A method for fixation of previously fresh-frozen human adult and fetal brains that preserves histological quality and immunoreactivity

doi:10.1016/0165-0270(92)90006-Y

Journal of Neuroscience Methods

Volume 44, Issues 2–3, September 1992, Pages 133-144

https://doi.org/10.1016/0165-0270(92)90006-Y Get rights and content

Abstract

A method is described that enables fixation of previously fresh-frozen and stored adult and fetal human or animal brains. The method involves fixing during thawing under controlled, cryoprotected conditions and is compatible with good histological quality and the preservation of enzymatic activity and immunoreactivity of many neural antigens. It offers considerable advantages for the storage of large amounts of tissue from which multiple samples can be taken and processed under fixation and other conditions that can be optimized for a variety of methods, many of which may be incompatible if the whole brain is fixed in a single fixative prior to storage.

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The UCIBB autopsy protocol is based largely on procedures validated by Kelly and Mann (1996) and includes questions concerning the decedents' demographics, medical history, psychiatric symptoms, medication use, hospitalizations, substance use, and physical health. The human brain dissection and freezing protocol are described in detail elsewhere (Jones et al., 1992). Briefly, after collection, frontal cortex samples were frozen in isopentane at −40 °C and then stored at −80 °C for downstream studies.
Clinical and preclinical studies suggest that some of the behavioral alterations observed in schizophrenia (SZ) may be mechanistically linked to synaptic dysfunction of glutamatergic signaling. Recent genetic and proteomic studies suggest alterations of cortical glutamate receptors of the AMPA-type (AMPARs), which are the predominant ligand-gated ionic channels of fast transmission at excitatory synapses. The impact of gene and protein alterations on the electrophysiological activity of AMPARs is not known in SZ. In this proof of principle work, using human postmortem brain synaptic membranes isolated from the dorsolateral prefrontal cortex (DLPFC), we combined electrophysiological analysis from microtransplanted synaptic membranes (MSM) with transcriptomic (RNA-Seq) and label-free proteomics data in 10 control and 10 subjects diagnosed with SZ. We observed in SZ a reduction in the amplitude of AMPARs currents elicited by kainate, an agonist of AMPARs that blocks the desensitization of the receptor. This reduction was not associated with protein abundance but with a reduction in kainate's potency to activate AMPARs. Electrophysiologically-anchored dataset analysis (EDA) was used to identify synaptosomal proteins that linearly correlate with the amplitude of the AMPARs responses, gene ontology functional annotations were then used to determine protein-protein interactions. Protein modules associated with positive AMPARs current increases were downregulated in SZ, while protein modules that were upregulated in SZ were associated with decreased AMPARs currents. Our results indicate that transcriptomic and proteomic alterations, frequently observed in the DLPFC in SZ, converge at the synaptic level producing a functional electrophysiological impairment of AMPARs.
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While downregulation of several growth factors in major depressive disorder is well established, less attention has been paid to the upregulation of other growth factors. Yet, upregulated growth factors may offer better therapeutic targets. We show that connective tissue growth factor (CTGF) represents a target based on its upregulation in major depressive disorder and studies in animal models implicating it in negative affect.
CTGF gene expression was first evaluated in the postmortem human amygdala. The findings were followed up in outbred rats and in two rat lines that were selectively bred for differences in novelty-seeking and anxiety behavior (bred low responders and bred high responders). We studied the impact of social defeat and early-life treatment with fibroblast growth factor 2 on CTGF expression. Finally, we assessed the ability of an anti-CTGF antibody (FG-3019) to alter CTGF expression and emotionality.
In the human amygdala, CTGF expression was significantly increased in major depressive disorder compared with control subjects. CTGF expression was also significantly increased in the dentate gyrus of adult bred low responders compared with bred high responders. Social defeat stress in bred low responders significantly increased CTGF expression in the dentate gyrus. Early-life fibroblast growth factor 2, a treatment that reduces anxiety-like behavior throughout life, decreased CTGF expression in the adult dentate gyrus. In outbred rats, CTGF administration increased depression-like behavior. Chronic treatment with FG-3019 decreased CTGF expression, and acute and chronic treatment was antidepressant.
This study is the first to implicate CTGF as a prodepressant molecule that could serve as a target for the development of novel therapeutics.
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Citation Excerpt :
As for other factors such as treatment and alcohol comorbidity, we acknowledge the possibility of this having an effect on the results, but the nature of the sample collection restrains the sample size so we chose to include all subjects available that passed the pH/agonal factor criteria to avoid losing analysis power. Following the method described by Jones et al (1992) the brains were removed during autopsy, cooled down and cut in the coronal plane into 0.75 cm slabs, which were then frozen and stored at −80 °C. To ensure the selection of samples adequate for gene expression analysis, brain tissue pH and agonal factors were evaluated following the procedure described by Tomita et al. (2004).
Glial cells are essential in maintaining synaptic function. In glutamatergic synapses astrocytes remove the products of neural activity, (i.e. potassium, glutamate and excess water) from the synaptic cleft and redistribute them across the glial network; these products of neural activity can then be recycled for neuronal use or released into the vascular compartment. This type of highly coupled cell network -or syncytium-maintains the balance of synaptic activity by restoring the basal levels of such molecules in the synaptic cleft. Previous studies have reported alterations of glia related genes in Major Depressive Disorder, including some genes related to syncytial function.
We used RNA isolated from hippocampal tissues of 13 MDD subjects and 10 healthy controls to broadly examine gene expression using microarrays. Hippocampal RNA samples were isolated by laser capture microdissection from human tissue sections carefully avoiding contamination from neighboring structures. Once RNA quality was validated RNA was labeled and hybridized to microarrays.
