Pre- and postnatal development of the catecholamine innervation of the hypoglossal nucleus in the rat: an immunocytochemical study.

The pre- and postnatal development of the catecholamine (CA) innervation to the hypoglossal nucleus (nXII) in the rat was investigated immunocytochemically with antisera to tyrosine hydroxylase (TH). Immunoreactive profiles positive for TH were first identified in nXII on gestational day (GD) 16. By GD 18, the adult-like distribution pattern was evident, characterized by the preferential targeting of the ventromedial region of nXII, but this pattern was not consistently found in all fetuses until GD 19. From GD 19 to postnatal day (PD) 180, the overall density of TH immunoreactivity, particularly in the ventromedial region, increased with further growth and maturation of nXII. These results establish the early prenatal CA innervation of nXII and support the hypothesis that CA are important in regulating motor tongue behavior in the newborn. Moreover, because the ventral compartment of nXII contains motoneurons that innervate protrusor muscles of the tongue, and tongue protrusor mechanisms play an essential role in suckling, deglutition, and respiratory (maintaining a patent upper airway) behaviors, it is further proposed that the CA innervation of nXII is critical to the survival of the newborn.

Effects of antioxidants on the blood-brain barrier and postischemic hyperemia.

The role of free oxygen radicals in blood-brain barrier (BBB) disruption and postischemic hyperemia was evaluated in the rabbit model of focal cerebral ischemia-reperfusion. Six groups of rabbits underwent clipping of the anterior cerebral, middle cerebral, and intracranial internal carotid arteries. Cerebral blood flow (CBF) was measured by using radiolabeled microspheres, before, during, and 15 minutes after 1-hour occlusion of these arteries. After 50 minutes of ischemia, Group 1 animals (control) received a placebo. Animals in Groups 2-4 received one of three drugs: catalase at 10 mg/kg, methimazole at 5 mg/kg, or indomethacin at 10 mg/kg. A fifth group received a tungsten-supplemented diet for 14 days before ischemia was induced, and a sixth group was sham operated. Microvascular integrity within the brain was determined by the presence or absence of Evan's Blue (EB)-albumin dye leakage across the BBB and was measured by microspectrofluorometry. In the control group during ischemia, CBF dropped to 14%, 7%, and 11% of preischemic levels in rostral, middle, and caudal sections of the brain, respectively, as characterized by extensive EB-albumin dye leakage through the BBB into the ischemic hemisphere. During early reperfusion, postischemic hyperemia was associated with an increase in CBF of 128%, 123%, and 129% of control in the rostral, middle, and caudal sections of the brain, respectively. In all treated groups and in the group receiving a tungsten-supplemented diet, BBB integrity was protected during reperfusion without inhibition of postischemic hyperemia. This study suggests that early disruption of the BBB to large molecules is mediated by free oxygen radicals, which inhibit rather than cause postischemic hyperemia.

Effect of superoxide dismutase on acute reperfusion injury of the rabbit brain.

To study the involvement of free oxygen radicals of the blood-brain barrier (BBB) disruption during early reperfusion, we isolated the distal internal carotid artery, and the middle and anterior cerebral arteries via the transorbital approach in anesthetized rabbits. Using radiolabeled microspheres, regional cerebral blood flow (rCBF) was measured before, during and after 1-hour unilateral occlusion of these vessels. Fifty-five minutes after ischemia, animals received intravenous saline placebo (control), superoxide dismutase (SOD) at 8 mg/kg = 30,000 U/kg, or weakened superoxide dismutase (wSOD) at 8 mg/kg = 30,000 U/kg. Integrity of the BBB was assessed by leakage of Evan's Blue-albumin dye (EB-albumin dye), which was given at 15 minutes of reperfusion and allowed to circulate for an additional hour. In the control and wSOD-treated groups, rCBF decreased (26% and 40% of control, respectively) within the blue-tinted tissue of the occluded hemisphere during ischemia; hyperemia was observed during early reperfusion. In the control and wSOD-treated groups, EB-albumin dye leakage across the BBB increased 49% within the occluded hemisphere. However, within the SOD-treated group, the BBB showed minimal dye leakage even though rCBF of the occluded hemisphere (so-called blue-tinted tissue) decreased by 38% during ischemia. We conclude that 1-hour focal cerebral ischemia and reperfusion produce a vascular endothelial injury at the BBB. Since SOD administration showed significant protection, free-oxygen-radical production during early reperfusion is associated with breakdown of the BBB to large molecules.

Sources of noradrenergic afferents to the hypoglossal nucleus in the rat.

