Distribution and stress-induced increase of glutamate and aspartate levels in discrete brain nuclei of rats

Concentrations of glutamate and aspartate have been measured in 45 microdissected brain areas and nuclei in rat. Both amino acids are ubiquitously present and distributed unevenly in the central nervous system. Very high glutamate levels were found in the cerebellum and the insular cortex, high levels in neocortical and limbic cortical areas, and in the nuclei of the medial hypothalamus. Aspartate is distributed rather uniformly with the highest concentration in the hypothalamic arcuate nucleus and the lowest in the midbrain central gray matter and the cerebellum. Acute formalin (pain) stress elevated glutamate and aspartate levels in the cortical areas and substantia nigra significantly, but had minor or no effects on other brain nuclei. Increased locomotor and behavioral activities due to a high dose of amphetamine resulted in a 2-5-fold increase of glutamate and aspartate concentrations, particularly in the biogenic amine-containing brain nuclei.     

Turnover and synthesis of biogenic amines in discrete brainstem nuclei of the rabbit

By use of a microtechnique and sensitive enzymatic isotopic assays norepinephrine (NE), dopamine (DA) and serotonin (5-HT) turnover rates were measured in 6 discrete brainstem regions. The results from young (3 days) and adult (1 year) rabbits were compared. In the dorsal raphe nucleus (dr) of the younger animals the slopes of disappearance of NE and 5-HT were significantly higher than in the adults. Whereas, in the dr the slope of the decline of DA was found to be significantly lower in younger animals. In the dr turnover rates of NE and 5-HT were higher in the young animals. However, higher turnover rates for DA were seen only in the LC-A6 region. The young animals had significantly lower turnover times for NE and 5-HT in the dr. The nts was the only nuclear group to reach significance for DA, and the young animals had longer turnover times when compared to adults. These results are indicative of the different roles the neurotransmitter systems play in maintaining homeostasis. The delicate balances in these systems in the brains of younger animals may contribute to their increased susceptibility to perturbations.     

Pro-opiomelanocortin-derived peptides (ACTH/beta-endorphin/alpha-MSH) in brainstem baroreceptor areas of the rat

Relatively high concentrations of adrenocorticotropic hormone (ACTH), beta-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) were determined by radioimmunoassay in the nucleus of the solitary tract (NTS) of rats. Dense networks of immunoreactive fibers for these peptides were most prominent in the commissural part of the nucleus, where immunostained perikarya (8-15 per section) were also seen in colchicine-treated rats. Moderate peptide levels and moderately dense immunoreactive networks of these peptides were found in the lateral reticular nucleus (including the A1 and A5-C1 catecholaminergic cell groups) and the nucleus ambiguus. Ten different types of surgical lesions or transections were performed in the hypothalamus and the lower brainstem to determine the origin of ACTH, beta-endorphin and alpha-MSH in the brainstem baroreceptor centers. Except the commissural part of the NTS, the baroreceptor areas receive ACTH, beta-endorphin and alpha-MSH innervations from both the hypothalamic arcuate cells and local neurons in the NTS. Fibers in the commissural part of the NTS seem to be of local origin. Hypothalamic fibers to the rostral part of the NTS and the vasomotor A5-C1 cell groups descend in both a medial (through the periaqueductal central gray) and a lateral (ventrolateral tegmental fibers) pathway, whereas fibers to the caudal lateral reticular nucleus (A1 cell group) and the nucleus ambiguus may run only in the lateral pathway. The descending fibers may decussate somewhere in the caudal hypothalamus-rostral midbrain, but caudal to that level they run and terminate ipsilaterally. Fibers from the ACTH-, beta-endorphin- and alpha-MSH-containing cells in the NTS form a bundle arching between the NTS and the ventrolateral medulla and partially (40-55%) innervate the vasomotor and the vasodepressor areas, as well as the nucleus ambiguus.     

Distribution of glutamate decarboxylase in discrete brain nuclei.

The L-glutamate decarboxylase (GAD) levels of 70 rat brain nuclei were determined. The distribution of GAD is uneven. High AAD activity was found in the substantia nigra, the colliculi, some hypothalamic nuclei (e.g. dorsomedial) some nuclei of the limbic system (e.g. tractus diagonalis and accumbens) and the medial forebrain bundle. The reticular part of the substantia nigra had the highest activity, two-fold higher than any other nucleus. Low GAD activity was found in the nuclei of the pons, the hypothalamic median eminence and the reticular formation. Moderate activity was measured in most areas studied. The biochemical mapping of GAD is discussed in relation to possible GABA pathways in the brain.     

