Quantitative organization in the mammillary bodies of adult cat

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BRAIN RESEARCH

Short Communications

Quantitative organization in the mammillary bodies of adult cat Neuron counts have been made in a number of brain steml,Z,5, 7-~ and spinal cord structures1,2, 8. Much of this counting has been done on normal material, although a few studies have been concerned with nuclear neuron population determinations after degeneration has occurred subsequent tolesion placement3, TM.Acharacteristic revealed by these previous studies, as well as those reported herein, is the relatively large variation in neuron population counts for the same normal nuclei from one animal to another within the same species. Variations of 20 ~ to 40 ~ are not uncommon. The data of this report indicate that for the mammillary bodies of the adult cat, a precisely quantifiable parameter characterizing organization is the ratio of the number of neurons in a nuclear subpopulation to the total unmodified neuron count in the nucleus. These ratios of neuron populations characterize group organization from animal to animal with essentially an order of magnitude better reproducibility than do total unmodified nuclear neuron count ratios. A subpopulation of a nucleus is described here as that neuron group which degenerates either in retrograde fashion with an efferent tract lesion or antrograde transneuronally due to deafferentation of any prescribed degree of severity. These studies indicate the existence of a precise degree of organization not topographically evident or morphologically readily definable and, as such, provide the basis for a quantitative biological model of this brain stem complex. How generally this type of organization is represented in other brain stem structures is not yet known, but presumably these nuclei are not unique in this regard. At this time, it is not entirely evident what significance this organizational pattern plays in regard to function, and speculation about the possibilities is reserved for more extensive publications. Adult cats (more than one year of age at start of the study), mostly female, provide the data for this report. Problems associated with nuclear boundary determinations, neuron identification and counting and lesion generation have been previously reported 3-6. All neurons involved in this study have been identified under the light microscope at oil immersion power ( × 1000) and each identified neuron has been documented in its position by a marking on a plastic overlay covering a photomicrograph ( × 250) of the stained slide section. This technique provides a permanent record for every identified neuron. Essentially all neurons were independently identified by two separate individuals and a third individual intercompared the work of the two original identifiers. To obtain absolute numbers of neurons from the neuron count data of this report, an Abercrombie number of 0.82 should be used. The ratios of numbers of neurons are not affected by using the neuron count data rather than absolute numbers of neurons. Table I contains the neuron count information on 11 normal animals for the Brain Research, 37 (1972) 115-122

