Modulatory roles of GABAergic mechanisms in post-rotatory nystagmus in the rabbit

1. To determine how the GABAergic mechanism operates in the generation of post-rotatory nystagmus, an experiment was performed with GABAergic drugs in rabbits. 2. Subconvulsive doses of picrotoxin (0.3-0.6 mg/kg, i.v.) and bicuculline (0.1 mg/kg, i.v.) decreased the number of post-rotatory nystagmus beats, whereas strychnine sulphate, at a subconvulsive dose (0.1 mg/kg i.v.), increased it. 3. Diazepam (1 mg/kg, i.v.) remarkably increased the number of post-rotatory nystagmus beats. Pretreatment with picrotoxin (0.45 mg/kg, i.v.), bicuculline (0.1 mg/kg, i.v.) or semicarbazide-HCl (180 mg/kg, i.v.) antagonized the effects of diazepam (1 mg/kg, i.v.). 4. GABAergic mechanisms may play a modulatory role in the production of nystagmus rhythm. Strychnine-sensitive neurons involved in the vestibular mechanism may behave in a different manner from picrotoxin-sensitive neurons.     

Effects of benzodiazepines and pentobarbitone on the gaba-ergic recurrent inhibition of hippocampal neurons

The actions of benzodiazepines and pentobarbitone on GABA-mediated recurrent inhibition of hippocampal pyramidal neurons were investigated in the immobilized unanaesthetized cat. Extracellular action potentials of single neurons were recorded in regions CA1 or CA2 with 4 M NaCl-containing glass micropipettes. Bicuculline (0.1 mg/kg i.v.) reduced the period of inhibition induced by stimulation of the fimbria and the septum, but fludiazepam and diazepam (0.3--1.0 mg/kg i.v.) and pentobarbitone (15--30 mg/kg i.v.) prolonged the inhibition. The prolongation produced by these compounds was antagonized by the administration of bicuculline (0.3 mg/kg i.v.). The results suggest that these two classes of compounds potentiate GABA-mediated recurrent inhibition in hippocampal neurons in a similar way.     

Pharmacological studies on the prolonged depressant effects of baclofen on lumbar dorsal horn units in the cat

The pharmacological basis of the prolonged depressant effect of baclofen on small diameter afferent inputs to dorsal horn neurons was studied because of the possibility that this depression represents, at the cellular level, the mechanism of the analgesic effects of baclofen. When l-baclofen (0.1–3.0 mg/kg, i.v.) was given, the ensuing prolonged depression could not be mimicked or modified by the GABA-mimetic drug, muscimol (0.5–5.0 mg/kg, i.v.). Administration of the GABA antagonist, bicuculline (1–5 mg/kg, i.v.), partially reversed the depression induced by baclofen but even after large doses of bicuculline, subsequent administration of baclofen produced depression. The opiate antagonist naloxone (0.1–0.3 mg/kg, i.v.) and the glycine antagonist strychnine (0.1–0.2 mg/kg, i.v.) failed to block the prolonged depression induced by baclofen. These results suggest that baclofen is acting in the dorsal horn via a mechanism which excludes opiate receptors and glycine receptors and which only partially includes bicuculline-sensitive GABA receptors. The results are consistent with a possible action of baclofen on the recently proposed ‘novel GABA receptor’, or GABA_B receptor, which is insensitive to bicuculline; if this indeed proves to be the case, then the present results suggest that at least in the spinal cord, these receptors are associated differentially with small diameter afferent fibres. However, regardless of the receptors involved, the most likely effect of baclofen is to reduce the amount of transmitter (or modulator) released from terminals of primary afferent fibres, and evidence is examined implicating glutamate and substance P. The transient, non-specific depressant effect (described in more detail in another publication) was prevented by bicuculline, and thus its effects are likely to be mediated by bicuculline-sensitive GABA receptors.     

