Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain  excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Quantitative assessment of new cell proliferation in the dentate gyrus and learning after isoflurane or propofol anesthesia in young and aged rats.

There is a growing body of evidence showing that a statistically significant number of people experience long-term changes in cognition after anesthesia. We hypothesize that this cognitive impairment may result from an anesthetic-induced alteration of postnatal hippocampal cell proliferation. To test this hypothesis, we investigated the effects of isoflurane and propofol on new cell proliferation and cognition of young (4 month-old) and aged (21 month-old). All rats were injected intraperitoneally (IP) with 50 mg/kg of 5-bromo-2-deoxyuridine (BrdU) immediately after anesthesia. A novel appetitive olfactory learning test was used to assess learning and memory two days after anesthesia. One week after anesthesia, rats were euthanized and the brains analyzed for new cell proliferation in the dentate gyrus, and proliferation and migration of newly formed cells in the subventricular zone to the olfactory bulb. We found that exposure to either isoflurane (p=0.017) or propofol (p=0.006) decreased hippocampal cell proliferation in young, but not in aged rats. This anesthetic-induced decrease was specific to new cell proliferation in the hippocampus, as new cell proliferation and migration to the olfactory bulb was unaffected. Isoflurane anesthesia produced learning impairment in aged rats (p=0.044), but not in young rats. Conversely, propofol anesthesia resulted in learning impairment in young (p=0.01), but not in aged rats. These results indicate that isoflurane and propofol anesthesia affect postnatal hippocampal cell proliferation and learning in an age dependent manner.

New approach to risk assessment of central neurotoxicity induced by 1-bromopropane using animal models.

1-Bromopropane (1-BP) induces central as well as peripheral neurotoxicity in workers. We have reported the dysfunction of feedback inhibition (i.e. disinhibition) in the rat hippocampus following exposure to 1-BP at concentrations of 1500 and 700 ppm. For risk assessment, we studied disinhibition of the CA1 region and the dentate gyrus in hippocampal slices obtained from control and 1-BP-exposed (200 and 400 ppm) rats, and determined the bromide concentration in the brain. Granule cell disinhibition was observed after inhalation exposure to 400 ppm 1-BP for 8 or 12 weeks, suggesting that the dentate gyrus was more sensitive than the CA1 region to 1-BP exposure. The lowest observed adverse effect level and the no observed adverse effect level of 1-BP inhalation for disinhibition were 400 and 200 ppm, respectively. The concentration of bromides in the brain increased from 2.9+/-1.5 to 85.0+/-25.4 microg/g-wet brain at week 4 of 400 ppm inhalation, and no further increase was observed even when the exposure period was extended for up to 12 weeks. The relationship between total dose (ppm-h) and the exposure concentration of 1-BP was investigated at different exposure concentrations. Disinhibition and death by inhalation depended on the total dose, and their occurrence appeared earlier as the exposure concentration increased. The results demonstrated a novel model for risk assessment of central neurotoxicity induced by 1-BP inhalation.

Quantitative morphological assessment reveals neuronal and glial deficits in hippocampus after a brief subtoxic exposure to chlorpyrifos in neonatal rats.

Neurochemical and behavioral studies indicate that the widely used organophosphorus insecticide, chlorpyrifos (CPF), evokes neurobehavioral teratogenicity with a wide window of vulnerability, ranging from embryonic life through postnatal development. Few studies have detailed morphological damage that corresponds to the operational deficits. We administered 5 mg/kg of CPF sc daily on postnatal days (PN) 11-14, a regimen that is devoid of systemic toxicity, but that elicits long-term cognitive impairment and disruption of cholinergic, catecholaminergic, and serotonergic synaptic function. On PN15 and 20, we conducted quantitative morphologic examinations of neurons and glia in CA1, CA3, and dentate gyrus regions of the hippocampus. Although hippocampal morphology after CPF exposure was normal on gross observation, morphometric analysis revealed a significant overall reduction in the total number of neurons and glia. Superimposed on this basic effect, CPF elicited a delayed-onset increase in the neuron/glia ratio that emerged by PN20, connoting selective gliotoxicity. The alterations in cell numbers were accompanied by significant perikaryal swelling and by enhanced development of astrocytic processes. Layer thickness also showed delayed-onset effects of CPF, with thinning of the CA1 and CA3 layers and enlargement of the dentate gyrus. Our results indicate that there are subtle morphological changes in the juvenile rat brain after neonatal CPF exposure that are detectable only with quantitative analysis and that correlate with regional and cell-specific targets identified earlier in neurochemical studies. The simultaneous targeting of neurons and glia by CPF is likely to play an important role in its developmental neurotoxicant effects.

Quantal amplitude and quantal variance of strontium-induced asynchronous EPSCs in rat dentate granule neurons.

1. Excitatory postsynaptic currents (EPSCs) were recorded from granule cells of the dentate gyrus in acute slices of 17- to 21-day-old rats (22-25 C) using tissue cuts and minimal extracellular stimulation to selectively activate a small number of synaptic contacts. 2. Adding millimolar Sr2+ to the external solution produced asynchronous EPSCs (aEPSCs) lasting for several hundred milliseconds after the stimulus. Minimally stimulated aEPSCs resembled miniature EPSCs (mEPSCs) recorded in the same cell but differed from them in ways expected from the greater range of dendritic filtering experienced by mEPSCs. aEPSCs had the same stimulus threshold as the synchronous EPSCs (sEPSCs) that followed the stimulus with a brief latency. aEPSCs following stimulation of distal inputs had a slower mean rise time than those following stimulation of proximal inputs. These results suggest that aEPSCs arose from the same synapses that generated sEPSCs. 3. Proximally elicited aEPSCs had a mean amplitude of 6.7 +/- 2.2 pA (+/- s.d., n = 23 cells) at -70 mV and an amplitude coefficient of variation of 0. 46 +/- 0.08. 4. The amplitude distributions of sEPSCs never exhibited distinct peaks. 5. Monte Carlo modelling of the shapes of aEPSC amplitude distributions indicated that our data were best explained by an intrasite model of quantal variance. 6. It is concluded that Sr2+-evoked aEPSCs are uniquantal events arising at synaptic terminals that were recently invaded by an action potential, and so provide direct information about the quantal amplitude and quantal variance at those terminals. The large quantal variance obscures quantization of the amplitudes of evoked sEPSCs at this class of excitatory synapse.

Serotonin transporter function in vivo: assessment by chronoamperometry.

Local application of selective serotonin reuptake inhibitors, fluvoxamine and citalopram, prolonged the clearance of exogenously administered serotonin (5-HT) in both the dentate gyrus and CA3 region of the dorsal hippocampus, as measured using in vivo chronoamperometry. These effects were abolished in rats pretreated with 5,7-dihydroxytryptamine. The NE uptake inhibitors, desipramine and protriptyline, did not alter the 5-HT signal in the CA3 region, but prolonged the clearance of 5-HT in the dentate gyrus; this effect was absent in rats pretreated with 6-hydroxydopamine. From these data, it is inferred that both the SERT and NET contribute to the active clearance of exogenously applied 5-HT in the dentate gyrus. In another experiment, cyanopindolol, an antagonist of the serotonin terminal autoreceptor, also prolonged the clearance of 5-HT from the CA3 region. These and other data have generated a working hypothesis that activation of the terminal serotonin autoreceptor enhances the kinetics of 5-HT uptake through an effect on the serotonin transporter.