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medial (MMN) and lateral mammillary nuclei (LMN). For 10 normal animals with M M N neuron counts, the average difference from right to left side is 1.74 ~ . The L M N requires a larger sample size than does the M M N to achieve comparable accuracy in right to left side counts. With a 20 ~ sample, the average difference is 5.3 for 7 normal animals, which is essentially 3.5 times as large as that for the 10~o sample size for the MMN. All brains are serially sectioned at 10/~m thickness. A 1 0 ~ sample is achieved by identifying and counting all circumscribed neurons on every tenth section. A 20 ~ sample is obtained by using every fifth section. For M M N the maximum neuron count is 83,400 and the minimum count is 69,500. These numbers represent a variation of approximately ± 9 ~o from the average. When the data from animals with lesions in the efferent system of the M M N and L M N are analyzed, the significance of the ratios of numbers of neurons in subpopulations of these nuclei to total unmodified nuclear neuron populations can be seen. Table II lists the neuron count information for the M M N of 8 modified brains. Lesions are restricted in each case to the efferent system of this nucleus. Where there is more than one animal with essentially similar lesions, the ratios of neuron populations can be directly intercompared, but the overall consistency of the data for lesions involving increasing numbers of efferent fibers needs also to be considered to evaluate the degree of reproducibility of this type of information. A case in point are data from cats 1166G, 1166C and 1235. These cats all had complete principal mammillary tract (PMT) transection. This transection severs the entire efferent system of the MMN. In each case the opposite M M N serves as the control, since there is no crossover of the efferent system in these nuclei. The losses of 88.5, 87.1 and 89 ~ yield an average of 88.2 ~ . This residual population is remarkably stable from animal to animal when interpreted as a ratio of neuron populations. Another series of animals showing a high degree of consistency in ratios of subpopulation to total unmodified nuclear neuron population are cats 1168, l144DA, 10591, 1111B and 826. These animals involve regional transection of the mammillothalamic tract (MThT) from its anterior to posterior extent. Cat 826 has a transection posterior to essentially all projections to the ventral medial nucleus of the thalamus (VMN) 3. Cat 1168 has a cut anterior to these fibers. Cats I144DA, 10591 and l l l l B have increasing numbers of VMN fibers cut as the lesions move more posteriorly. Essentially 50 ~ of the neurons o f the M M N are dependent on projections to the anterior ventral (AV) and anterior medial (AM) thalamic nuclei for survival, while essentially 15 ~ are additionally dependent on projections to the VMN ~. It should be noted that the modified animals show a somewhat larger spread of neuron population counts in the control M M N than do the normal animals. The average neuron count value for these control nuclei is 78,400 compared to 76,250 for the normal animals. For the LMN, the average normal neuron count is 5,500 and the maximum to minimum variation is 36 ~o (Table I). In Table III, a series of lesions is summarized which result in retrograde degeneration in the LMN. A series of 4 animals (cats 836, 10621, 1168 and 1144DA) with lesions in the M T h T severing connections to the anterior dorsal thalamic nuclei (AD) have losses of 14 ~ , 13.3 ~ , 13.9 ~ and 18 ~ . The average loss is 14.8~ with a variation o f - - 1 . 5 ~ and -k2.2~. These ratios are an Brain Research, 37 (1972) 115-122

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order o f magnitude more reproducible than are the total unmodified nuclear neuron population count ratios for the normal animals studied (36 %). Lesions in the afferent system of these nuclei appear to produce anterograde transneuronal degeneration, yielding neuron population losses which presumably can be quantified in a manner similar to that shown for efferent tract transection. Since the major afferent system for the mammillary complex projects bilaterally, no one mammillary nucleus can be used without further consideration as a control for the contralateral nucleus in terms o f the absolute number o f neurons present before deafferentation. Transneuronal loss is indicated when there is a smaller neuron count for a modified nucleus than for the minimum neuron count on normal animals. Equal bilateral loss can be presumed if the bilateral neuron counts are essentially equal and the residual counts are below that for the minimum count on normal animals. Unequal losses can be accurately assessed by comparing the relative loss in one nucleus to the contralateral nucleus. Data from a series o f 6 animals with various survival periods after different degrees o f deafferentation are suggestive o f quantification in the transneuronally affected subpopulations of neurons (Table IV). The temporal sequence o f events in

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Fig. 1. M M N neuron counts for adult cats with lesions. Points are plotted for the right and left brain neuron counts for each cat listed in Table IV. The horizontal dashed lines describe the range of M M N neuron counts for the 10 normal animals listed in Table I. Below each pair of points is the cat number and survival time. The number adjacent to each point refers to the percent loss (Table IV). Fxt, Fornix; MFbT, Medial forebrain bundle tract.

