Sensory neuron number in neonatal and adult rats estimated by means of stereologic and profile-based methods.

Postnatal neuron addition, if it occurred, would have profound implications both for the conceptualization of developmental processes and for efforts directed at replacing neurons that were lost to injury or disease. Although dorsal root ganglia (DRGs) offer the advantages of clear boundaries and functional homogeneity, studies comparing neuron number in the DRGs of animals of different ages or sizes have yielded conflicting results. In the present study, neuron number in DRGs L3-L6 was compared in neonatal (approximately 11 days old, mean weight of 24.5 g, mean volume of 25 cm3) and adult (approximately 80 days old, mean weight of 373.5 g, mean volume of 346 cm3) male Sprague-Dawley rats. Estimates of neuron number were derived by using both stereological (physical disector) and profile-counting (one or more nucleoli within a nucleus) methods. The reliability and validity of the two methods were evaluated by comparing estimates of neuron number with those derived from three-dimensional reconstruction of a subset of neurons. The recommended protocol for using the physical disector was found to give accurate estimates of neuron number, but the heterogeneous distribution of neurons in the ganglion led to sampling errors of up to 50%. Reliability was improved by increasing the number of disector pairs examined. Counts of nuclear/nucleolar profiles were more reliable, but introduced a bias that worked against the experimental hypothesis in that estimates of neuron number in neonates exceeded actual values. Nonetheless, both methods indicated that adult rats had more DRG neurons than did neonates. Profile counts were 19% higher in adults (P < .01, two-tailed t-test); and data obtained by using the physical disector showed that adult rats had 28% more neurons than did neonates (P < .05). The difference in neuron number between adults and neonates could be due either to neuron proliferation or to late differentiation of neurons that do not assume a typical appearance until adulthood.

Use of cryostat sections from snap-frozen nervous tissue for combining stereological estimates with histological, cellular, or molecular analyses on adjacent sections.

Adequate tissue preparation is essential for both modern stereological and immunohistochemical investigations. However, combining these methodologies in a single study presents a number of obstacles pertaining to optimal histological preparation. Tissue shrinkage and loss of nuclei/nucleoli from the unprotected section surfaces of unembedded tissue used for immunohistochemistry may be problematic with regard to adequate stereological design. In this study, frozen cryostat sections from hippocampal and cerebellar regions of two rat strains and cerebellar and cerebral regions from a human brain were analyzed to determine the potential impact of these factors on estimates of neuron number obtained using the optical disector. Neuronal nuclei and nucleoli were clearly present in thin sections of snap-frozen rat (3 microm) and human (6 microm) tissue, indicating that neuronal nuclei/nucleoli are not unavoidably lost from unprotected section surfaces of unembedded tissue. In order to quantify the potential impact of any nuclear loss, optical fractionator estimates of rat hippocampal pyramidal cells in areas CA1-3 and cerebellar granule and Purkinje cells were made using minimal (1 microm) upper guard zones. Estimates did not differ from data reported previously in the literature. This data indicates that cryostat sections of snap-frozen nervous tissue may successfully be used for estimating total neuronal numbers using optical disectors.

Reliability and validity of the physical disector method for estimating neuron number.

The physical disector was proposed as an unbiased and efficient means to estimate neuron number; however, the validity and reliability of this method have been examined only infrequently. Estimates of neuron number in the dorsal root ganglia (DRG) of bullfrogs (Rana catesbeiana) were compared to nucleolar counts based on 3-dimensional reconstructions. Accuracy of disector estimates were not affected by size of the animal. Similarly, disector estimates were not systematically altered when area measurements were limited to cellular regions of the DRG versus inclusion of the entire cross-sectional area. However, the recommended protocol for applying the disector resulted in sampling errors that introduced considerable variability in repeated estimates of neuron number from a single ganglion. In addition to this lack of reliability, disector estimates were consistently lower than those obtained by means of a nucleolar counting method that was calibrated against 3-dimensional reconstructions of neuronal profiles. The systematic error of the disector method was greater when ganglia were cut parallel to the long axis of the DR than when they were cut perpendicular to this axis. Increasing the sample size beyond what was recommended increased the reliability of estimates obtained with the disector; however, the bias associated with the plane of section was not reduced. These results emphasize the need for empirical validation of methods used to estimate neuron number in the tissue to which they are to be applied.

Subnucleus-specific loss of neurons in medial thalamus of schizophrenics.

The hypoactivity of dorsolateral prefrontal cortex in schizophrenics is well known. One cause of this hypoactivity may be defective corticocortical or thalamocortical connections. Recent imaging studies of the thalamus suggest reductions in volume of the whole thalamus and reduced activity in the medial group of thalamic nuclei, which may indicate loss of functional input to the cortex. Using stereological techniques in six pairs of individually matched brains from schizophrenics and controls, we measured the volumes and obtained estimates of the number of neurons in the three subnuclei (parvocellular, pc; densocellular, dc; magnocellular, mc) of the mediodorsal nucleus (MD) and from the ventral posterior medial nucleus. There was a significant reduction in total neuron number in MD as a whole but this neuron loss was largely restricted to MDpc and MDdc [-30.9 and -24.5%, respectively (P