Faculty Bibliography

The intrahippocampal distribution of axon collaterals of individual CA3 pyramidal cells was investigated in the rat. Pyramidal cells in the CA3 region of the hippocampus were physiologically characterized and filled with biocytin in anesthetized animals. Their axonal trees were reconstructed with the aid of a drawing tube. Single CA3 pyramidal cells arborized most extensively in the CA1 region, covering approximately two-thirds of the longitudinal axis of the hippocampus. The total length of axon collaterals in the CA3 region was less than in CA1 and the axon branches tended to cluster in narrow bands (200-800 microns), usually several hundred microns anterior or posterior to the cell body. The majority of the recurrent collaterals of a given neuron remained in the same subfield (CA3a, b, or c) as the parent cell. CA3a neurons innervated predominantly the basal dendrites, whereas neurons located proximal to the hilus (CA3c) terminated predominantly on the apical dendrites of both CA1 and CA3 cells. Two cells, with horizontal dendrites and numerous thorny excrescences at the CA3c-hilus transitional zone, were also labeled and projected to both CA3 and CA1 regions. All CA3 neurons projected some collaterals to the hilar region. Proximal (CA3c) neurons had numerous collaterals in the hilus proper. One CA3c pyramidal cell in the dorsal hippocampus sent an axon collaterals to the inner third of the molecular layer. CA3c pyramidal cells in the ventral hippocampus had extensive projections to the inner third of the dentate molecular layer, as well as numerous collaterals in the hilus, CA3, and CA1 areas, and several axon collaterals penetrated the subiculum. The total projected axon length of a single neuron ranged from 150 to 300 mm. On the basis of the projected axon length and bouton density (mean interbouton distance: 4.7 microns), we estimate that a single CA3 pyramidal cell can make synapses with 30,000-60,000 neurons in the ipsilateral hippocampus. The concentrated distribution of the axon collaterals ('patches') indicates that subpopulations of neurons may receive disproportionately denser innervation, whereas innervation in the rest of the target zones is rather sparse. These observations offer new insights into the physiological organization of the CA3 pyramidal cell network

Four reliable silver methods for the visualization of acute or advanced phases of neuronal damage have been modified so that they can be performed with good results on materials freshly fixed with transcardial perfusion of buffered formaldehyde for other silver methods and immunocytochemical techniques. The four modified methods respectively stain: (1) the somata and dendrites of 'dark' (impaired) neurons, (2) the perikarya of all neurons, (3) microglial cells, and (4) capillaries. Without the present modifications the 'dark' neuron method is only effective with glutaraldehyde fixation while the other three methods predominantly stain myelin in freshly fixed mammalian nervous tissue

In this study we investigated the involvement of the cerebellum in high voltage spike-and-wave spindles, a rodent model of petit mal epilepsy. High voltage spindles, recorded epidurally from the sensorimotor neocortex, were correlated with single or multiple unit activity in the cerebellar cortex and deep cerebellar nuclei. The majority of neurons or neuronal groups in the cerebellum (77.9%) fired rhythmically and phase-locked with the high voltage spindles, either during the spike (43.2%; n = 41) or during the wave (34.7%; n = 33) component of the high voltage spindle. Tremor of the head and neck musculature, recorded with an accelero-meter, occurred during the high voltage spindle in approximately half of the rats. When present, rhythmic movement occurred predominantly during the wave phase of the high voltage spindle. The remaining half of the rats did not show tremor during high voltage spindles but, nevertheless, had cerebellar units that burst during the spike or wave phase of the high voltage spindle. These latter results demonstrate that phase-locked bursting of cerebellar units during high voltage spindle is independent of rhythmic movement. The findings suggest that rhythmic output from the cerebellum may contribute to the maintenance of generalized petit mal seizures

Much of the work on forebrain ischemia in the hippocampus has focused on the phenomenon of delayed neuronal death in CA1. It is established that dentate granule cells and CA3 pyramidal cells are resistant to ischemia. However, much less is known about interneuronal involvement in CA3 or ischemic injury in the dentate hilus other than the fact that somatostatin neurons in the latter lose their immunoreactivity. We combined two sensitive methods--heat-shock protein (HSP72) immunocytochemistry and a newly developed Gallyas silver stain for demonstrating impaired cytoskeletal elements--to investigate the extent of ischemic damage to CA3 and the dentate hilus using the four-vessel-occlusion model for inducing forebrain ischemia. HSP72-like immunoreactivity was induced in neuronal populations previously shown to be vulnerable to ischemia. In addition, a distinct subset of interneurons in CA3 was also extremely sensitive to ischemia, even more so than the CA1 pyramidal cells. These neurons are located in the stratum lucidum of CA3 and possess a very high density of dendritic spines. In silver preparations, they were among the first to be impregnated as 'dark' neurons, before CA1 pyramidal cells; microglial reaction was also initiated first in the stratum lucidum of CA3. Whereas CA1 damage was most prominent in the septal half of the hippocampus, hilar and CA3 interneuronal damage had a more extensive dorsoventral distribution. Our results also show a far greater extent of damage in hilar neurons than previously reported. At least four hilar cell types were consistently compromised: mossy cells, spiny fusiform cells, sparsely spiny fusiform cells, and long-spined multipolar cells. A common denominator of the injured neurons in CA3 and the hilus was the presence of spines on their dendrites, which in large part accounted for the far greater number of mossy fiber terminals they receive than their non-spiny neighbors. We suggest that the differential vulnerability of neuronal subtypes in these two regions may be attributed to their extremely dense innervation by the mossy fibers and/or the presence of non-NMDA receptor subtypes that are highly permeable to calcium. In addition, early impairment of these spiny CA3 cells and hilar neurons after ischemia may be causal to delayed neuronal death in the CA1 pyramidal cells

