Rosalinda C. Roberts

An electron microscopic study of GABAergic neurons and terminals in the central nucleus of the inferior colliculus of the rat

SummaryNeurons and terminals in the ventral lateral portion of the central nucleus of the inferior colliculus (ICCN) of the rat were labelled immunocytochemically with antisera to GABA or to its synthesizing enzyme, glutamic acid decarboxylase. Four types of GABAergic neuron are described: small, medium-sized and large multipolar neurons, as well as medium-sized bipolar neurons. All sizes of GABAergic multipolar neurons are characterized by highly infolded nuclei, many mitochondria and both asymmetric and symmetric axosomatic synapses. A dense plexus of terminals occurs on the proximal dendrites of GABAergic neurons, and most of these terminals form asymmetric axodendritic contacts. Small GABAergic neurons (diameter < 15 μm) are multipolar, and have a large nucleus to cytoplasm ratio, prominent nucleoli and usually two to five axosomatic synapses per thin section, with the majority of these contacts being symmetric. Medium-sized GABAergic neurons (15–25 μm in diameter) display both multipolar and fusiform shaped somata, have a more abundant cytoplasm than the small neurons and show about ten axosomatic contacts per thin section. Large GABAergic neurons (diameters > 25 μm) have eccentrically located, highly infolded nuclei, abundant cytoplasm and a denser plexus of terminals that form axosomatic synapses than the other cell types. These results indicate that four of the six major cell types in the ICCN are probably GABAergic inhibitory neurons.The axon initial segments of GABAergic neurons in the ICCN all have similar features in that they are contacted by only one or two terminals that form symmetric synapses on their proximal portions and are invested by a glial sheath from 3 to 20 μm from the cell body. Many immunoreactive myelinated axons (approximately 0.5 μm in diameter) are observed and some terminals that arise from these axons form synapses with small neuronal somata. Both these and other labelled terminals are shown to form symmetric synapses. These data suggest a complex circuitry for the GABAergic neurons within the ICCN.

Increased numbers of GABAergic neurons occur in the inferior colliculus of an audiogenic model of genetic epilepsy

The numbers of GABAergic neurons as determined by GAD immunocytochemistry and total neurons as determined from Nissl preparations were counted and classified at the light microscopic level in the inferior colliculus (IC) of the genetically epilepsy-prone rat (GEPR) and the non-epileptic Sprague-Dawley (SD) strain of rat. GAD-positive neurons are abundant in the IC in all 3 subdivisions. Several sizes of multipolar neurons as well as medium-sized bipolar or fusiform neurons are GAD-positive. GAD-positive punctate structures that were interpreted to be axon terminals and transversely-sectioned dendrites and preterminal axons are abundant in the IC of both the GEPR and SD. A dramatic increase in the number of GAD-positive neurons occurs in the GEPR as compared to the SD. This increase is most evident in the middle of the rostrocaudal extent of the IC. Although the increase is statistically significant in all subdivisions of the IC, it is most pronounced in the central nucleus, especially the ventral lateral portion. Within the central nucleus, the increase in the number of GAD-positive neurons is due to a selective increase in the small (200%) and medium (90%) cell body size populations (10-15 micron and 15-25 micron in diameter, respectively). Concomitant with this increase in the number of GAD-positive neurons, an increase in total numbers of neurons occurs as determined from Nissl preparations. A 100% increase in the number of small neurons and a 30% increase in the number of medium-sized neurons occur in the GEPR as compared to the SD rat. The proportion of GAD-positive neurons to total neurons is also increased in the GEPR. Approximately 25% of the neurons in the IC in SD rat are GAD-positive, while about 35% of the neurons in the GEPR are GAD-positive. These data demonstrate an anatomical difference in the IC of the GEPR as compared to the SD which appears to be preferential for the GABAergic system.

The ultrastructure of the central nucleus of the inferior colliculus of the Sprague-Dawley rat