Analysis of microarray data identified mRNA transcripts involved in glial syncytial function that were downregulated in MDD subjects compared to controls, including potassium and water channels (KCNJ10, AQP4), gap junction proteins (GJA1) and glutamate transporters (SLC1A2, SLC1A3). These gene expression differences were confirmed by qPCR.
The downregulation of these genes related to the syncytial network activity of glial cells is consistent with the hypothesis that synaptic homeostasis is disrupted thereby disrupting hippocampal synaptic function in MDD patients. Such glial gene expression changes could contribute either to the onset or perpetuation of depressive symptoms and hence, represent targets for novel therapeutics.
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All subjects that were clinically characterized with the psychological autopsy method died suddenly without prolonged agonal state. The human brain dissection and freezing protocol are described in detail elsewhere (Jones et al., 1992). RNA was prepared from 80 to 100 mg of frozen tissue samples, with Omni Prep Multi-Sample Homogenizer (Omni International, Kennesaw, GA).
The rs1344706, an intronic SNP within the zinc-finger protein 804A gene (ZNF804A), was identified as one of the most compelling risk SNPs for schizophrenia (SZ) and bipolar disorder (BD). It is however not clear by which molecular mechanisms ZNF804A increases disease risk. We evaluated the role of ZNF804A in SZ and BD by genotyping the originally associated rs1344706 SNP and an exonic SNP (rs12476147) located in exon four of ZNF804A in a sample of 422 SZ, 382 BD, and 507 controls from the isolated population of the Costa Rica Central Valley. We also investigated the rs1344706 SNP for allelic specific expression (ASE) imbalance in the dorsolateral prefrontal cortex (DLPFC) of 46 heterozygous postmortem brains.
While no significant association between rs1344706 and SZ or BD was observed in the Costa Rica sample, we observed an increased risk of SZ for the minor allele (A) of the exonic rs12476147 SNP (p = 0.026). Our ASE assay detected a significant over-expression of the rs12476147 A allele in DLPFC of rs1344706 heterozygous subjects. Interestingly, cDNA allele ratios were significantly different according to the intronic rs1344706 genotypes (p-value = 0.03), with the rs1344706 A allele associated with increased ZNF804A rs12476147 A allele expression (average 1.06, p-value = 0.02, for heterozygous subjects vs. genomic DNA).
In conclusion, we have demonstrated a significant association of rs12476147 with SZ, and using a powerful within-subject design, an allelic expression imbalance of ZNF804A exonic SNP rs12476147 in the DLPFC. Although this data does not preclude the possibility of other functional variants in ZNF804A, it provides evidence that the rs1344706 SZ risk allele is the cis-regulatory variant directly responsible for this allelic expression imbalance in adult cortex.
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Citation Excerpt :
Control subjects had no history of psychiatric or neurological disorders nor any in their first degree relatives. The brains were removed during autopsy and cut in the coronal plane into 0.75 cm slabs (Jones et al., 1992). The slabs were then frozen and stored at −80 °C until needed.
Glutamatergic therapies are emerging as the new path for the treatment of Major Depression Disorder. Recent reports reviewing the use of glutamate activity modulators in the treatment of resistant depression advocate the importance of understanding the alterations of the diverse components of this complex system in mood disorders. In this postmortem study we used in situ hybridization and microarray analysis to evaluate the gene expression of the membrane transporters SLC1A2 and SLCA3 and the vesicular transporter SLCA17A7 in the hippocampus of Major Depressive Disorder (MDD) and Bipolar Disorder (BPD) subjects.
Samples from 8 controls, 11 MDD and 6 BPD subjects were processed for in situ hybridization using cRNA probes for SLC1A2, SLC1A3 and SLC17A7. Laser capture microdissection was used to collect tissue from adjacent sections for microarray analysis. The results showed that the expression of the membrane transporters SLC1A2 and SLC1A3 was diminished in the MDD group compared to controls. The expression of the vesicular glutamate transporter SLC17A7 on the other hand was increased in MDD subjects. As for the BPD group, all three transporters showed trends similar to those observed in MDD, but the changes observed did not reach significance. We hypothesize that the decreased expression of the membrane glutamate transporters and the increased expression of the vesicular transporter in the hippocampus would affect the balance of the glutamatergic circuitry of the hippocampus, and that this effect may be a major contributor to depressive symptoms.
Analysis of miR-137 expression and rs1625579 in dorsolateral prefrontal cortex
2013, Journal of Psychiatric Research
MicroRNAs (miRNAs) are small non-coding RNAs that act as potent regulators of gene expression. A recent GWAS reported the rs1625579 SNP, located downstream of miR-137, as the strongest new association with schizophrenia [Ripke S, Sanders AR, Kendler KS, Levinson DF, Sklar P, Holmans PA, et al. Genome-wide association study identifies five new schizophrenia loci. Nat Genet 2011;43:969–76.]. Prior to this GWAS finding, a schizophrenia imaging-genetic study found miR-137 target genes significantly enriched for association with activation in the dorsolateral prefrontal cortex (DLPFC) [Potkin SG, Macciardi F, Guffanti G, Fallon JH, Wang Q, Turner JA, et al. Identifying gene regulatory networks in schizophrenia. Neuroimage 2010;53:839–47.].
We investigated the expression levels of miR-137 and three candidate target genes (ZNF804A, CACNA1C, TCF4) in the DLPFC of postmortem brain tissue from 2 independent cohorts: (1) 26 subjects (10 control (CTR), 7 schizophrenia (SZ), 9 bipolar disorder (BD)) collected at the UCI brain bank; and (2) 99 subjects (33 CTR, 35 SZ, 31 BD) obtained from the Stanley Medical Research Institute (SMRI). MiR-137 expression in the DLPFC did not differ between diagnoses. We also explored the relationship between rs1625579 genotypes and miR-137 expression. Significantly lower miR-137 expression levels were observed in the homozygous TT subjects compared to TG and GG subjects in the control group (30% decrease, p-value = 0.03). Moreover, reduced miR-137 levels in TT subjects corresponded to increased levels of the miR-137 target gene TCF4. The miR-137 expression pattern in 9 brain regions was significant for regional effect (ANOVA p-value = 1.83E-12), with amygdala and hippocampus having the highest miR-137 expression level. In conclusion, decreased miR-137 expression is associated with the SZ risk allele of rs1625579, and potential regulation of TCF4, another SZ candidate gene. This study offers additional support for involvement of miR-137 and downstream targets as mechanisms of risk for psychiatric disorders.