We examined Fisher 344 female rats aged 6, 27, and 33 months old. Prior to sacrifice and morphometric analyses of forebrain cholinergic neurons all rats underwent behavioral characterization in a spatial learning task using the Morris water maze. Performance on the spatial task permitted subsequent grouping of the 27- and 33-month-old animals into impaired or nonimpaired groups. Importantly, the percentage of animals that displayed spatial impairments increased sharply with advancing age. Quantitative assessment of the size and density of choline acetyltransferase (ChAT)-positive neurons throughout the basal forebrain revealed a significant enlargement of forebrain cholinergic neurons within 27-month-old nonimpaired rats compared to 6-month-old rats and 27- and 33-month-old impaired animals. This increase in size was most noted in the medial septum and nucleus of the diagonal band. Significant decreases in the density of ChAT-positive neurons was observed only in the nucleus of the diagonal band of 27-month-old impaired rats compared to 6-month-old controls. Although the significance of enlarged forebrain cholinergic neurons is unclear, we discuss the possibility that within aged rodents neuronal swelling is an active event and represents an early manifestation of the aging process and may constitute a restorative and/or compensatory event in that these rats are relatively asymptomatic with respect to their behavioral deficits. In addition, we discuss in some detail various technical and life effect issues which may vary the outcome of investigations of aged rodents

A back-propagation network was trained to recognize high voltage spike-and-wave spindle (HVS) patterns in the rat, a rodent model of human petit mal epilepsy. The spontaneously occurring HVSs were examined in 137 rats of the Fisher 344 and Brown Norway strains and their F1, F2 and backcross hybrids. Neocortical EEG and movement of the rat were recorded for 12 night hours in each animal and analog data were filtered (low cut: 1 Hz; high cut: 50 Hz) and sampled at 100 Hz with 12 bit precision. A training data set was generated by manually marking durations of HVS epochs in 16 representative animals selected from each group. Training data were presented to back-propagation networks with variable numbers of input, hidden and output cells. The performance of different types of networks was first examined with the training samples and then the best configuration was tested on novel sets of the EEG data. FFT transformation of EEG significantly improved the pattern recognition ability of the network. With the most effective configuration (16 input; 19 hidden; 1 output cells) the summed squared error dropped by 80% as compared with that of the initial random weights. When testing the network with new patterns the manual and automatic evaluations were compared quantitatively. HVSs which were detected properly by the network reached 93-99% of the manually marked HVS patterns, while falsely detected events (non-HVS, artifacts) varied between 18% and 40%. These findings demonstrate the utility of back-propagation networks in automatic recognition of EEG patterns

Degeneration within the hippocampus was examined at the light microscopic level using the Gallyas silver stain two, four or nine months after bilateral transection of the fimbria-fornix and commissural connections. At two or four months after the lesion the strata oriens and radiatum of the subicular end of the CA1 subfields were strongly argyrophilic as was the inner third of the molecular layer of the dentate gyrus. At nine months post-lesion argyrophilia diminished but clearly persisted in the same layers. Electron microscopic examination revealed a large number of electron-dense axon terminals in the argyrophilic areas, most of them making asymmetric synaptic contacts with dendritic spines. These findings suggest that at least a portion of the Schaffer collaterals of the CA3 pyramidal cells and associational collaterals of hilar neurons were in a process of acute degeneration at all time points after the initial surgical trauma. This persistent synaptic reorganization of intrahippocampal circuits may be related to abnormal electrical activity observed several months after fimbria-fornix transection

Neuronal damage induced by 15-min forebrain ischemia was investigated in adult rats 1-5 days (short-term group) and 1-5 months (long-term group) after the initial ischemic attack. In addition to the vulnerable areas reported previously, we observed that the optic tract was also very susceptible. Degeneration of the optic tract and subsequent transsynaptic cell death in the superior colliculus developed slowly and was observed only in the long-term group. The delayed, progressive neuronal damage in this sensory system may serve as a suitable model to investigate the mechanisms of long-term changes in the injured brain