Involvement of central GABA receptors in the regulation of the urinary bladder function of anaesthetized rats

Summary Cystometric recordings were performed in pentobaribitone anaesthetized rats and the effects of gammaaminobutyric acid (GABA) mechanisms on urinary bladder function were evaluated as their influence on a bladder hyperactivity induced by 1-dihydroxyphenylalanine (l-DOPA) after peripheral decarboxylase inhibition. The bladder response was inhibited by intracerebroventricular (i.c.v., 4th ventricle) injections of GABA (250 g), muscimol (0.2 g) and glycine (1,000 g) as well as by systemically administered muscimol (4 mg/kg) and diazepam (2 mg/kg). Intravenous (i.v.) bicuculline, but not i.v. strychnine, antagonized the inhibitory actions of intraperitoneal (i.p.) and i.c.v. muscimol and i.v. diazepam while the opposite was true for the inhibitory action of i.c.v. glycine. In rats not pretreated with l-DOPA, i.p. administration of bicuculline (4 mg/kg) after 15 min caused prominent detrusor contractions that were prevented by an infracollicular brain transection.It is suggested that GABA synapses in the pontinemesencephalic brain region may be involved in the modulation of urinary bladder function.     

Benzodiazepine antagonist flumazenil reduces bicuculline-induced enhancement of neuronal activity in the spinal cord.

The interaction between the GABAA receptor antagonist bicuculline and the benzodiazepine receptor (BR) antagonist flumazenil was studied in the lumbosacral cord of spinal cats. Bicuculline (0.8 mg/kg i.v.) evoked epileptiform bursting of motoneurons in parallel with a depression of peripherally elicited dorsal root potentials (DRP) and dorsal root reflexes (DDR). A low dose of flumazenil (0.03 mg/kg i.v.) reduced the bursting activity of motoneurons as well as partially reversed the depression of DRP and DRR induced by bicuculline. It is suggested that an endogenous BR ligand down-regulating GABAA receptors is released during the bicuculline-evoked enhancement of neuronal activity and may contribute to the epileptiform discharge of motoneurons in the spinal cord.     

Inhibitory effects of beta-phenylethylamine on photic-evoked potentials in the visual cortex of the cat

We investigated the effects of beta-phenylethylamine (PEA) on photic-evoked potentials recorded from the cerebral cortex (visual cortex) in the anesthetized cat. The photic-evoked potential consisted of negative (N) and positive (P) components in the cerebral cortex, with peak times of 39.4 +/- 15.4 msec and 57.5 +/- 16.3 msec, respectively. Intravenous administration of PEA (6.5, 12.5 and 25 mg/kg) induced a dose-dependent decrease in the amplitude of the N component of photic-evoked potentials. PEA-induced decreases in the amplitude of photic-evoked potentials (at 12.5 mg/kg) were antagonized by yohimbine (2 mg/kg). Prazocin (2 mg/kg, i.v.) and propranolol (0.5 mg/kg, i.v.) did not change the PEA-induced decrease of photic-evoked potentials. Methysergide (2 mg/kg, i.v.), haloperidol (1 mg/kg, i.v.) and naloxone (0.5 mg/kg, i.v.) also failed to alter the inhibitory effect of PEA. Pretreatment with alpha-methyl-p-tyrosine or reserpine did not completely attenuate the PEA-induced decrease of photic-evoked potentials. According to these results, PEA inhibits the photic-evoked potentials in the visual cortex partially through the noradrenergic alpha 2-receptor.     

Antagonism of L-glycine to seizures induced by L-kynurenine,quinolinic acid and strychnine in mice

L-glycine (1–12.5 μg, intracerebroventricularly, i.c.v.) completely prevented seizures induced by i.c.v. administration of L-kynurenine, and practically did not modify those induced by another convulsant quinolinic acid, a metabolite of tryptophan, and by strychnine. L-Glycine administered intraperitoneally (i.p.) (1000 mg/kg) decreased lethality after K-kynurenine and quinolinic acid; at doses of 3000 and 4000 mg/kg which are sedative and hypothermic it prolonged the latency of strychnine and L-kynurenine seizures. The convulsant action of pentylenetetrazol was not modified. Kynurenine seizures are suggested to be related to the action of kynurenine on glycine receptors in the central nervous system.     