The sources of noradrenergic (NA) innervation to the hypoglossal nucleus (nXII) in the rat were investigated with double-labeling histochemical/immunocytochemical and lesion/degeneration techniques. Following injection of wheat germ-agglutinin conjugated to horseradish peroxidase into nXII, brain stem sections were reacted with tetramethylbenzidine, stabilized, and incubated in antiserum to tyrosine hydroxylase (TH). Double-labeled neurons were observed in three pontine sites bilaterally, although mainly ipsilaterally, that included the nucleus subceruleus (nSC; 68.75%) and the A7 (21.09%) and A5 (10.15%) cell groups. Confirmation of the above results and identification of the course taken by descending NA-nXII projections was accomplished by lesioning the rostral pons, the nSC, or the medullary catecholamine bundle (MB), the suspected route by which NA afferents reach nXII. Quantitative estimates of the reduction of TH immunoreactivity on the lesioned compared to nonlesioned side of nXII were made densitometrically. In each case, TH immunostaining was significantly decreased (75%) in the ipsilateral caudoventromedial district of nXII, the predominant target area of NA input. The results from this study establish that multiple NA sources in the pons project to nXII in the rat, the majority of NA-nXII afferents are derived from the nSC, and descending NA-nXII projections course in the MB. These data are discussed relative to tongue control.

Effects of MK-801, a non-competitive NMDA antagonist, on linguopharyngeal events in rats.

The effects of MK-801 at doses from 0.005 to 1 mg/kg IP on linguopharyngeal events (protrusions, retrusions and swallows) were determined in rats to find out whether MK-801 resembles ketamine in its capacity to increase the frequency of recurrence of such events that we have demonstrated in previous studies. All rats receiving a dose of 0.05 mg/kg or higher showed an increase in linguopharyngeal event frequency within 5 min and this enhancement (3-fold from baseline level) was maintained for longer than 1 h. At the lowest dose of 5 micrograms/kg the effect lasted only very briefly. A general increase in motor behavior was also observed within 10 min of drug administration. More complex patterns of motor behavior, consisting of stereotypical head bobbing, paw movements reminiscent of walking activity, nystagmus, and ataxia were observed with doses of 0.25 mg/kg and higher. All rats showed a marked startle response at early stages post-injection and hypersensitivity to external stimuli such as noise or movement in the room. However, there was an absolute lack of coordinated avoidance responses normally associated with such startle responses or arousing stimuli.

Catecholamine-containing axon terminals in the hypoglossal nucleus of the rat: an immuno-electronmicroscopic study.

A correlative light and electron microscopic investigation was undertaken to determine the morphology and distribution of catecholamine (CA)-containing axon terminals in the hypoglossal nucleus (XII) of the rat. This was accomplished immunocytochemically with antibody to tyrosine hydroxylase (TH). The major findings in this study were the following: 1) Immunoreactive profiles were found throughout XII and included unmyelinated axons, varicosities, axon terminals and dendrites; 2) Nonsynaptic immunoreactive profiles (preterminal axons, varicosities) were more frequently observed (55.2%) than synaptic profiles (43.5%); 3) CA-containing axon terminals ending on dendrites were more numerous (71.8%) than those synapsing on somata (25.4%) or non-labeled axon terminals (2.7%); 4) The morphology of labeled axon terminals was variable. Axodendritic terminals typically contained numerous small, round agranular vesicles, a few large dense-core vesicles and were associated with either a symmetric or no synaptic specialization, axosomatic terminals were often associated with a presynaptic membrane thickening or a symmetric synaptic specialization and contained small, round and a few elliptical-shaped vesicles, while axoaxonic synapses formed asymmetric postsynaptic specializations; and 5) CA-positive dendritic processes were identified in XII. These findings confirm the CA innervation of XII, and suggest a complex, multifunctional role for CA in controlling oro-lingual motor behavior.

Serotonin-containing axon terminals in the hypoglossal nucleus of the rat. An immuno-electronmicroscopic study.

The morphology and distribution of serotonin-containing axon terminals in the rat hypoglossal nucleus (XII) was investigated immunocytochemically at the electron microscopic level. Serotonin-positive profiles were found throughout all regions of XII and included unmyelinated axons, varicosities and axon terminals. Most labeled profiles (68.1%) were nonsynaptic unmyelinated axons and varicosities, while synaptic profiles, ending on dendrites and somata, were seen less frequently (28.7%). The majority of labeled axon terminals (76.9%) ended on small-to-medium-sized dendrites. Most axodendritic terminals contained small, round agranular vesicles (20-55 microns), several large (60-100 microns) dense core vesicles, and were associated with a pronounced asymmetric postsynaptic specialization. By contrast, labeled axosomatic terminals were seen less often than those ending on dendrites (23.0%). Axosomatic terminals typically contained small, round, agranular and large dense core vesicles and were associated with a symmetric or no postsynaptic specialization. These results provide the structural substrates for elucidating the functional role of serotonin in tongue control.

