Betty L. Hamilton

GABA and specific GABA binding sites in brain nuclei associated with vagal outflow

Abstract GABA levels and specific (3H) GABA binding were determined in several nuclei of cat brain. Since previous pharmacological studies (DiMicco et al. [3]) suggested that nucleus ambiguus (NA) may be the site of a GABA-receptor mediated inhibition of vagal outflow to the heart, we were interested in comparing the GABA content and density of (3H) GABA binding in NA with that of other nuclei known to contain GABAergic synapses. The GABA content of NA was 21.2 ± 2.4 nmol/mg protein, similar to that found in the caudate nucleus (28.4 ± 2.9 nmol/mg protein) and 2.5 fold higher than the GABA content of the surrounding reticular nuclei. In frozen-thawed and Triton X-100 treated membranes prepared from NA, specific GABA binding was 98 ± 28 fmol/mg protein when measured using 30 nM (3H) GABA. This was more than 3 fold higher than binding obtained in surrounding reticular tissue and approximately half the value obtained in substantia nigra. GABA content of paired right and left NA was nearly equal; however specific GABA binding of paired right and left NA differed markedly, with the right NA usually exhibiting greater specific binding than the left NA. Retrograde degeneration of vagal fibers of NA by intracranial sectioning of the right vagal trunk decreased the asymmetry in GABA binding of paired right and left NA. Asymmetry was also noted in the percent of the reflex-induced bradycardic response mediated by each vagus nerve. These results suggest that GABAergic synapses may be present in NA, and that some of the postsynaptic receptors for GABA may be associated with vagal efferents. The bilateral asymmetry in the physiological reflex-induced response coupled with the bilateral asymmetry in GABA binding in NA suggests that the degree of vagal activity emanating from NA may be determined by the density of GABA receptors.

Importance of CNS GABAergic mechanisms in the regulation of cardiovascular function

Abstract The purpose of these studies was to assess the role of CNS GABA in regulating central parasympathetic and sympathetic outflow to the cardiovascular system. Studies were performed by monitoring arterial pressure, cardiac rate and rhythm in anesthetized cats treated with drugs that either counteract the CNS effects of GABA (picrotoxin and bicuculline) or activate CNS GABA receptors (GABA and muscimol). The agents were given intravenously, intra-arterially (vertebral artery), intracerebroventrically (lateral and third cerebroventricles, and fourth cerebroventricle), and by microinjection into specific brain stem nuclei. The antagonist drugs were found to produce central sympathetic stimulation by acting primarily in forebrain areas, and central parasympathetic stimulation by acting primarily in the medulla, specifically at nucleus ambiguus. The antagonist drugs were also found to act centrally to enhance reflex-induced vagal bradycardia. The agonist drugs were found to decrease sympathetic and parasympathetic outflow, and to counteract reflex-induced changes in heart rate and arterial pressure. The effect on parasympathetic outflow occurred in the medulla whereas an effect to counter sympathetic activity occurred in both forebrain and hindbrain regions. Results obtained suggest the existence of a tonically active GABAergic system present in the forebrain which exerts inhibitory control over sympathetic activity influencing arterial pressure and heart rate. Results also suggested the presence of a tonically active GABAergic system in the CNS that exerts inhibitory control over spontaneously active parasympathetic neurones in nucleus ambiguus. The latter system could be considered as an example for “disinhibition as an organizing principle in the nervous system”.

Three-dimensional computer reconstruction and display of neuronal structure

Shaded, three-dimensional images of microscopic neuronal structures are generated using a special computer graphics system known as THREAD (Three-Dimensional Reconstruction Aand Display). This system uses both a computer and its associated image processing hard-ware and specialized computer software to produce both line-drawings (contour-grams) and shaded images obtained from 35 mm film negatives of cross-sectional images of microscopic neuronal structures. The resulting image is displayed on a TV monitor in either color or black and white and can be rotated to view all surfaces. Structural volume and planar surface areas are also evaluated during the procedure. Examples of a brainstem region and of a neuron nucleolus are presented.

Evidence for a role of central serotonergic neurones in digitalis-induced cardiac arrhythmias.

EVIDENCE indicates that digitalis drugs administered intravenously increase central sympathetic outflow and that this in turn results in cardiac arrhythmias1–6. The central nervous system transmitter(s) that mediates this effect is not known. According to Saito et al., noradrenaline, the primary transmitter studied so far, is not involved in the case of guinea pigs7. 5-Hydroxytryptamine (5-HT) however, may be an important chemical mediator regulating central sympathetic outflow8,9, and it is involved in the respiratory arrest induced in rats by intravenous administration of digitoxigenin10. We now report evidence that suggests that brain 5-HT is involved in digitalis-induced cardiac arrhythmias.

Stress-induced changes in the function of the parasympathetic nervous system are mimicked by blocking GABA in the CNS of the cat

The purpose of this study was to determine the effect of blockade of receptors for gamma-aminobutyric acid (GABA) in the forebrain, on vagal activity to the stomach and heart. This was done by injecting bicuculline (50 micrograms) into the lateral ventricle of the brain and restricting the drug to the forebrain ventricles by cannulating the cerebral aqueduct. Studies were performed in chloralose-anesthetized cats and gastric motility was monitored using extraluminal force transducers, sutured to the antrum and pylorus. Cardiac vagal activity was determined by noting the sinus bradycardia that developed from activation of the baroreceptor reflex induced by phenylephrine. Administration of bicuculline into the lateral ventricle of 7 animals produced increases in the minute motility index of 5.3 +/- 0.8 (antrum) and 13.9 +/- 2.1 (pylorus). This was associated with inhibition of baroreceptor-induced vagal bradycardia (i.e. -38 +/- 6.4 beats/min before bicuculline and -7.7 +/- 5.7 beats/min after bicuculline). These data indicate that a GABAergic mechanism in the forebrain may be important for controlling vagal outflow to both the stomach and the heart.