Effects of central GABA receptor agonism and antagonism on evoked diencephalic cardiovascular responses

The administration of the GABA agonists diazepam (0.3 mg/kg i.v.) and muscimol (0.3 μg/kg, i.c.v.) to chloralose-anaesthetized cats caused significant inhibition of the pressor responses elicited by electrical diencephalic stimulation. The inhibition was apparently a result of a reduction in centrally emanating sympathetic discharge to vasoconstrictor nerves. The GABA antagonist bicuculline (0.5 mg/kg i.v.) reversed the inhibition caused by muscimol but not by diazepam; however, pretreatment with bicuculline prevented the inhibition caused by diazepam. Muscimol caused significant and marked reductions in blood pressure and heart rate by decreasing central sympathetic nerve discharge; this action was also completely reversed by bicuculline. It is suggested that central activation of GABA receptors results in an inhibition of centrally evoked cardiovascular responses by preventing increases in sympathetic outflow. Furthermore, basal as well as evoked blood pressure, heart rate and sympathetic nervous discharge can be reduced by small doses of the directly acting GABA receptor stimulant muscimol.     

Cardiovascular effects of intracerebroventricular GABA,glycine and muscimol in the rat

Summary Intracerebroventricular (i.c.v.) injections of gammaaminobutyric acid (GABA, 125–2,000 g), glycine (250–2,000 g) and muscimol (0.5–4 g) caused dose-dependent reductions of blood pressure and heart rate in conscious rats. Low doses of muscimol (<0.12 g) were hypertensive. The cardiovascular depression induced by GABA was antagonized by bicuculline (3.5 mg/kg i.p.), while that induced by glycine was counteracted more effectively by strychnine (0.7 mg/kg i.v.) than by bicuculline. The cardiovascular response to GABA, but not to glycine, was potentiated by pretreatment with pentobarbitone (50 mg/kg i.p.). It was prolonged by diazepam (5 mg/kg i.p.) and aminooxyacetic acid (AOAA, 25 mg/kg i.p., 4h) but not by AOAA (1 h) or nipecotic acid (1–100 g i.c.v.). Following treatment with d-amphetamine (5 mg/kg i.p.) or reserpine (10 mg/kg i.p., 6h) the cardiovascular depression induced by GABA was attenuated. Propranolol (2.5+2.5 mg/kg i.v. + s.c.) or atropine (2 mg/kg i.v.) alone partially counteracted and in combination completely prevented the negative chronotropic response to GABA. Neither central noradrenaline depletion by means of -MMT (after carbidopa) and -MT nor pretreatment with phenoxybenzamine (20 g i.c.v.) influenced the response to GABA. The arterial hypotension induced by GABA was prolonged after a high dose of atropine (10 mg/kg i.v.) but appeared attenuated by a low dose (2 mg/kg i.v.) or by physostigmine (0.1 mg/kg i.v.). Central serotonergic activation by means of 5-hydroxytryptophan after benzerazid antagonized the cardiovascular actions of GABA. Spiroperidol (0.05 mg/kg i.v.) and apomorphine (1 mg/kg i.v.) did not affect the blood pressure reduction in response to GABA.It is concluded that i.c.v. GABA causes cardiovascular depression by activation of GABA receptors and that the response is mediated by the sympathetic and vagal systems. There were no indications for an involvement of central noradrenergic of dopaminergic mechanisms while a decreased central cholinergic and an increased serotonergic activity possibly contribute to the cardiovascular effects of GABA.     

Blockade of bicuculline-induced pressor and tachycardic responses by forebrain administration of muscimol

To determine whether i.v. administered bicuculline acts in the forebrain to increase arterial blood pressure and heart rate, this agent was administered i.v. to chloralose anesthetized cats that had muscimol injected into and restricted to the forebrain ventricles. Bicuculline (0.5 mg/kg i.v.) given alone increased arterial pressure by 56 +/- 8 mm Hg and heart rate by 45 +/- 11 beats/min. Bicuculline given to animals exposed to muscimol exhibited no increase in either of these parameters. Muscimol localized to the forebrain did not alter the pressor response to a non-GABA antagonist agent, strychnine, indicating a specific interaction of the drugs with GABA receptors in the forebrain.     