Effects of clozapine on ketamine-induced linguopharyngeal events in rats.

We tested the effects of clozapine (0.02-20 mg/kg i.p.) on ketamine-induced linguopharyngeal events in rats anesthetized with i.m. injections of ketamine hydrochloride (100 mg/kg) and mounted on a stereotaxic with the tip of the tongue tied to a force displacement transducer monitoring tongue protrusions, retrusions and swallows. Reduction began at the 0.04 mg/kg dose. At 4.8 mg/kg there was total suppression of events. At 20 mg/kg, suppression lasted for 1 h. Notably clozapine doses causing total suppression of events in our model were much lower than those usually reported to alter dopamine turnover.

Cortical control of tongue contractility in the rat under ketamine anaesthesia.

1. Cortico-lingual and linguo-cortical interconnectivity was investigated in ketamine-anaesthetized rats mounted onto a stereotaxic apparatus. The tip of the tongue was tied to a force displacement transducer to monitor tongue retrusions. The tongue cortical area was exposed in one or both hemispheres to record evoked potentials or spontaneous electroencephalographic (EEG) activity, or to stimulate electrically with single square pulses of up to 50 V and 0.25 ms pulse width. 2. The results showed that (i) tongue retrusions elicited by electrical stimulation were identical to those induced by ketamine; (ii) ketamine-induced tongue contractions correlated in time with cortical EEG potentials which were easily distinguished from the background noise; (iii) haloperidol (2.5 mg/kg, i.m.) suppressed the cortically evoked tongue contractions as well as the ketamine-induced contractions. 3. These results suggest that ketamine-induced tongue retrusions may involve the cortex in their mediation if not their generation and that this may provide a basis for the suppression of dyskinetic activity during sleep or unconsciousness.

Metoclopramide fails to suppress linguopharyngeal events in a rat dyskinesia model.

The effects of metoclopramide on ketamine-induced linguopharyngeal events exemplified by tongue retrusions, protrusions, and swallowing acts were tested in ketamine-anesthetized rats mounted on a stereotaxic frame with the tip of the tongue tied to a force displacement transducer to monitor retrusion and protrusion and a pressure transducer inserted into the mouth at the level of the soft palate to monitor swallowing. Metoclopramide at doses ranging from 0.5 to 50 mg/kg im failed to decrease protrusions, retrusions, or swallowing. It rather increased all three events for up to 2.5 hours. The significance of these findings is discussed.

Ketamine-induced tongue protrusions in rats.

1. The purpose of this study was to demonstrate that ketamine anesthesia (100 mg/kg) induces tongue protrusions (P) in addition to retrusions (R) and swallows (S) in adult rats. 2. These linguo-pharyngeal events occur alone or combined in various sequential patterns. 3. The SPR sequence is not the predominant pattern in all preparations suggesting profound disruption of physiological linkages by ketamine. 4. Haloperidol administration suppresses these events for 1-120 min depending on the dose (0.75-2.5 mg/kg). 5. Swallows are the least vulnerable to haloperidol. 6. This and previous findings provide further evidence that ketamine induced linguo-pharyngeal activity can serve as a model for acute or tardive dyskinesia better than stereotypies.

Prenatal ethanol exposure impairs lesion-induced plasticity in a dopaminergic synapse after maturity.

This study examined the consequences of alcohol (ethanol) exposure during fetal life on lesion-induced dopaminergic synapse responsiveness (plasticity) in the olfactory tubercle of the adult rat. Normally, in the olfactory tubercle, olfactory bulbectomy elicits alterations in pre- and postsynaptic dopaminergic markers, including, respectively, (1) increased tyrosine hydroxylase activity and immunoreactivity, which is associated with dopaminergic axon sprouting, and (2) increased dopaminergic receptor density and potentiated dopamine activation of adenylate cyclase. We have utilized biochemical and quantitative immunocytochemical methodology to examine these synaptic markers in olfactory bulbectomized or sham-operated adult rats. These animals were offspring of dams which were administered one of the following diets during pregnancy: (1) liquid diet containing 35% ethanol-derived calories ad libitum; (2) liquid diet containing an isocaloric amount of maltose-dextrin instead of ethanol, pair-fed; or (3) unaltered liquid diet ad libitum. The results show that prenatal alcohol exposure leads to suppression of the lesion-elicited dopaminergic synapse responsiveness in the olfactory tubercle. There were no significant differences between offspring born to control and pair-fed animals, indicating that the observed abnormalities were not due to alterations in their nutritional status. In conclusion, the present data are a biochemical and quantitative immunocytochemical demonstration of impaired lesion-induced synaptic responsiveness. This renders a new dimension in support of previous evidence indicating that prenatal alcohol exposure leads to altered neuroanatomical, neuroendocrinological and behavioral responsiveness to various challenges. Such impaired synaptic responsiveness may underlie brain functional abnormalities characteristic of fetal alcohol syndrome.