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transneuronal degeneration are delineated by these data, which are plotted in Fig. 1, to show the modified cell counts compared with the range of counts for 10 normal animals. Cat 903HB has a bilateral hypothalamic fornix (FxT) transection, which suggests the possibility that the bilateral lesion produces no additional cell loss above that of a unilateral fornix transection. A step function representing transneuronal degeneration (cell loss) is in evidence between 12 and 14 months after lesion generation and the data suggest that this neuron loss might remain reasonably stable for up to 39 months, with a small additional neuron loss from 14 months to 39 months (9 ~). The percentage of loss shown in Fig. 1 compares the residual neuron count of the nucleus to the average neuron count of the normal animals. The accuracy of the average percentage loss determined by this comparison method depends on the number of animals available with comparable lesions. Other methods may offer possibilities for determining neuron count information for the MMN of a given animal before deafferentation occurred. A consideration of the ratios of the neuron populations of the LMN to MMN in 7 normal animals, shown in Table I, shows a considerable spread; however, the numbers are oriented in specific small groups. This grouping may be purely a function of the complexity of boundary determination for the LMN, which is considerable. The available quantitative data suggest the use of the LMN as a control for the MMN in cases involving fornix and medial forebrain bundle (MFbT) lesions provided the ratios of the LMN to MMN neuron populations are accurately quantified and reproducible for the normal animals. The LMN and MMN, although adjacent, show minimal evidence for direct connectivity, although they share a common efferent tract (PMT) and their projection fields presumably partially overlap in the VMN and the tegmental nuclei. The significance of the neuron population ratios reported here for related nuclei remains to be determined. The data presented in this report are part of a larger volume of information involving lesion complexes in the efferent and afferent system of the mammillary body complex. A quantitative organization characterized by the invariance of ratios of neuron counts in subpopulations to total unmodified nucleus neuron counts is indicated in this brain stem complex. This type of information, when combined with individual neuron dendritic and synaptic morphology in the various residual neuron populations, should permit the generation of an essentially complete quantitative circuit diagram of a nuclear complex. Analysis of the broader implication of the available larger volume of quantitative information on these and related nuclei must be done in more comprehensive manuscripts. Studies in other brain stem systems are indicated to see if such quantification is more generally represented. Advances in automated means of neuron identification, sizing and counting in normal and modified nuclei potentially provide the means for more rapid and economical data acquisition for this type of study of quantitative neuronal structural organization. Research supported in part by NB 05821 of the U.S. Public Health Service. Bioacoustics Research Laboratory, Department of Electrical Engineering, University of lllinois, Urbana, Ill. 61801 (U.S.A.)

FRANCIS J. FRY

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1 BLINKOV, S. M., AND GLEZER, I. I., The Human Brain in Figures and Tables, (Engl. Transl.), Plenum Press, New York, 1968. 2 CALARESU,F. R., AND HENRY, J. L., Sex difference in the number of sympathetic neurons in the spinal cord of the cat, Science, 173 (1971) 343-344. 3 FaY, W. J., Quantitative delineation of the efferent anatomy of the medial mammillary nucleus of the cat, J. camp. Neurol., 139 (1970) 321-336. 4 FRY, W. J., Mammillary complex of cat brain - - aspects of quantitative organization, Anat. Rec., 154 (1966) 175-184. 5 FRY, W. J., FRY, F. J., MALEK, R., AND PANKAU, J. W., Quantitative neuroanatomic studies implemented by ultrasonic lesions - - mammillary nuclei and associated complex of cat brain, J. acoust. Soc. Amer., 36 (1964) 1795-1835. 6 FRY, W. J., KRUMINS, R., FRY, F. J., THOMAS,G., BORBELY,S., AND ADES, H., Origins and distribution of some efferent pathways from the mammillary nuclei of the cat, J. comp. Neurol., 120 (1963) 195-258. 7 GUILLERY, R. W., A quantitative study of the mammillary bodies and their connexions, J. Anat. (Land.}, 89 (1955) 19-32. 8 KONIGSMARK,B. W., Methods for the counting of neurons. In W. J. H. NAUTA AND S. O. E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, New York, 1970, pp. 334-339. 9 POWELL, T. P. S., GUILLERY, R. W., AND COWAN, W. M., A quantitative study of the fornix mammillothalamic system, J. Anat. (Lond.), 91 (1957) 419-437. 10 POWELL, T. P. S., AND ERULKAR, S. D., Transneuronal cell degeneration in the auditory relay nuclei of the cat, J. Anat. (Lond.), 96 (1962) 249-268. (Accepted November 12th, 1971)

Brain Research, 37 (1972) 115-122