Enhanced 5-hydroxytryptamine and dopamine-mediated behavioural responses following convulsions—I the effects of single and repeated bicuculline-induced seizures

Rats were injected daily with vehicle (controls) or bicuculline (0.375 mg/kg, i.v.) for 10 days. The bicuculline administration produced a major convulsion. Twenty-four hours after the final injection it was found that the bicuculline-treated rats displayed enhanced behavioural responses following the administration of either tranylcypromine (TCP; 5 mg/kg) followed by l-DOPA (50 mg/kg), or of TCP followed by l-tryptophan (75 mg/kg), when responses were compared to controls.The responses following apomorphine or TCP/l-DOPA were also enhanced 24 hr after a single bicuculline injection, whilst those following tranylcypromine/l-tryptophan were unchanged. A single sub-convulsive dose of bicuculline (0.2 mg/kg, i.v.) failed to enhance the TCP/l-DOPA response.When a bicuculline-induced seizure was produced during halothane anaesthesia enhanced TCP/l DOPA responses were not seen 24 hr later, in agreement with the failure of a single electroconvulsive shock (ECS) given during halothane anaesthesia to enhance this response. However an enhanced response was seen 24 hr after a single ECS given without anaesthetic.The possibility that altered monoamine function is a general consequence of repeated convulsions is discussed and the possible role of anaesthetics in retarding these changes is suggested and examined further in the subsequent paper (Cowen, Nutt and Green, Neuropharmacology, 19, 901–906).     

Anticonvulsant actions of the putative gamma-aminobutyric acid (GABA)-mimetic, ethylenediamine.

1 Ethylenediamine, 31.6-1000 mg/kg intraperitoneally, inhibited the convulsive effects of pentylenetetrazol, 100 mg/kg (i.p.) in mice. 2 Ethylenediamine, 100-1000 mg/kg (i.p.) increased the convulsion threshold to the intravenous infusion of three convulsants in the order pentylenetetrazol greater than bicuculline greater than strychnine. 3 The benzodiazepine antagonist R0 15-1788, 10 mg/kg (i.p.), significantly inhibited the anticonvulsant action of diazepam, 50 micrograms/kg, but not ethylenediamine, 1000 mg/kg. 4 These results clearly indicate that ethylenediamine has anticonvulsant properties and are consistent with the hypothesis that ethylenediamine is a gamma-aminobutyric acid (GABA)-mimetic.     

Involvement of NMDA receptors in thiopental-induced loss of righting reflex, antinociception and anticonvulsion effects in mice

Potentiation of GABA(A) receptor-mediated inhibitory neurotransmission contributes to the anesthetic action of thiopental. However, the inhibiting action of general anesthetic on excitatory neurotransmission also purportedly underlies its effects. The aim of the study was to elucidate the role of glutamate receptors (NMDA and AMPA receptors) in thiopental-induced anesthesia. Intracerebroventricular (i.c.v.) NMDA (50 ng) significantly increased the induction time of loss of righting reflex and decreased sleep time induced by intraperitoneal injection (i.p.) of thiopental (50 mg/kg). Furthermore, NMDA at 50 ng i.c.v. increased the 50% effective dose values for thiopental to produce loss of righting reflex and immobility in response to noxious tail clamp by 25% and 21% (p < 0.05), respectively. However, intrathecal (IT) administration of NMDA or both of i.c.v. or IT administration of AMPA did not show such antagonizing effects on thiopental action at subconvulsive dose. Finally, thiopental (25 mg/kg i.p.) inhibited convulsions induced by NMDA (0.4 microg i.c.v.) or bicuculline (0.6 microg i.c.v.). However, i.p. muscimol (1 mg/kg) blocked the convulsions induced by bicuculline, but not those induced by NMDA at 3 mg/kg. Similarly, i.p. MK-801 (0.1 mg/kg) antagonized NMDA-induced convulsions, but not bicuculline-induced convulsions at 0.3 mg/kg. Therefore, we suggest that the effects of the selective GABA(A) and NMDA receptors on convulsive behavior are special to their sites of action, and that the inhibitory action of thiopental on NMDA receptors is possibly not mediated by secondary effects of its GABA(A) receptors agonism. These results above indicate the involvement of NMDA receptors in thiopental-induced anesthesia in mice.     

The differential effects of post-session administration of amineptine and imipramine on memory processes in mice

The effects of post-trial administration of amineptine, a dopaminergic antidepressant drug, were compared with those of memory-facilitating (strychnine, piracetam) or impairing drugs (phenobarbital, imipramine) on an experimental model of memory. Mice were given two sessions in open-field test and the decrease in activity at the second session (habituation) served as an index of retention. The good retention observed with a 1-day inter-session interval was impaired by post-session administration of phenobarbital (10 mg/kg i.p.) or imipramine (5.0 mg/kg i.p.). The poor retention observed with a 5-day inter-session interval was enhanced by post-session administration of strychnine (0.20 mg/kg i.p.), piracetam (1000 mg/kg i.p.) and amineptine (10 mg/kg i.p.). These findings show that different profiles of cognitive and psychomotor effects were produced by imipramine and amineptine. Amineptine, lacking sedative and anticholinergic properties which are characteristic of imipramine, interferes positively with learning and memory, in a manner similar to piracetam and strychnine.     