Distribution of HCN1 and HCN2 in rat auditory brainstem nuclei

Auditory brainstem neurons that are involved in the precise analysis of the temporal pattern of sounds have ionic currents activated near the resting potential to shorten membrane time constants. One of these currents is the hyperpolarization-activated current (Ih). Molecular cloning of the channels underlying Ih revealed four different isoforms (HCN1-4). HCN1 and HCN2, which are widely distributed in the brain, differ in their activation kinetics, voltage dependence and sensitivity to cAMP. We determined the distribution of the HCN1 and HCN2 isoform in the auditory brainstem and midbrain of young rats (P20-30), using standard immunohistochemical techniques. HCN1 antibodies gave rise to punctate staining on the somatic and dendritic membrane. Strong HCN1 staining was present on octopus and bushy cells of the ventral cochlear nucleus, principal neurons of the lateral and medial superior olive, and neurons of the ventral nucleus of the lateral lemniscus. No HCN1 staining was observed in the dorsal cochlear nucleus and the medial nucleus of the trapezoid body (MNTB). In contrast, HCN2 staining was strongest in the MNTB and the dorsal nucleus of the lateral lemniscus. Strong HCN2 antibody labelling was also observed in bushy cells of the ventral cochlear nucleus. In the central nucleus of the inferior colliculus only a subpopulation of neurons showed HCN1 or HCN2 immunolabelling. This differential distribution of HCN1 and HCN2 channels is in agreement with the physiologically observed Ih currents in corresponding neuronal populations and might represent the basis for functional heterogeneity and diverse sensitivity to neuromodulators.     

Distribution and brainstem origin of cholecystokinin-like immunoreactivity in the opossum cerebellum

In order to determine the distribution of the peptide cholecystokinin (CCK) within the cerebellum and medullary precerebellar nuclei of the adult opossum, sections of these brain regions were processed for peroxidase-antiperoxidase immunohistochemistry. Within the inferior and superior cerebellar peduncles, fine-beaded fibers are evident and a beaded plexus of fibers is present in all the cerebellar nuclei. In the overlying cerebellar cortex, CCK-positive mossy fiber rosettes are present in all lobules, where their morphology varies from simple enlargements to more complex rosettes. However, their distribution varies particularly in vermal lobules II, III, VII, and IX where they are organized in parasagittal bands. Climbing fibers that are positive for CCK are present in very restricted areas of vermal lobules IV, VII, and VIII. After colchicine pretreatment, CCK-positive cell bodies are seen in restricted regions of the posterior interposed and fastigial nuclei as well as within several precerebellar nuclei known to give rise to mossy fibers. Such nuclei include the lateral cuneate nucleus, the nucleus prepositis hypoglossi, the nucleus reticularis lateralis, the nucleus raphe obscurus, the paramedian reticular nucleus, the nucleus reticularis gigantocellularis, and the medial vestibular nucleus. To localize the brainstem origin(s) of the CCK fibers in the cerebellum, a double-label paradigm employing a retrograde tracer and CCK immunohistochemistry was used. These experiments indicate that CCK mossy fibers originate primarily within the lateral cuneate nucleus, the perihypoglossal complex, and the lateral reticular nucleus. Some also originate within the medial vestibular nucleus and the nucleus reticularis gigantocellularis. In addition, double-labeled cell bodies are present within the caudal medial accessory inferior olive, the likely source of the CCK-positive climbing fibers. These data indicate that specific populations of climbing fibers and mossy fibers may utilize CCK to alter the firing rate of their target neurons.     

Acoustic brainstem nuclei express Fos after flurothyl-induced generalized seizures in rats

The inferior colliculus (IC) plays a key role in modulating audiogenic seizures (AS) in rats. We investigated whether acoustic brainstem nuclei express Fos-like immunoreactivity (FLI) after flurothyl-induced generalized seizures in rats. Compared to controls, experimental animals showed significantly (P<0.05) more FLI in the dorsal and external cortex of the IC, as well as in the medial part of the medial geniculate body (MGB), perigeniculate area, and dorsal cochlear nucleus. No significant increase of FLI was observed in the central nucleus of the IC, ventral and dorsal parts of the MGB, dorsal nucleus of the lateral lemniscus, or ventral cochlear nucleus. Because this pattern of FLI closely resembles that observed after AS in previous studies, these results suggest that Fos expression in acoustic brainstem nuclei is not specific for AS.     