A Device for the Precise Transfer of Serial Sections for Electron Microscopy

A device based on a standard stereotaxic instrument, in which a Formvar-dipped loop carrying serial thin sections, and a rotatable grid holder are mounted in separate electrode holders, thus permitting accurate positioning of each with respect to the other and the precise transfer of the sections to the grid, is described and illustrated.

GABAergic transmission sites important in cardiovascular function

Abstract Various brainstem sites have been shown to be important for mediating changes in cardiovascular function induced by GABA agonists and antagonist drugs. These will be discussed in terms of their cellular morphology, cytoarchitectonics, efferent and afferent connections, and finally, their exact stereotaxic locations in a living cat. This will encompass both a review of the existing literature and some unpublished work from our laboratory. The general framework for this presentation was based on computerized axial tomographic scans of the head of a cat held in true stereotaxic position. These scans were correlated with both gross slices and histological sections to provide better coordinates for approaching brain stem, particularly medullary, structures stereotaxically. The individual areas include the ventral surface of the medulla oblongata, the chemosensitive areas of Mitchell [30], Schlafke [34], and Loeschske [28]; the lateral reticular nucleus; nucleus ambiguus; nucleus of the tractus solitarius; dorsal nucleus of the vagus; nucleus raphe dorsalis and the periaqueductal gray matter.

Modification of the Carmen-Faull-Pullar Racks for Free-Floating Serial Sections

The original staining racks designed by Carmen et al. (Stain Techn., 43: 157-60) have been redesigned to use inside plates of 0.06 inch thickness and outside (top and bottom) ones of 3/16 inch. The greater thicknesses permit freer circulation about the sections and avoid the need for outer clamps to hold the assembly together. As in the original racks acrylic sheeting has been used, but with 25 × 45 mm holes for sections in each plate. Ordinary fiberglass window screening was cemented to one side of each plate. The assembly of 12 inner and the 2 outer plates was held together by 2 bolts made of 1/4 inch acrylic rod. Since clamps on the edges of the assembly were not needed, smaller staining dishes could be used, with coincident economy in volume of staining solutions.

A simplified decerebration technique in cats and its applicability to neuro-cardiovascular drug studies.

interrupted by periods of exaggerated gnawing and sniffing. On the other hand, when the SP was injected either immediately dorsal to, or anterior to the zona reticulata the animals always responded with a postural bias or turning in the opposite direction. SP injections made posterior to the zona reticulata, or injections of saline given into any of these regions, failed to elicit any form of stereotyped behaviour. Although there was considerable variation between animals in the striatal concentrations of dopamine (range 0 . 9 1 . 9 ~ g g ~ wet wt) and HVA (range 0.321.02 pg g-’ wet wt), bilateral differences in the striatal contents of these substances in saline-injected controls were less than 10%. In the low dose employed here SP had no effect on the steady-state level of dopamine in the striatum. However, Table 1 clearly indicates that SP-induced contralateral turning was accompanied by a pronounced increase in ipsilateral striatal HVA concentrations (P < 0.001), and that turning towards the injected side was characterized by a significant decrease in this metabolite ipsilaterally (P < 0.001). SP injections at sites which did not elicit turning caused no significant change in striatal HVA concentrations. Since the neurons in the substantia nigra are known to be susceptible to excitation by electrophoreticallyapplied SP (Davies & Dray, 1976), it seems reasonable to speculate that the SP-induced contralateral turning observed here results from the unilateral stimulation of the ascending nigrostriatal dopaminergic pathways, especially as the HVA concentrations in the corresponding striata were raised accordingly. The critical placement of the SP injection is also interesting, because it emphasises that the application of SP to other cells in the neighbourhood of the zona reticulata ultimately reduces impulse traffic in the ascending dopaminergic neurons on that side and leads to ipsilateral circling. The nature of the synaptic connections in the region of the substantia nigra is not fully understood, but it is possible that in this case SP may be acting indirectly upon the zona reticulata through the intervention of an inhibitory interneuron. Although the mode of action of SP remains to be determined, this preliminary study serves to illustrate that SP possesses demonstrable physiological activity when injected in small doses into the substantia nigra, an area of the brain which possesses high concentrations of SP and which could conceivably utilize this peptide as an endogenous synaptic transmitter. January 12, 1977

An oriented plastic slicing guide for facilitating stereotaxic interpretations in brain sections.

A rigid plastic cutting guide for whole brains in which the interior conforms to the contours of the interior base of the cranial cavity was molded from plastic that sets at room temperature. The mold was poured in a glass staining dish surrounding a wax-coated plaster cast of the floor of the skull. Both the casting and the molding processes were carried out in a stereotaxic apparatus to insure proper alignment. The molded box supports the brain in correct stereotaxic alignment, and vertical slits sawed at 5 mm intervals along the width of the box allow the passage of a blade through the brain. The resulting tissue slabs are of uniform thickness and can be easily correlated with the coronal planes shown in stereotaxic atlases. This inexpensive cutting device reduces the number of thin sections lost between tissue blocks, an important consideration when tracking degenerating nerves.