Previous studies from this laboratory have indicated that increased numbers of GABAergic neurons, as well as total neurons, occur in the central nucleus of the inferior colliculus (IC) of genetically epilepsy-prone rats (GEPRs) as compared to non-seizuring Sprague-Dawley rats. Since electron microscopic studies of the IC have not been reported for rats, we wanted to determine the ultrastructure of neurons and their processes in this brain region to serve as a basis for future studies on neuronal circuitry in the GEPRs. Both disc-shaped and stellate types were found for each of three size categories: large, medium and small. Thus, six types of neuron were distinguished by differences in somatic size, shape, organelles and dendritic orientation. Large neurons (longest diameter greater than 25 micron), which are the least frequent cell type, contained vast perikaryal cytoplasm, eccentrically located nuclei and abundant granular endoplasmic reticulum (GER) adjacent to the nucleus as well as clustered in the cytoplasm; many axosomatic, symmetric synapses were present. Medium-sized neuronal somata (15-25 micron in diameter) had smooth as well as infolded nuclear membranes and clusters of GER in their cytoplasm but no GER adjacent to the nucleus; synapses were sparse along the surface of their somata. Small neurons (10-15 micron in diameter), which are the most frequent cell type, had scant perikaryal cytoplasm, usually infolded nuclei, frequently two nucleoli, and few or no stacked cisternae of GER in the perikaryal cytoplasm; only infrequent axosomatic synapses were found. Based on previous retrograde and immunocytochemical studies, most large disc-shaped and stellate cells project to the medial geniculate body and are probably excitatory, but some large stellate neurons have been shown to be GABAergic and it is doubtful that such neurons participate in this projection. A dense plexus of terminals that form symmetric synapses covers the soma and proximal dendrites of large neurons, and may provide a strong GABAergic inhibition of this type of projection neuron. Small and medium-sized disc-shaped cells also project to the thalamus but they lack this dense axosomatic plexus. The stellate cells from these same two size categories probably do not project to the thalamus and may be GABAergic local circuit neurons. Other ultrastructural features of IC neurons that were analysed include dendrites, dendritic spines, axon hillocks, initial segments and terminals, as well as the laminae of myelinated axons. Dendrites were either beaded or smooth and few spines were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Anatomical and behavioral analyses of the inheritance of audiogenic seizures in the progeny of genetically epilepsy-prone and Sprague-Dawley rats

Our previous studies have shown an increase in the number of GABAergic and total neurons in the inferior colliculus (IC) of the genetically epilepsy-prone rat (GEPR-9) as compared to the non-seizing Sprague-Dawley (SD) rat. To determine whether an increase in neuron number in the IC is genetically associated with seizure behavior, seizing and non-seizing offspring of GEPR-9 and SD progenitor strains were studied as well as offspring from backcrosses made with F1 and either GEPR-9 or SD rats. In addition, the ontogeny of seizure behavior was studied in seizing rats from these same backgrounds. The development of seizure behavior in GEPR-9s was shown to be dependent on age and the number of exposures to sound stimulus up until the age of 9 weeks. The F1 and F2 generations displayed different audiogenic seizure profiles than those of the two progenitor strains. In the F1 generation, the ratio of seizing to non-seizing rats was always greater than 3:1, and the distribution of seizure scores was similar for males and females. In addition, the off-spring from backcrosses made with F1 rats (high or low seizing) and GEPR-9s displayed maximal audiogenic response scores (ARS) of 9, a characteristic of the GEPR-9s used in this study. The results of these genetic studies indicate a polygenetic inheritance of this autosomal dominant trait of audiogenic seizure susceptibility. For the quantitative study of neuronal density in the IC, neurons were counted from cresyl violet-stained preparations from seizing and non-seizing F1 and F2 rats, backcrosses from different categories and age-matched SD rats. Statistically significant increases in the number of both small (70% increase) and medium-sized (14% increase) neurons occurred in the high seizing animals (ARS = 7-9) as compared to either the non-seizing F2 or SD rats. In addition, a significant increase in the number of small neurons (77% increase) occurred in the high seizing offspring of the F1 X GEPR-9 backcross as compared to that of the non-seizing offspring of the F1 X SD backcross. The data from 25 rats generated a 0.9 coefficient of linear correlation between ARS and the number of small neurons. The results from the anatomical studies suggest that the inheritance of audiogenic seizures appears to be closely linked to the increase in cell number. Therefore, the increase in cell number in the IC may be an important determinant of seizure behavior for GEPR-9s.

IV. Anatomical changes of the GABAergic system in the interior colliculus of the genetically epilepsy-prone rat

The number of GABAergic neurons as determined by GAD immunocytochemistry and total neurons as determined from Nissl preparations were counted and classified at the light microscopic level in the inferior colliculus (IC) of the genetically epilepsy prone rat (GEPR) and the non-epileptic Sprague-Dawley (SD) strain of rat. GAD-positive neurons are abundant in the IC and a significant increase in the number of GAD-positive neurons occurs in the GEPR as compared to the SD in all three subdivisions. However, the most pronounced difference occurs in the ventral lateral portion of the central nucleus, where there is a selective increase in the small (200%) and medium-sized (90%) GABAergic somata (10-15 microns in diameter and 15-25 microns in diameter, respectively). As determined from Nissl preparations an increase in total numbers of neurons also occurs. Thus, a 100% increase in the number of small neurons and a 30% increase in the number of medium-sized neurons occur in the adult GEPR as compared to the SD rat. A statistically significant increase in the numbers of small neurons also occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found, and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compensatory to the seizure activity, but may either be genetically programmed or be a failure of cell death. Based on other studies of genetic models of epilepsy, we propose that the additional GABAergic neurons may disinhibit excitatory projection neurons in the IC.

