Lois Winsky

Summary of a National Institute of Mental Health workshop: Developing animal models of anxiety disorders

Abstract. Rationale: There exists a wide range of animal models and measures designed to assess anxiety or fearfulness. However, the relationship between these models and clinical anxiety symptoms and syndromes is unclear. The National Institute of Mental Health convened a workshop to discuss the relationship between existing behavioral models of anxiety and the clinical profile of anxiety disorders. A second goal of this workshop was to outline various approaches towards modeling components of anxiety disorders. Objectives: To briefly describe epidemiological and behavioral manifestations of clinical anxiety syndromes and how they relate to commonly employed animal models of anxiety. To describe approaches and considerations for developing, improving, and adapting anxiety models to better understand the neurobiology of anxiety. Methods: Clinicians, psychiatrists and clinical and basic neuroscientists presented data exemplifying different approaches towards understanding anxiety and the role of animal models. Panel members outlined what they considered to be critical issues in developing and employing animal models of anxiety. Results: This review summarizes the discussions and conclusions of the workshop including recommendations for improving upon existing models and strategies for developing novel models. Conclusions: The probability of developing comprehensive animal models that accurately reflect the relative influences of factors contributing to anxiety disorder syndromes is quite low. However, ample opportunity remains to better define and extend existing models and behavioral measures related to specific processes that may be disrupted in anxiety disorders and to develop new models that consider the impact of combined factors in determining anxious behaviors.

Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium

Abstract: The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF‐hand calcium‐binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane‐associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.

Antibody Recognition of Calcium-Binding Proteins Depends on Their Calcium-Binding Status

Abstract: Previous studies have revealed changes in immunohistochemical stains for calcium‐binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium‐induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium‐free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium‐dependent conformational changes. A calcium‐insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium‐binding proteins preferentially recognize particular calcium‐induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium‐binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.

Calretinin distribution in the thalamus of the rat: Immunohistochemical and in situ hybridization histochemical analyses

The distribution of calretinin-containing cells was examined by in situ hybridization histochemistry and compared with the immunohistochemical mapping of calretinin in the thalamus of the rat. Results revealed a close correspondence between the immunohistochemical localization of cell bodies and the messenger RNA label produced by the calretinin oligonucleotide probe. Calretinin cells were most prominent in the midline (paraventricular, reuniens, rhomboid) and intralaminar (central medial, paracentral) nuclei and in a group of cells along the rostral central gray which appeared continuous with the caudal extent of the midline nuclei. A subpopulation of calretinin cell bodies was also identified in the reticular nucleus. The mediorostral lateral posterior nucleus, subparafascicular, lateral geniculate and habenular nuclei also contained calretinin messenger RNA probe label. In contrast, no positive cells were found in the anterior, ventral or posterior thalamic nuclei. The distribution of calretinin cells did not correspond directly with that of other histochemical markers. Thus, the in situ hybridization histochemical and immunohistochemical results revealed calretinin as a unique identifying marker for distinct sets of thalamic neurons.

Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat: distribution and coexistence with putative transmitter agents

Immunoreactivity for the calcium binding protein, calretinin (calretinin-ir), was demonstrated in cell bodies of vagal and glossopharyngeal sensory ganglia (jugular, petrosal, and nodose ganglia) and in associated nerve fibers. In the jugular and petrosal ganglia, many calretinin-ir neurons were also immunoreactive for calcitonin gene-related peptide and substance P. In the nodose ganglion, most of the calretinin-ir neurons lacked these peptides. None of the calretinin-ir neurons in these ganglia were also immunoreactive for tyrosine hydroxylase.

Calretinin expression in the chick brainstem auditory nuclei develops and is maintained independently of cochlear nerve input

The expression of the calcium‐binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR‐IR) was localized on adjacent tissue sections. CR mRNA signal in the auditory nuclei was expressed at comparable levels at embryonic day (E)9 and E11 and increased thereafter to reach the highest levels in posthatch chicks. CR‐IR neurons were apparent in NM and NA at E11 and in NL by E13, and CR‐IR increased in all three auditory nuclei thereafter. Neither unilateral nor bilateral otocyst removal caused detectable changes in the intensity of CR mRNA expression or CR‐IR in the auditory nuclei at any of the several ages examined. Similarly, columella removal at posthatching day 2 or 3 failed to significantly affect CR mRNA or CR‐IR levels at 3 hours, 1 day, or 3–4 days survival times. We conclude that cochlear nerve input is not necessary for expression of either calretinin mRNA or protein and that the profound decrease in sound‐evoked activity caused by columella removal does not affect the maintenance of CR expression after hatching. J. Comp. Neurol. 383:112–121, 1997.

Calretinin mRNA and immunoreactivity in the medullary reticular formation of the rat: colocalization with glutamate receptors

Calretinin-positive cells were identified in the medullary reticular formation of the rat by both immunohistochemistry and in situ hybridization histochemistry. In addition, double immunocytochemical labeling was used to examine the degree of colocalization of calretinin with GluR2/R3, GluR4 and GluR5-7 glutamate receptor subtypes. Results indicated regional variation in calretinin expression across reticular formation regions with the exception of the largest cells which were mostly calretinin-positive. Calretinin mRNA was particularly abundant in the parvocellular reticular nucleus. Most calretinin-immunoreactive cells also expressed at least one of the glutamate receptor subtypes examined with the exception of the smallest calretinin-positive cells of the parvocellular reticular formation which were generally not immunoreactive for any of the glutamate receptors examined. Calretinin immunoreactivity was colocalized with immunoreactivity for all three glutamate receptor subtypes examined in most of the large cells of the reticular formation. Immunoreactivity for the GluR4 antibody was least abundant in the reticular formation and GluR4 immunoreactive cells were least likely to co-express calretinin. These results suggest that calretinin and glutamate receptor antibodies may be used to identify specific subsets of reticular formation neurons.

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH. In cochlear and vestibular ganglia, calretinin mRNA was localized in subpopulations of neurons with patterns of distribution similar to those shown by immunocytochemistry. By contrast, the observations in the sensory epithelia differed with the two techniques, ISH revealing less positive structures than immunocytochemistry. Rat inner hair cells and guinea pig inner hair cells, Hensens cells and Deiters cells, which had been described strongly immunoreactive, appeared positive with radioactive but not with non-radioactive ISH. On the other hand, rat vestibular type II hair cells and guinea pig interdental cells of the spiral limbus which were faintly immunoreactive were not positive with both ISH techniques.

Radioimmunoassay of calretinin in the rat brain

Abstract A radioimmunoassay was developed for the quantitation of the brain specific calcium binding protein, calretinin, in micropunch samples of the rat brain. The assay was sensitive, capable of detecting 2–100 ng calretinin and was specific in that no significant cross reactivity was observed with calbindin D-28k. Results indicated several brain regions with high concentrations of calretinin including the ventral cochlear nucleus (6400 ng/mg total protein), periventricular nucleus of the thalamus (4400 ng/mg), optic chiasm (3600 ng/mg), medial habenula (3400 ng/mg) and lateral lobules of the cerebellum (3100 ng/mg). Low concentrations of calretinin were found in, for example, the cerebral cortex and hippocampus. The development of this assay will be useful in future studies of experimental and physiological factors influencing calretinin content in the brain.

Quadruple colocalization of calretinin, calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.