Catecholamine innervation of the rat hypoglossal nucleus.

The catecholamine innervation of the hypoglossal nucleus (XII) was investigated immunocytochemically by comparing the distribution patterns of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the rat. Numerous TH- and DBH-positive profiles were found throughout XII, while only occasional PNMT immunoreactivity was observed. Significantly, the distribution patterns of TH and DBH immunoreactivity were coextensive with the most intense staining found ventromedially along the caudal half of XII. We conclude, therefore, that the catecholamine innervation of XII is largely noradrenergic, and that motoneurons innervating the genioglossi muscles, the principal protrusors of the tongue, are the primary targets of this input.

Interconnections between substantia nigra reticulata and medullary reticular formation.

Injections of wheat germ agglutinin-HRP into the medullary reticular formation (MRf) or the substantia nigra reticulata (SNr) revealed the presence of reciprocating fiber connections between the two areas. Large injections in the MRf demonstrated the existence of labeled neurons in the lateral portions of the SNr. Isolated injections into the parvocellular nuclei of the MRf resulted in the presence of terminal fields in the SNr particularly its lateral portions. Injections in the SNr resulted in the presence of labeled cells in the parvocellular nuclei. The significance of these findings is discussed in terms of oro-facial dyskinesias.

An electrographic characterization of ketamine-induced linguopharyngeal motor activity.

Ketamine-induced buccolinguopharyngeal motor activity was studied in rats visually and by means of force displacement transduction of tongue retrusions, electromyogram (EMG) of motor units of tongue muscles, and pressure transduction of swallowing acts. Each animal was anesthetized with an intramuscular injection of ketamine hydrochloride (100 mg/kg body weight). Through a tracheotomy the airway was intubated and the animal was mounted on a stereotaxic frame in a supine position for monitoring of the above parameters. Four varieties of events were demonstrated: (a) swallowing acts followed by tongue retrusion, (b) tongue retrusions in isolation, (c) tongue retrusions followed by swallowing events, and (d) swallowing events in isolation. All four types of events were vulnerable to intramuscular injection of haloperidol 0.75-2.5 mg/kg within 5 to 10 min and the suppression endured for at least several hours. We conclude that there is a parallel between ketamine-induced oral motor activity and neuroleptic-induced dyskinesia in that both are temporarily suppressed by neuroleptic drugs.

Distribution of glutamic acid decarboxylase and gamma-aminobutyric acid in the hypoglossal nucleus in the rat.

Immunocytochemistry was used to investigate the distribution of glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the hypoglossal nucleus (XII) of the adult rat. The distribution of GAD and GABA was found to be co-extensive throughout XII. Although immunoreactivity was moderately dense in all regions, the intensity of staining was greatest in the ventral district of XII particularly ventromedially in the caudal half of the nucleus. Immunoreactive terminal-like profiles were observed around motoneuron somata and dendrites. There also was evidence of sparse mediolaterally-oriented densities of immunoreactivity at the junction of XII and the dorsal vagal nucleus and between dorsal and ventral districts of XII. In addition, neurons staining positive for GABA were found scattered within XII laterally and immediately outside of XII in and around the Nucleus of Roller. These observations suggest a complex, differential distribution of GABA in XII and are discussed in relation to tongue motor behavior.

Ketamine as a pharmacological model for tongue dyskinesia.

The purpose of this article is to document that ketamine hydrochloride, administered at an anesthetic dosage of about 100 mg/kg, produces tongue contractile activity in the rat. The methods for monitoring and quantitating ketamine-induced tongue contractions (KITCs) are described. We also found that neuroleptic agents consistently and readily abolish KITCs. On the basis of these observations and other pharmacological properties of ketamine, we propose that KITCs may be a useful model for studying neuroleptic-induced oral dyskinesia, e.g., tardive dyskinesia. Additional findings in support of this model are presented.