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Research paperA method for fixation of previously fresh-frozen human adult and fetal brains that preserves histological quality and immunoreactivity

Abstract

J. Neurosci. Methods

Neuroscience

Neuron

Trends Neurosci.

Neuroscience

Brain

Picric and formaldehyde fixation for immunoferritin studies

Histochemistry

Localization of the growth-associated phosphoprotein GAP-43 (B-50, F1) in the human cerebral cortex

J. Neurosci.

Parvalbumin in the human brain

J. Neurochem.

Monoclonal antibody to neurofilament protein (SMI-32) labels a subpopulation of pyramidal neurons in the human and monkey neocortex

J. Comp. Neurol.

Calcium-binding protein parvalbumin as a neuronal marker

Nature

Calbindin immunoreactivity alternates with cytochrome c-oxidase-rich zones in some layers of the primate visual cortex

Nature

1. Cytology and distribution in normal human cerebral cortex of neurons immunoreactive with antisera against neuropeptide Y

J. Comp. Neurol.

Glutamate-positive neurons in the somatic sensory cortex of rats and monkeys

J. Neurosci.

Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones

Nature

Morphology, distribution and synaptic relations of somatostatin and neuropeptide Y immunoreactive neurons in rat and monkey neocortex

J. Neurosci.

Numbers and proportions of GABA immunoreactive neurons in different areas of monkey cerebral cortex

J. Neurosci.

Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities

Exp. Brain Res.

Morphology of tyrosine hydroxylase-immunoreactive neurons in the human cerebral cortex

Exp. Brain Res.

Research paper
A method for fixation of previously fresh-frozen human adult and fetal brains that preserves histological quality and immunoreactivity