Increased branching of basal dendrites on pyramidal neurons in the occipital cortex of homozygous Brattleboro rats in standard and enriched environmental conditions: A Golgi study

As a continuation of our previous reports in a series of studies on the brain of Brattleboro rats, the branching of basal dendrites of pyramidal neurons in the upper layers of the occipital cortex was quantified in three groups of male heterozygous and homozygous Brattleboro rats. One group raised in standard environmental conditions was killed at 60 days of age, and another from standard conditions was killed at 90 days of age. A third group from enriched environmental conditions was killed at 90 days of age after 30 days of enrichment. Comparing the two types of Brattleboro rats, the homozygous rats showed significantly more total dendritic branching segments per neuron in both the 60-day-old standard condition group and the 90-day-old enriched group. A similar measure (segments per primary branch) was also significantly greater in homozygous than in heterozygous rats at 60 days of age. In the 90-day-old enriched group, the homozygous rats showed a trend toward more segments per primary branch than the heterozygous rats. The results suggest that the complete absence of vasopressin produces metabolic effects which, at certain ages or in certain environmental conditions, increase the branching of basal dendrites of pyramidal neurons in the upper layers of the occipital cortex.     

Increase in thickness of cerebral cortex in response to environmental enrichment in brattleboro rats deficient in vasopressin

Changes in cortical thickness were observed in male Brattleboro rats killed at 60 and 90 days of age from standard environmental conditions, and at 90 days of age after 30 days in an enriched condition. When 60- and 90-day-old standard animals were compared, both homozygous Brattleboro rats with diabetes insipidus and also heterozygous nondiabetic rats showed reduction in cortical thickness with age. When 90-day-old enriched animals were compared with the 90-day-old standard group: the homozygous rats with enrichment showed statistically significant thickening in frontal, parietal, and occipital cortices, and the heterozygous rats showed statistically significant thickening with enrichment in parietal and occipital regions. In both heterozygous and homozygous Brattleboro rats the most significant increases were in the occipital cortex. These results are in general agreement with previously reported developmental and enrichment studies in the normal Long-Evans strain of rats, with an important difference: both heterozygous and homozygous animals showed greater and more generalized response to enrichment. We speculate that this greater responsiveness to environmentally induced cortical thickening in the Brattleboro rats is due to increased arousal, and it may be related to alterations in norepinephrine concentration and/or turnover associated with absence of, or abnormalities in, vasopressin in the brain.     

Correlation between water intake and dendritic branching in homozygous Brattleboro rats: A Golgi study

This report is the fourth in a series on the brains of three groups of male Brattleboro rats. The present study concerns only homozygous Brattleboro rats. One group raised in standard environmental conditions was killed at 60 days of age, another raised in standard conditions was killed at 90 days of age, and a third group raised in standard conditions for 60 days was killed at 90 days of age after 30 days of environmental enrichment. Water intake (measured between 33 and 40 days of age) was compared with the number of branching segments on basal dendrites of pyramidal neurons in the upper layers of the parietal and occipital cortices. A significant negative correlation between water intake and the number of dendritic branching segments per primary branch was found in both parietal and occipital cortices in the 90-day-old standard condition group. In contrast, the 90-day-old enriched group showed a significant positive correlation between water intake and segments per primary branch in the parietal cortex, and a similar but nonsignificant response to enrichment in the occipital cortex. These results may reflect a cumulative effect of the congenital absence of vasopressin which is apparent by 90 days of age and which is modified by environmental enrichment. In the light of our previous data linking vasopressin and dendritic branching, we speculate that the neuropeptide, oxytocin, may be a physiologic correlate linking the behavioral parameter (water intake) and the anatomic parameter (dendritic branching) in homozygous Brattleboro rats.     