Serotonin turnover in discrete hypothalamic nuclei and mesencephalic raphe nuclei of young and adult spontaneously hypertensive rats

Serotonin levels and turnover were analyzed in discrete forebrain and mesencephalic nuclei of young (4-week-old) and adult (14-week-old) spontaneously hypertensive rats and age-matched normotensive control Wistar Kyoto rats. Most changes observed were age-dependent, and occurred only in young, early hypertensive rats. Both serotonin levels and the accumulation rate of 5-hydroxy-tryptophan after L-amino acid decarboxylase inhibition were higher in the nuclei periventricularis and paraventricularis of the hypothalamus of young hypertensive rats than in controls. In addition, 4-week-old spontaneously hypertensive rats showed higher 5-hydroxytryptophan accumulation rates in the nuclei supraopticus and dorsomedialis of the hypothalamus than controls. The only difference in serotonin metabolism found in adult hypertensive rats was high serotonin concentration in the median eminence of the hypertensive animals. Our results suggest the presence of anatomically specific, age-dependent alterations in serotonin metabolism, localized to selected hypothalamic nuclei in young hypertensive rats. These data support a role for the hypothalamic serotonin in the development of the spontaneous (genetic) hypertension in the rat.     

Strain differences in pituitary beta-endorphin and ACTH content in inbred mice

The whole pituitary contents of beta-endorphin and ACTH were found to vary widely among 5 inbred strains of mice. beta-endorphin values were 2.5-fold different and ACTH values 1.5-fold. Strains low in beta-endorphin were also low in ACTH. The existence of genetic differences raises the possibility that there exist, or can be developed, strains with extremely low or high levels of these peptides that would aid research directed at elucidating the physiology of opioid peptides.     

Somatostatin in catecholamine-rich nuclei of the brainstem

Somatostatin (SRIF) concentrations in catecholamine-rich nuclei of the rat brainstem were measured by radioimmunoassay. The study was performed both in control or sham operated animals and after transecting the major projections of hypothalamic SRIF-containing neurons. Concentrations of the peptide were found to be relatively high in the locus coeruleus, the parabrachial nucleus and the nucleus of the solitary tract; they were intermediate in the lateral reticular nucleus (A1 cell group) and low in the substantia nigra. Transection of hypothalamic periventricular efferents resulted in a 58% depletion of SRIF content in the locus coeruleus, while concentrations of the peptide in other areas were unaffected. Transection of the medial forebrain bundle at the level of the lateral hypothalamus decreased SRIF content by 55% in the substantia nigra, but not in the other nuclei tested. It is concluded that the hypothalamus contributes significantly to the somatostatinergic innervation of the locus coeruleus and the substantia nigra, whereas SRIF in the other nuclei is intrinsic or originates outside the hypothalamus.     

Norepinephrine content of discrete brain nuclei in acutely and chronically stressed borderline hypertensive rats

Forty-three male borderline hypertensive rats were subjected to either 3 days, or 4, 10, or 16 weeks of daily stress. An additional 43 animals served as unstressed, age-matched controls. At the end of study, animals were sacrificed, brains were removed, and cardiovascularly-important nuclei in the brainstem and hypothalamus were removed by micropunch. Assays revealed that norepinephrine (NE) levels were initially elevated in the brainstem in animals stressed for 3 days. As stress continued, NE levels were significantly lower in the brainstem, and eventually in the hypothalamus, of stressed animals. The relationship of these observations to environmentally-induced hypertension is discussed.     

Calcium and myokymia of brainstem origin

In five patients with Guillain-Barré syndrome, clinical myokymia increased and myokymic burst amplification occurred when ionized Ca++ was lowered by hyperventilation. Myokymia decreased when ionized Ca++ was increased after IV infusion of CaCl2. These responses were absent or diminished in the four patients with myokymia due to brainstem lesions, suggesting that the blood-brain barrier impedes the effects of altered serum ionized Ca++ on axonal excitability. Altering serum Ca++ can distinguish peripheral and central myokymia.     

Glutamic acid decarboxylase activity in discrete hypothalamic nuclei during the development of rats

We have assessed the activity (nmol/mg protein/h) of glutamic acid decarboxylase (GAD) in discrete hypothalamic nuclei before and after sexual maturation in the developing female rat. Activity in other brain regions including the cortex, septum and caudate-putamen was also assessed. While there appears to be a general rise (approximately 30%), with age, in GAD activity, the rise is most marked, and highly significant (P less than 0.001), in the anterior portion of the hypothalamus (56%). In contrast, no significant increase of GAD activity was found in the medical basal hypothalamus.     

Increased density of substance P binding sites in specific brainstem nuclei of spontaneously hypertensive rats

Substance P binding sites were quantitated in 16-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats by incubation of brain sections with¹²⁵I-Bolton-Hunter substance P, autodiography and microdensitometry. The maximum binding capacity (B_max) was significantly increased in the dorsal motor nucleus of the vagus, in the hypoglossal, ambiguus and inferior olivary nuclei, and in lobes 9 and 10 of the vermis cerebelli of SHR.     