Effects of cholinergic and anticholinergic drugs on ketamine-induced linguopharyngeal motor activity.

Benztropine mesylate (Cogentin) and physostigmine salicylate (Antilirium), were tested for changes in tongue protrusions, retrusions, and swallowing acts in rats anesthetized with a 100 mg/kg IM injection of ketamine hydrochloride. These ketamine-induced linguopharyngeal events were monitored by means of a force displacement transducer fed onto a polygraph. Benztropine (0.05-1 mg/kg) caused mild to moderate reductions in the rate of these events for a short period of time, up to about 30 min. With physostigmine (5-25 micrograms/kg), linguopharyngeal activity was markedly increased, up to 50-fold by the highest dose within 5 min and returned almost to the baseline within 60 min. With lower doses, more moderate responses were obtained. If methscopolamine (1.4, 3, 6 mg/kg IM) preceded physostigmine, the physostigmine enhancement was preserved.

Perinatal, not adult, hypothyroidism suppresses dopaminergic axon sprouting in the deafferented olfactory tubercle of adult rat.

We reported recently that chronic thyroid deficiency in rat, beginning in utero and terminating after maturity, suppresses lesion-induced central catecholaminergic axon sprouting in the adult brain [Gottesfeld et al, 1985]. The present work was undertaken to define the critical period of hypothyroidism on subsequent neuronal sprouting. Thyroid hormones deficiency was induced in rats by methimazole during (a) gestational days 8-21 (20 mg/kg/day in the drinking water); (b) postnatal days 1-15 (0.2 or 0.4 mg/pup/day; i.p.), or (c) in the mature animal for 4 weeks (20 mg/kg/day in the drinking water). The olfactory tubercles (OTs) were used as a model to study sprouting of dopaminergic (DA) nerve terminals, elicited by olfactory bulbectomy. Animals in each group received lesions or sham operations as adults, and sacrificed 3 weeks after the operation. Thus, for each of the above treatments four subgroups were formed: (a) euthyroid/sham-operation, (b) euthyroid/lesion, (c) hypothyroid/sham-operation, and (d) hypothyroid/lesion. Sprouting of DA axon terminals in the OTs was identified by biochemical assays and quantitative immunofluorescent microscopy, using tyrosine hydroxylase (TH) as a marker. Serum thyroxine levels served as an index of the thyroid status. The results demonstrate that lesion-induced sprouting of DA axon terminals in OTs of adult rats is suppressed by hypothyroidism induced prenatally or during the early postnatal period, but not after maturity. Thus, there is a perinatal critical period during which altered thyroid function exerts long-term effects on neuronal plasticity.

Characterization of fimbria input to nucleus accumbens.

The hippocampal input to the nucleus accumbens was studied by correlative electrophysiological and anatomical techniques in acutely prepared rabbits. Field and extracellular unitary potentials were recorded in the nucleus accumbens following ipsilateral fimbria stimulation. Analysis of the components of the field response was based on the relevant correlations with extracellular unitary activity. The cellular types that are the recipients of the hippocampal projection were determined by combined intracellular horseradish peroxidase (HRP) and Golgi analyses. The distribution of the hippocampal input was determined by combined field potential and current source density analyses. It was found that the ipsilateral fimbria projection was distributed to the dorsal two-thirds of the nucleus, with the projection being heaviest in the more caudal portions of the nucleus. The negative (N) component of the field response was studied by correlating its behavior with the appropriate extracellular unitary recordings. It was concluded that the N-component represented an envelope of monosynaptically activated action potentials. The positive (P) component of the field response throughout the nucleus accumbens was studied pharmacologically with the iontophoretic administration of bicuculline. The P-components, in both the dorsal and ventral regions of the nucleus, were diminished by bicuculline application, indicating that this potential results from the activation of gamma-aminobutyric acid (GABA) mechanisms. The cell populations that are the targets for the hippocampal projections were studied by the technique of intracellular staining with HRP. These results were correlated with the findings of a Golgi analysis. Two distinct cell types were found to respond in a monosynaptic manner to ipsilateral fimbria stimulation. The most common of the two were the small-to medium-sized spiny neurons, and they were distributed throughout the nucleus. These cells have a spherical dendritic arrangement. The second, and most distinctive, of the cell types were the large aspiny neurons. These cells were distributed medially and caudally in the nucleus. Two of the outstanding features of these cells were the expanse of their dendritic domains and the fact that axons originated from relatively remote portions of the dendrites.