Expression of somatostatin receptors is impaired in the cerebellum of developing Brattleboro rats

Somatostatin (SRIF) receptors are expressed in the external granule cell layer of the rat cerebellum during early postnatal life. The aim of the present study was to investigate the distribution and biochemical characteristics of SRIF binding sites in the cerebellum of homozygous (vasopressin deficient) Brattleboro rats, which exhibit a selective impairment of their granule cell layer. This study has been conducted in 13-day-old rats by means of membrane-binding assay and autoradiography using [125I-Tyr0,DTrp8]S14 as a radioligand. In the cerebellum of homozygous Brattleboro rats, Scatchard plot analysis revealed the existence of a single class of SRIF receptors with similar Kd values as in Long-Evans or heterozygous Brattleboro rats (180-200 pM). Conversely, a marked reduction of the concentration of SRIF binding sites was observed in Brattleboro rats as compared to heterozygous or Long-Evans rats. In homozygous Brattleboro rats, autoradiographic studies revealed that the concentration of SRIF receptors was reduced in all lobules of the cerebellum as compared to Long-Evans. In addition, the magnitude of the decrease of receptor concentration was greater than the loss of granule cells observed in the homozygous Brattleboro rat. These results indicate that the expression of SRIF receptors by immature granule cells of the cerebellum is markedly reduced in Brattleboro rats. Whether the impairment of SRIF receptors in diabetes insipidus rats can directly be ascribed to vasopressin deficiency remains to be determined.     

Impaired development of individual cerebellar lobules in the diabetes insipidus Brattleboro rat

The size differences in the midsagittal cerebellum, its individual lobules and its layers are studied in homozygous diabetic (HOM) and heterozygous Brattleboro rats on postnatal days 12, 24 and 180. As has been reported for total cerebellar weight, no significant reduction in cerebellar size in HOM rats occurs at day 12, whereas from day 12 onwards the cerebellar growth in HOM rats is significantly stunted. The HOM cerebellum shows a particular structural etiology: not all cerebellar lobules are equally affected and in 180-day-old rats a sexually dimorphic effect is found.     

Amygdala and pyriform cortex kindling in vasopressin deficient rats (Brattleboro strain)

Homozygous and heterozygous Brattleboro rats and Long--Evans control rats were subjected to repeated electrical stimulation of the amygdala or pyriform cortex in a kindling paradigm. The homozygous Brattleboro group stimulated in the amygdala was retarded in its kindling rate relative to heterozygous Brattleboros and Long--Evans controls. The retarded kindling rate of the homozygous Brattleboros stimulated in the amygdala is attributed to a delay in seizure development at stages 1 and 2 which suggests that vasopressin may be necessary for normal kindling from the amygdala to take place.     

Lack of the suckling-induced prolactin release in homozygous Brattleboro rats: the vasopressin-neurophysin-glycopeptide precursor may play a role in prolactin release

Suckling stimulus did not induce significant release of prolactin (PRL) in lactating homozygous Brattleboro rats, whereas it did it in heterozygous animals. Daily treatment of homozygous rats with vasopressin partly restored the PRL response to suckling. Findings suggest that vasopressin-neurophysin-glycopeptide precursor missing in homozygous Brattleboro rats may play a role in suckling-induced PRL release.     

Ontogeny of Diabetes Insipidus in the Brattleboro Rat: Morphological and Physiological Correlates

The homozygous Brattleboro rat is a mutant of the Long Evans rat which fails to produce assayable quantities of vasopressin. Somata of supraoptic magnocellular neurons from adult Brattleboro rats are hypertrophied relative to those from normally hydrated adult Long Evans rats. We have investigated, by light microscopic morphometric analysis of immunoperoxidase-labelled vibratome sections, the postnatal growth of magnocellular neurons in normal Long Evans rats, and the relative hypertrophy of these cells in Brattleboro rats. Morphometric analysis of the somata of immunoidentified oxytocinergic and vasopressinergic supraoptic magnocellular neurons from Long Evans rats aged between 1 and 140 days postnatum revealed that their somata increased rapidly in size only after 14 days; a time that coincides with the start of weaning, with a transient increase in serum osmolality, and with the onset of ability to produce hyperosmotic urine. Oxytocin- and vasopressin-containing neurons in Long Evans rats achieved adult dimensions by 45 days postnatum. By contrast, somata of oxytocin neurons in the Brattleboro rat already showed significant hypertrophy relative to those in Long Evans rats at 7 days postnatum; hypertrophy continued until at least 140 days. The hypertrophy in the Brattleboro rat at 7 days was associated with markedly raised serum osmolality relative to that of age-matched Long Evans rats between 1 and 14 days.     

