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.     

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.     

Effect of age and enrichment on certain brain dimensions in Brattleboro rats deficient in vasopressin

As a sequel to our first report in the series of studies on the brains of Brattleboro rats, measurements in caudal diencephalon, subcortical telencephalon, hippocampus, and pyriform cortex were made in three groups each of male heterozygous and homozygous Brattleboro rats. One group raised in standard 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 enrichment. Comparing 60- and 90-day-old standard condition animals, the heterozygous rats showed a significant increase in height of the caudal diencephalon. Comparing 90-day-old enriched animals with the 90-day-old standard group, significant increases occurred in 8 of 12 comparisons. In comparing the two types of Brattleboro rats, heterozygous rats had significantly greater brain dimensions than homozygous rats in width of the diencephalon in the 60-day-old standard group, and in witth of the telencephalon in both the 90-day-old standard and enriched groups. Differences in brain measures between heterozygous and homozygous Brattleboro rats tended to increase with age. Enrichment appeared to prevent this age-related increase in the difference between brain dimensions in the two types of Brattleboro rat. The same pattern was reflected in body weight differences, and it may be due to a greater anabolic response to enrichment in homozygous rats than in heterozygous rats. We suggest that brain abnormalities associated with congenital absence of vasopressin increase with age, and may be ameliorated by repetitive arousal as occurs in enrichment.     

Plasticity in the 904-day-old male rat cerebral cortex

Ten pairs of male Long-Evans rats living in nonenriched environments (3 rats per small cage) were transferred to either enriched environments (10 rats per large cage plus "toys") or nonenriched environments (2 rats per small cage) at 766 days of age. One hundred and thirty-eight days later, at 904 days of age, the cerebral cortical thickness from these animals was measured on projected, 10-micron, thionine-stained, transverse sections. Although the thickness in the enriched rats was greater than in the nonenriched rats in all sections through the frontal, parietal, and occipital cortices, the 4 to 10% differences were statistically significantly different in only the frontal and occipital cortices. Right greater than left cortical thickness differences were not statistically significant in either the enriched or the nonenriched animals by 904 days of age. Neuron and glial counts were made on enlarged photographs of area 18 in the occipital cortex on 6-micron-thick, luxol fast blue-stained sections. No significant differences in cell counts were noted between the enriched and nonenriched animals. No significant differences in neuronal counts were found among 108-, 650- (from previous experiments), and the 904-day-old nonenriched rats. The notable findings were the plasticity of the extremely old, enriched rats' occipital cortex and the lack of the loss of neurons in cerebral cortical area 18, whether or not the environments were enriched. These results showed that the cerebral cortex remained structurally plastic throughout the lifetime of the organism.     

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.     

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.     

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.     

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.     

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.     

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.     

Rat cortical morphology following crowded-enriched living conditions

This experiment studied cerebral cortical morphology in rats living in a crowded-enriched condition. Three groups of 60-day-old, male Long-Evans rats were divided accordingly: 12 rats, 3 per small cage (32 X 20 X 20 cm), standard colony condition; 12 rats in a single, large, enrichment cage with "toys" (70 X 70 X 45 cm), enriched condition; and 36 rats in a large, single, enrichment cage with "toys", crowded-enriched condition. Matched toys for the two enriched cages were changed twice a week at the time of cage cleaning. Measurements on 20-micron, transverse brain sections showed that in both the crowded-enriched and enriched groups the thickness of the medial occipital cortex increased by 4 to 6% compared with the cortex from animals in the standard colony condition. In addition, the crowded-enriched group demonstrated a 4% (P less than 0.05) increase in thickness in area 39 in the left hemisphere compared with the standard control. However, the thickness in area 39 in the crowded group was not significantly different from that of the enriched area 39. These results indicate that the cortex increases in thickness as much with "crowding" and enrichment as with enrichment alone. We hypothesize that diversion through interaction with "toys" mitigates the stress of crowded conditions.     

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.     

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.     

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.     

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.     

High histamine levels in specific hypothalamic nuclei of Brattleboro rats lacking vasopressin

Histamine levels were determined in whole hypothalamus and specific hypothalamic nuclei of male 9-week-old homozygous Brattleboro rats lacking vasopressin and presenting the syndrome of diabetes insipidus (DI rats). These levels were compared to those in heterozygous Brattleboro rats with a partial deficit in vasopressin (HZ rats) and those in Long Evans control rats (LE rats).In whole hypothalamus, histamine levels in DI rats were higher than those found in HZ and LE rats. DI rats showed histamine levels higher than those present in LE rats in the nuclei supraopticus, paraventricularis and suprachiasmatis and in the area retrochiasmatica. Vasopressin replacement produced a selective decrease in histamine levels of DI rats, restricted to the area retrochiasmatica and nuclei supraopticus and paraventricularis. In contrast, vasopressin increased histamine levels in the area retrochiasmatica and eminentia mediana of HZ rats.In other areas normally rich in vasopressin such as the nuclei arcuatus and the eminentia mediana, the histamine content was not different among LE, HZ or DI rats.Our results suggest a physiological interaction between vasopressin and histamine systems in specific hypothalamic areas of the rat and support the hypothesis of a role of brain shitamine on the central control of water balance.     

Age-related changes of vasopressin content of microdissected areas of the rat brain

Vasopressin contents of posterior pituitaries and intra- and extrahypothalamic nuclei of brains from 3-month and 24-month-old Long-Evans rats and from Brattleboro rats heterozygous (HEDI) and homozygous (HODI) for diabetes insipidus were measured by radioimmunoassay.The pituitary content of vasopressin was highest in young rats and was significantly lower in HEDI tissue. The peptide was not detected in HODI pituitaries.In the brain regions studied both aged and HEDI rats showed reduced vasopressin contents as compared to young animals. In light of evidence of memory deficiencies in both HEDI and HODI rats, the possibility arises that memory decrements associated with senescence may be related to altered vasopressin synthesis and/or secretion occurring with aging of these neuronal systems.     

Alterations in Cyclic AMP Concentration and Adenylate Cyclase Activity in Specific Brain Areas of Rats with Inherited Hypothalamic Diabetes Insipidus (Brattleboro Rats)

The concentration of cyclic AMP (cAMP) and the activity of sodium-fluoride-stimulated adenylate cyclase was measured in 29 microdissected brain areas of homozygous Brattleboro rats and their Long-Evans control rats. In ten of the investigated brain areas a decreased cAMP level was measured in Brattleboro rats. It was particularly decreased in the supraoptic nucleus, cingulate and parietal cortex, hippocampus, habenula and organum vasculosum laminae terminalis. Significantly lower cAMP levels were also found in the periventricular nucleus, bed nucleus of the stria terminalis, area postrema and locus coeruleus. An increased cAMP concentration was detected only in the subcommissural organ of Brattleboro rats. In most brain areas, where cAMP was decreased, sodium fluoride-stimulated adenylate cyclase activity was significantly increased (supraoptic nucleus, parietal cortex, periventricular nucleus, bed nucleus of the stria terminalis, locus coeruleus) or unchanged (hippocampus, habenula, organum vasculosum laminae terminalis). The coincidence of alterations in cAMP concentration and adenylate cyclase activity in brain areas of Brattleboro rats with relatively dense vasopressinergic innervation and/or vasopressin receptor population in control rats, suggests an influence of brain vasopressin on the cAMP-adenylate cyclase second messenger system.     

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.     

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.