were able to acutely suppress kindled seizures, but consistent antiepileptic effects were obtained only with high, toxic dosages. Tolerance to the antiepileptic effects of THC developed very rapidly when the drugs were give repeatedly, and there was evidence that the repeated administration of a high dosage of delta9-THC resulted in a state of acute physical dependence. Administration of the isomers of THC during seizure development resulted in a suppression of kindling, suggestive of a prophylactic effect. The rate of rekindling after withdrawal of the drugs was not significantly different from that of vehicle-treated control rats, however, indicating that a genuine prophylactic effect was not obtained.