Increased concentrations of immunoreactive cathepsin D in supraoptic nucleus of the Brattleboro rat

The distribution of cathepsin D, an aspartyl endopeptidase, was measured in selected, discrete nuclei of the forebrain of the Brattleboro rat by means of microdissection and radioimmunoassay. The results indicate that cathepsin D is widely distributed, but in varying amounts among nuclear groups in this region of the brain. High concentrations were detected in the supraoptic and paraventricular nuclei. In studies of the vasopressin-deficient Brattleboro rat, an increased content of cathepsin D in the supraoptic nucleus was observed compared to the heterozygous control. No differences were detected between homozygous and heterozygous Brattleboro rats in the caudate, medial preoptic, suprachiasmatic or paraventricular nuclei or globus pallidus. These results raise the possibility that brain cathepsin D may be involved in the physiological events related to fluid homeostatis.     

Anterior neocortical kindling in vasopressin-deficient rats

Kindling of seizures with stimulation of anterior neocortex was examined in control rats and in Brattleboro rats deficient in arginine-vasopressin (AVP). There were no significant differences between control rats, homozygous Brattleboro rats, and heterozygous Brattleboro rats in the rate and pattern of kindling of generalized seizures. Thus AVP is not critically involved in anterior neocortical kindling.     

Age-dependent changes in adenosine A1 receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats

This study shows the distribution and density of adenosine A1 receptor (A(1)R) within the nucleus tractus solitarii (NTS) of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) from birth to adulthood (1, 15, 30 and 90 days old). The NTS shows heterogeneous distribution of A(1)R in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. A(1)R decrease from rostral to caudal within dorsomedial/dorsolateral subnucleus in 15-, 30- and 90-day-old WKY and SHR. A(1)R increase from rostral to caudal subpostremal subnucleus in 30- and 90-day-old WKY, and in 15-, 30- and 90-day-old SHR. Furthermore, A(1)Rs are increased in SHR as compared with WKY within dorsomedial/dorsolateral in 30- and 90-day-old and within subpostremal of 15-, 30- and 90-day-old rats. Finally, A(1)Rs increase from 1- to 30-day-old rats. Medial/intermediate did not show any changes in A(1)R from rostral to caudal levels, age or strain. In summary, our result highlights the importance of A1 adenosine system regarding the neural control of blood pressure and the development of hypertension.     

Metabolic alterations in the hypothalamus of the Brattleboro rat demonstrated with cytochrome oxidase histochemistry

Metabolic activity in the hypothalamus of homozygous and heterozygous Brattleboro rats and Long-Evans control rats was studied using cytochrome oxidase histochemistry. Increased metabolic activity was observed in the paraventricular nucleus (PVH), supraoptic nucleus (SON) and nucleus circularis (NC) of homozygous Brattleboro rats, and in the PVH of heterozygous rats. These results suggest that the metabolic activity of PVH and SON neurons is altered because of the inability of magnocellular neurosecretory neurons to produce vasopressin. In addition, the hyperactivity of neurons in the NC is probably related to the chronic dehydration present in these animals.     

Kindling of the hippocampus and septum in vasopressin-deficient rats (Brattleboro strian)

Homozygous and heterozygous Brattleboro rats deficient in central vasopressin and Long-Evans control rats were electrically kindled in the ventral hippocampus or lateral septum. The homozygous and heterozygous Brattleboro groups stimulated in the ventral hippocampus or lateral septum kindled significantly faster than the respective Long-Evans control groups. The accelerated kindling rate of the Brattleboro groups is attributed to faster seizure development during the early seizure stages. These results suggest that vasopressin may be involved in the kindling of convulsions from limbic structures where it is normally found.     

Occipital cortical morphology of the rat: Alterations with age and environment

With the use of Golgi impregnation, this study demonstrated dendritic proliferation in layers II and III in the medial occipital cortex of 444- and 630-day-old male rats. Increases occurred in animals housed in either enriched or standard colony conditions. Specifically, third- and fourth-order basal dendrites increased significantly in frequency from 414 to 444 to 630 days of age. This study approximated a longitudinal study because the different age groups were littermates. The finding of an increase in dendritic branching in old age was not new, but our study was the first to note this increase in the rat's cerebral cortex. In a separate study the cortical morphology was examined in 90- and 630-day-old rats which had been living together in a single enriched environment for 30 days. The brains of these rats were compared with littermates living with their respective age groups in standard colony conditions. The dendritic pattern was similar in those two age groups irrespective of environment. Only the sixth order significantly differed, with the frequency of branching being greater in the 630-day-old animals. An increase in cortical thickness in the enriched animals was apparent compared with controls, but the differences were not significant. Because previous results showed the 60- to 90-day-old enriched rats without old companions developed a significantly thicker cortex than standard control littermates, it is possible that when young rats live with old rats they do not interact with their environment as musch as when living with other young rats.     