Morphine-induced sensitization in mice: changes in locomotor activity by prior scheduled exposure to GABAA receptor agents

This study investigated the effects of a gamma-amino-butyric acid type A (GABAA) receptor agonist and antagonist on morphine-induced locomotor sensitization in male albino mice. Subcutaneous administration to mice of a high dose of morphine (30 mg/kg), but not lower doses (5, 10 and 20 mg/kg) increased locomotion. The maximum locomotor activity was achieved during a 20-min measurement period. The locomotor response to a low dose of morphine (5 mg/kg, subcutaneously) given on day 9 was enhanced in mice pretreated with morphine (7.5, 15 and 30 mg/kg/day x 3 days), indicating that sensitization had developed. Three-day intracerebroventricular (i.c.v.) administration of the GABAA receptor agonist, muscimol (0.025, 0.05, 0.1 and 0.2 microg/mouse/day) significantly decreased both morphine-induced motor stimulation and locomotor sensitization. On the other hand, a 3-day pretreatment with the GABAA-receptor antagonist, bicuculline (0.25, 0.5 and 1 microg/mouse/day) reduced morphine (15 mg/kg)-induced locomotor sensitization. Repeated i.c.v. injections of a lower dose of bicuculline (0.25 microg/mouse/day x 3 days) by itself also decreased morphine-induced locomotion. Furthermore, repeated i.c.v. administration of bicuculline (0.25, 0.5 and 1 microg/mouse/day x 3 days) decreased the effect of i.c.v. injection of muscimol (0.1 microg/mouse/day x 3 days) on locomotor activity induced by morphine (5 mg/kg) in both control and sensitized mice. The magnitude of this response was, however, variable. The results indicate that GABAA receptors might be involved in the acquisition of morphine-induced sensitization.     

The role of GABA and glycine in the physostigmine-induced alterations of spinal cord reflexes.

The purpose of this study was to determine which inhibitory pathway(s) mediate the alterations in the monosynaptic (MSR) and polysynaptic (PSR) reflexes after two different doses of physostigmine. It was found previously that 0.8 mg/kg physostigmine facilitated the MSR and 2.0 mg/kg initially depressed and then facilitated the MSR. Both doses facilitated the PSR. In this study, the animals were pretreated with either strychnine (0.1 mg/kg) or bicuculline (0.5 mg/kg), prior to the administration of either dose of physostigmine. It was found that both strychnine and bicuculline blocked the facilitation produced by the small dose of physostigmine, while bicuculline alone blocked the depression of the MSR produced by the large dose of physostigmine. Strychnine partially blocked the effects of both doses of physostigmine on the PSR, while bicuculline only partially blocked the effects of the small dose of physostigmine. These data suggest that the depression of the MSR was the result of a GABA-mediated pathway, while the facilitation of MSR involved both glycine and GABA.     

GABA(A) but not GABA(B) receptor stimulation induces antianxiety profile in rats.

The effect of GABA receptor agonists and antagonists on anxiety behavior in rats in the elevated-plus-maze has been investigated. The increase in two parameters of %open arm entries (%OAE) and %time spent in the open arms (%OAT) and decrease in the %time spent in closed arm (%CAT) was considered as antianxiety effects. Intracerebroventricular (i.c.v.) injection of different doses of the GABA(A) receptor agonist muscimol (0.25, 0.5, and 1 microg/rat) increased %OAE and %OAT and decreased %CAT in rats dose-dependently. The higher response was obtained with 1 microg/rat of the drug. Neither icv (0.05, 0.1, and 0.2 microg/rat) nor intraperitoneal (i.p.) (1, 2, and 4 mg/kg) injection of the GABA(B) receptor agonist baclofen altered %OAE, %OAT, and %CAT. However, the GABA(B) receptor antagonist CGP35348 (5, 10, and 30 microg/rat i.c.v.) increased %OAE and %OAT and decreased %CAT in the animals. The response induced by injection of muscimol (0.5 microg/rat i.c.v.) or administration of CGP35348 (10 microg/rat i.c.v.) was reduced by i.c.v. (1, 2, and 4 microg/rat) or i.p. (0.25, 0.5, and 0.75 mg/kg) injection of the GABA(A) receptor antagonist bicuculline, except the effect of CGP35348 on %CAT which was not significantly altered by i.p. administration of bicuculline. Ip but not i.c.v. administration of bicuculline by itself reduced both %OAE and %OAT but did not alter %CAT. None of the drugs altered the locomotor activity of the animals. The current findings support our hypothesis that the anxiolytic effects of GABA(B) antagonist are mediated by autoreceptor blockade-induced release of endogenous GABA, which in turn activates postsynaptic GABA(A) receptors.     