Cortisol, ACTH, and beta-endorphin after dexamethasone administration in Parkinson's dementia

The dexamethasone suppression test (DST) has been suggested as an effective tool for differentiating between depression and dementia. After administering 1 mg dexamethasone, we measured cortisol, ACTH, and beta-endorphin levels in 32 nondepressed patients with idiopathic Parkinson's disease (PD) (14 also with dementia) and 20 healthy, age-matched controls. Four of the 20 controls, 9 of the 18 with PD alone, and 8 of the 14 with PD and dementia were dexamethasone nonsuppressors (cortisol value greater than or equal to 5 micrograms/100 ml). PD patients without dementia (nonsuppressors) showed higher basal plasma values of cortisol (22.06 +/- 5.30 micrograms/100 ml) compared with the suppressors (13.38 +/- 3.30 micrograms/100 ml). Plasma ACTH and beta-endorphin responded in a coupled way to dexamethasone challenge. Higher basal levels of both peptides were found among PD patients (demented and nondemented), nonresponders to DST. Thus, the DST does not appear to be effective in differentiating between depression and dementia in PD. In addition, PD nonsuppressors showed higher basal values of plasma ACTH, beta-endorphin, and cortisol (similar to patients with major depression). This suggests that although the depression is clinically undetectable, both disorders may share some pathophysiological features at the hypothalamic hypophyseal adrenal level.     

Norepinephrine levels in discrete brain nuclei in borderline hypertensive rats exposed to compound stressors

The borderline hypertensive rat (BHR) appears to be an appropriate model for investigating the role of the environment in producing hypertension. Previous studies have demonstrated that the BHR shows chronic blood pressure elevations to both stress and high salt intake. Other studies suggest that interactions between the brain and kidney play an important role in initiating this hypertension. The central noradrenergic system has been implicated in these effects, especially in the hypothalamus. Because exercise has been found to attenuate stress-induced hypertension in the BHR, the current study sought to examine the impact of stressors paired with exercise (salt intake or stress) with those combining stress and high salt. Male BHR were exposed to either control, salt plus stress, salt plus exercise, or stress plus exercise conditions for either 2 or 6 months, beginning at 2 months of age. Following sacrifice, brain nuclei in the brain stem and hypothalamus were removed using the Palkovits micropunch technique. Punches were analyzed for NE content via liquid chromatography with electrochemical detection. Compared with the control condition, chronic salt plus stress led to reductions in NE content, especially in the hypothalamus. Compared with salt plus stress, the exercise conditions were associated with elevated NE levels, especially in the early phases of exposure to the treatment. The possible role of exercise training in preventing a central nervous system trigger from inducing hypertension in the BHR is discussed.     

Electrical stimulation of the cochlear nerve in rats: analysis of c-Fos expression in auditory brainstem nuclei

We investigated functional activation of central auditory brainstem nuclei in response to direct electrical stimulation of the cochlear nerve using c-Fos immunoreactivity as a marker for functional mapping. The cochlear nerve was stimulated in the cerebellopontine angle of Lewis rats applying biphasic electrical pulses (120-250 muA, 5 Hz) for 30 min. In a control group, bilateral cochlectomy was performed in order to assess the basal expression of c-Fos in the auditory brainstem nuclei. The completeness of cochlear ablations and the response of auditory brainstem nuclei to electrical stimulation were electrophysiologically verified. C-Fos immunohistochemistry was performed using the free floating method. In anaesthetized animals with unilateral electrical stimulation of the cochlear nerve, increased expression of c-Fos was detected in the ipsilateral ventral cochlear nucleus (VCN), in the dorsal cochlear nucleus bilaterally (DCN), in the ipsilateral lateral superior olive (LSO) and in the contralateral inferior colliculus (IC). A bilateral slight increase of c-Fos expression in all subdivisions of the lateral lemniscus (LL) did not reach statistical significance. Contralateral inhibition of the nuclei of the trapezoid body (TB) was observed. Our data show that unilateral electrical stimulation of the cochlear nerve leads to increased expression of c-Fos in most auditory brainstem nuclei, similar to monaural auditory stimulation. They also confirm previous studies suggesting inhibitory connections between the cochlear nuclei. C-Fos immunoreactivity mapping is an efficient tool to detect functional changes following direct electrical stimulation of the cochlear nerve on the cellular level. This could be particularly helpful in studies of differential activation of the central auditory system by experimental cochlear and brainstem implants.