High, vasopressin-reversible, immunoreactive somatostatin in specific hypothalamic nuclei of rats with diabetes insipidus (Brattleboro rats)

Somatostatin content was studied in brain nuclei and pituitary lobes of homozygous Brattleboro rats lacking vasopressin (DI rats) with and without vasopressin replacement, and their corresponding controls, heterozygous Brattleboro (HZ) rats and Long-Evans (LE) rats. High, vasopressin-reversible, somatostatin levels occurred in DI rats. The changes in somatostatin were localized to the nucleus periventricularis and to brain areas receiving somatostatin projections from this nucleus, including the eminentia mediana. In contrast, vasopressin administration failed to alter somatostatin levels in HZ rats. In brain areas not belonging to the somatostatin periventricular system, the peptide concentration in DI rats was not higher than that of LE controls. High somatostatin levels were present in the intermediate lobe of the pituitary gland. Somatostatin was higher in DI and HZ rats when compared to LE rats. In the posterior pituitary lobe, HZ rats had higher, and DI rats lower, somatostatin than control LE rats. These results suggest that rat brain and pituitary vasopressin and somatostatin systems are interrelated.     

Vasopressin-reversible increase in angiotensin-converting enzyme in specific hypothalamic nuclei of Brattleboro rats

The activity of the angiotensin-converting enzyme (ACE) (kininase II, EC 3.4.15.1) was examined in 5 discrete hypothalamic nuclei of rats lacking vasopressin (homozygous Brattleboro rats, DI, di/di) and their corresponding controls (heterozygous Brattleboro rats, HZ, di/+, and Long Evans, LE, +/+ rats), with and without hormonal replacement with arginine-vasopressin (AVP). DI rats showed a vasopressin-reversible increased ACE activity when compared with LE controls, HZ rats showing intermediate activity. These changes occurred only in the supraoptic and periventricular hypothalamic nuclei, and were absent in other hypothalamic areas studied, including the paraventricular nucleus. These results provide biochemical evidence in support of previous anatomical and physiological data, for an interaction between the brain vasopressin and angiotensin systems in discrete hypothalamic nuclei, and suggest that vasopressin could regulate the formation of brain angiotensin II by modulating the activity of the converting enzyme.     

Rat brain 5'-nucleotidase: developmental changes in myelin and activities in subcellular fractions and myelin subfractions

The activities of 5'-nucleotidase, measured in brain homogenates and myelin isolated from rats at 21, 60 and greater than 90 days of age, were compared to values for two other myelin-associated enzymes, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and carbonic anhydrase. Whereas the activities of all 3 enzymes were higher in brain homogenates from 60-day-old rats than in those from 21-day-old rats, only 5'-nucleotidase increased significantly in specific activity in both homogenates and myelin after the age of 60 days. The ratios of 5'-nucleotidase to the myelin basic and proteolipid proteins in subcellular fractions from adult rat brain suggested that the microsomal fraction was the only fraction containing 5'-nucleotidase levels not attributable to contamination by myelin membranes. Like carbonic anhydrase, 5'-nucleotidase had a greater distribution than CNP into microsomes of adult rats. When purified myelin was fractionated on a density gradient, the specific activity of 5'-nucleotidase was highest in the heaviest subfraction, with recovery of significant activity occurring, however, in all 3 subfractions. In rats over 60 days of age the recovery of 5'-nucleotidase in myelin was almost as high as that of the relatively myelin-specific enzyme CNP, suggesting that myelin may be the predominant, although not exclusive locus of 5'-nucleotidase in the adult rat brain.     