In vivo inhibitory effects of stimulation at the central end of the pelvic nerve severed from urinary bladder on urinary bladder contraction in rats

Two- or five-Hz electrical stimulation of the central end of the left pelvic nerve severed from the urinary bladder in rats inhibited bladder contraction induced by intravesical infusion of Tyrode's solution. Inhibition of bladder motility by 2-Hz nerve stimulation appeared after pretreatment with strychnine (0.3 mg/kg, i.v.), naloxone (1 mg/kg, i.v.) and picrotoxin (1 mg/kg, i.v.). Hypogastric nerve stimulation, however, did not affect bladder contraction. These results suggest the presence of an inhibitory mechanism on the pelvic motoneuron activated by contralateral pelvic nerve stimulation in rats.     

The effect of baclofen on the urinary bladder contraction accompanying micturition in anesthetized rats

We studied the effects of baclofen on the bladder contraction induced by infusion of Tyrode's solution into the urinary bladder in anesthetized rats. Baclofen (5 mg/kg, i.v.) completely inhibited bladder contraction and abolished the efferent discharges recorded from the left pelvic nerve, causing the bladder pressure to rise until solution leaked from the penis. The inhibitory effect of baclofen (5 mg/kg, i.v.) could not be reversed by picrotoxin (1 mg/kg, i.v., twice with an interval of 10 min) or naloxone (1 mg/kg, i.v.). In parallel with convulsion, strychnine (1 mg/kg, i.v.) contracted the bladder which had been inhibited by baclofen and generated electrical activities consisting of efferent discharges and electromyograms. The dose of intracerebroventricularly or intrathecally injected baclofen which completely inhibited the bladder contraction was 0.1 or 10 micrograms, respectively. After the inhibition of bladder contraction by i.v. injection of baclofen, electrical stimulation of the sacral cord could contract the bladder and cause a fall in bladder pressure to around the level existing after micturition. From these results, the active site of baclofen which is related to the inhibition of bladder contraction is thought to be the micturition center in the brain stem.     

Inhibitory effect of inaperisone hydrochloride (inaperisone), a new centrally acting muscle relaxant, on the micturition reflex.

We examined the effects of inaperisone hydrochloride (inaperisone), a new centrally acting muscle relaxant, on bladder function in anesthetized rats and isolated rat tissues. We also investigated its mechanism of action. When a balloon inserted into the bladder was expanded, rhythmic bladder contractions were observed; inaperisone (4 mg/kg i.v.) abolished these contractions, in both normal and decerebrated rats. The bladder tonus or bladder contraction induced by peripheral stimulation of the pelvic nerve was barely inhibited by inaperisone (4 mg/kg i.v.), but this dose of inaperisone abolished the efferent discharge from the pelvic nerve that accompanied the rhythmic bladder contractions. The doses of intracerebroventricularly (i.c.v.) and intrathecally injected inaperisone which abolished the rhythmic bladder contractions were 10 and 100 micrograms, respectively. The inhibitory effects of inaperisone (4 mg/kg i.v.) were not diminished by naloxone (1 mg/kg i.v.) or by bicuculline (0.5 mg/kg i.v.), but were diminished by phaclofen (30 mg/kg i.v. or 300 micrograms i.c.v.). The specific binding of [3H]baclofen to rat brain synaptosomal membranes was barely inhibited by inaperisone (up to 1 mM). From these results, it is speculated that, among other possible mechanisms, inaperisone inhibits the micturition reflex by acting indirectly on GABAB receptors in the brainstem.