Increased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone rat

To determine if the increase in the number of neurons observed in the inferior colliculus (IC) of the adult genetically epilepsy-prone rat (GEPR) as compared to the Sprague-Dawley rat was present in the young GEPRs prior to the time at which seizure activity commences, brains from both types of rats 4-10 days of age were studied. A statistically significant increase in the numbers of small neurons occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found in the GEPR as compared to the Sprague-Dawley rat and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compensatory to the seizure activity, but is genetically programmed.

The ultrastructure of the central nucleus of the inferior colliculus of the genetically epilepsy-prone rat.

The inferior colliculus of the genetically epilepsy-prone rat (GEPR) was examined at the ultrastructural level to determine if any abnormalities exist in the inferior colliculus of the GEPR as compared to the non-epileptic Sprague-Dawley rat. Both routine electron microscopic preparations and glutamate decarboxylase (GAD) and GABA immunocytochemical preparations were examined in the GEPR and compared to previous studies from this laboratory that described the normal ultrastructure of the Sprague-Dawley rat. Cell counts from 2 micron semi-thin sections confirmed our previous observations that showed a large, significant increase in the number of neurons in the inferior colliculus of the GEPR as compared to the Sprague-Dawley rat. Many of the small neurons in the inferior colliculus of the GEPR were found to be smaller than those in the inferior colliculus of the Sprague-Dawley rat. Moreover, the small neurons in the GEPR were frequently clumped in clusters of 3-5. Several ultrastructural abnormalities present in the inferior colliculus of the GEPR have been observed at epileptic foci or in brain regions along the pathway of seizure spread in other experimental models of epilepsy. These changes included the presence of dendrites which are almost completely devoid of organelles, hypertrophy of glial processes, and terminals that contain either swollen vesicles or very few vesicles. Other features that were frequently observed in the GEPR but were rarely found in preparations of Sprague-Dawley rats included an abundance of extra membranes, whorl bodies and multivesicular bodies within somata, dendrites and axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cartilage Metabolism during Growth Retardation following Irradiation of the Head of the Neonatal Rat

Abstract The heads of 2-day-old male and female rats were irradiated with a single dose of 600 rads X irradiation, a dose which is known to stunt body weight, tibial length, and tail length, in order to ascertain its effect on synthesis by cartilage of sulfated proteoglycans, DNA, chondroprotein, and collagen as determined by utilization of [35S]sulfate, [Me-3H]thymidine, [1-14C]leucine, and [3,4-3H]proline, respectively. Data have been collected at 20-21, 23, 41-45, and 70-71 days of age. In comparison to controls, growth in body weight, tibial length, and tail length was significantly retarded in irradiated rats of both sexes. Although slow catch-up growth was observed with respect to tail length in both sexes and tibial length in females, a significant deficit in body weight in irradiated rats in both sexes remained at 70-71 days. Cartilage metabolism as evidenced by incorporation of the labeled substances showed no significant disturbance just prior to weaning (20-21 days) or after completion of the principal growth surge (70-71 days). Reduced sulfate and thymidine incorporation attributable to a brief period of undernutrition associated with weaning occurred in head-irradiated rats immediately following weaning (23 days). Increased isotope incorporation occurred at 41-45 days of age in cartilage of irradiated rats incubated with labeled sulfate, leucine, and proline; it did not increase with labeled thymidine. We conclude that neonatal head irradiation slows the rate of growth through the age of most rapid postnatal growth in normal rats. The pattern of cartilage metabolism during this time can be the result either of stimulation by a factor other than somatomedin, or selective inhibition of cartilage thymidine incorporation acting in combination with somatomedin.

432 CHONDROCYTE FUNCTION IS DISTURBED IN RATS WITH STUNTED GROWTH FOLLOWING NEONATAL X-IRRADIATION LIMITED TO THE HEAD

Stunted growth after irradiation of the head only of the neonatal rat is unexplained. Prior studies show no relationship to food intake and no response to growth hormone and/or thyroxine. Male and female rats were X-irradiated with 600 rads limited to the head at 2 days of age. Controls were non-irradiated littermates. Irradiation resulted in retarded growth of body weight and tail length beginning prior to weaning; catch-up growth did not occur. Costal cartilage at ages 21 days (d), 40 d and 70 d was incubated 20 h at 37C in serum-free medium (M) consisting of phosphosaline buffer, pH 7.4, amino acids, glucose and antibiotics or in medium with 10 percent pooled normal rat serum (MS). 35S-sulfate (S), 3H-thymidine (T), 14C-leucine (L) or 3H-proline (P) were added to media. In M irradiated rats had significantly increased incorporation of P at 21 d and 40 d and L at 40 d; there was no change in S. In MS irradiated rats had significantly increased incorporation of S and L at 21 d and 40 d. T incorporation was not increased over controls at any age in M or MS. We conclude that in stunted head-irradiated rats synthesis in cartilage of sulfated proteoglycans and collagen is increased and/or hyperresponsive to normal serum. That T incorporation is not increased is compatible with the possibility that failure in catch-up growth of bones after head irradiation results from inhibition of mitosis of chondrocytes.