Levels of pro-neo-endorphin/dynorphin-derived peptides in the hypothalamo-posterior pituitary system of male and female Brattleboro rats

Immunoreactivities for leucine-enkephalin and the related opioid peptides α-Neo-endorphin, dynorphin(1–17) and dynorphin(1–) were measured in hypothalamus and poterior-intermediate pituitary extracts from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous(able to produce vasopressin) for daibetes insipidus. In hypothalamus no differences were found in peptide levels among the 4 groups of animals. In contrast, striking, but variable differences were found in posterior intermediate pituitary. In homozygous male animals dynorphin(1–17) immunoreactivity was not significantly different from levels measured in heterozygous male animals. Immunoreactivities for the 3 other peptides, however, showed greatly reduced levels in homozygous male animals compared to heterozygous male animals. Female homozygous animals had greatly reduced levels of all 4 peptides (including dynorphin(1–17) compared to female heterozygous controls. In addition, female heterozygous animals had considerably higher levels of all peptides than male heterozygous animals. In contrast, no sex differences were found in normal Long-Evans rats of the strain from which Brattleboro rats were derived.The following conclusions could be drawn from these findings. (1) Homozygous Brattleboro rats have reduced levels of Leuenkephalin-related opioid peptides in posterior pituitary due to defect or alteration of secretion or turnover but dot due to a defect in biosynthesis. (2) This defect is partially sex dependent and is directly or indirectly linked to the vasopressin deficiency. (3) Since normal rats do not show sex differences in peptides levels, the partial sex dependence of the opioid peptide defect in posterior pituitary seems to be X-chromosomally linked to the vasopressin deficiency.     

Effects of quisqualic acid and glutamate on subsequent learning, emotionality, and seizure susceptibility in the immature and mature animal

To compare the long-term behavioral effects of chronic administration of excitatory amino acids in the mature and immature brain quisqualic acid (QA) and glutamate (GLU) were administered intraventricularly by osmotic pumps over 7 days in 20- and 60-day-old rats. Both age groups received identical dosages of QA or GLU. At age 90 days, all animals were assessed for abnormalities of learning and memory using the Morris water maze, emotionality using the handling test, and seizure threshold using flurothyl inhalation. No significant differences were found in either the water maze or handling test. However, 60-day-old rats receiving QA or GLU had more spontaneous seizures than the 20-day-old rats. In both age groups histological damage following QA and GLU was limited to the ipsilateral hippocampus, was maximum at the site of the catheter tube, and was similar in the two age groups studied. The adverse effects of long-term effects of chronic exposure to excitatory amino acids are similar in the immature and mature brain.     

Age-Dependent Accumulation of Hybrid Vasopressin-Oxytocin Gene Products but not Hybrid Oxytocin-Vasopressin Products in the Endoplasmic Reticulum of Brattleboro Rats

The age-dependence of the incidence of magnocellular neurosecretory neurons containing abnormal accumulations of peptide in the rough endoplasmic reticulum was examined in homozygous Brattleboro rats and in their wild-type Long Evans counterparts. Neurons in which the immunophenotype of the peptide aggregates indicate that somatic cross-over mutations involving the 5' end of the vasopressin gene and the 3' end of the oxytocin gene have occurred, increased with age in homozygous Brattleboro rats, reaching a maximum of 24 cells per hypothalamus (approximately 0.6% of the vasopressin neurons). The increase occurred in both male and female animals but was significantly greater in females. The average incidence of such cells was 6 times greater in the supraoptic than in the paraventricular nucleus. No such cells could be detected in either nucleus of Long Evans rats despite the evidence for hybrid mRNA in these animals. Moreover, no accumulation of peptide translated from the hybrid mRNAs derived from the 5' end of the oxytocin gene and the 3' end of the vasopressin gene could be detected in either Brattleboro or Long Evans animals. These results strongly suggest that the accumulation of peptide in the rough endoplasmic reticulum of vasopressin neurons in homozygous Brattleboro rats is due to an abnormality other than the somatic crossing-over mutation. A second type of abnormal magnocellular neuron with accumulations of peptide in the rough endoplasmic reticulum, in which the immunophenotype of the peptide reveals products derived only from the oxytocin precursor, was present in both Long Evans and Brattleboro rats, but did not increase with age in Brattleboro rats. The incidence of these cells was similar in the supraoptic and paraventricular nuclei.