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Single cell nuclei were investigated using two-dimensional angularly and spectrally resolved scattering microscopy. We show that even for a qualitative comparison of experimental and theoretical data, the standard Mie model of a homogeneous sphere proves to be insufficient. Hence, an accelerated finite-difference time-domain method using a graphics processor unit and domain decomposition was implemented to analyze the experimental scattering patterns. The measured cell nuclei were modeled as single spheres with randomly distributed spherical inclusions of different size and refractive index representing the nucleoli and clumps of chromatin. Taking into account the nuclear heterogeneity of a large number of inclusions yields a qualitative agreement between experimental and theoretical spectra and illustrates the impact of the nuclear micro- and nanostructure on the scattering patterns.

In the real world, task-relevant, conditioned stimuli are often embedded in a varying background, from which they have to be segregated. Besides sensory mechanisms, associative learning assumingly plays an important role for the segregation of the conditioned from the background stimuli, especially if conditioned and background stimuli are spectro-temporally structured, and psychophysically similar. We therefore investigated the influence of spectro-temporally structured background tones on associative learning of conditioned tones depending on the complexity of the behavioral task and the psychophysical similarity between conditioned and background tones. Frequency-modulated tone sweeps were used as conditioned stimuli, and persisting frequency-modulated tones as background. In a shuttle-box, Mongolian gerbils were subjected to a simple detection task, or to a more complex discrimination task. In contrast to detection learning, introduction or change of background tones affected discrimination performance both during learning and at the stage of retrieval, especially when conditioned and background tones were spectro-temporally similar. The change from a familiar to a new background tone at the stage of retrieval caused a prefrontal dopamine increase and lead to relearning of task-relevant associations. We conclude that conditioned stimuli and background stimuli are processed concomitantly, which might provide contextual information, but requires additional cognitive processing.

Changes of cortical activity were examined with electrocorticograms sampled from gerbils during learning. Animals were subjected to tone-conditioned avoidance training in a shuttle-box. Electrocorticograms were recorded from an electrode placed over medial prefrontal cortex. Temporal patterns of theta activity were analyzed across 180 successive trials. With the start of conditioning strong theta activity occurred in each trial in the phase immediately after hurdle crossing. With reliable occurrence of conditioned responses, that is at the stage of retrieval of the avoidance response, the theta activity became reduced. A negative correlation exists between the theta power and the development of learning progress. Theta reduction thus could reflect decreasing demands on information processing in the course of avoidance success monitoring across trials.

Synapse maturation includes the shortening of postsynaptic currents, due to changes in the subunit composition of respective transmitter receptors. Patch clamp experiments revealed that GABAergic inhibitory postsynaptic currents (ISPCs) of superior colliculus neurons significantly shorten from postnatal day (P)1 to P21. The change started after P6 and was steepest between P12 and P15, i.e. around eye opening. It was accompanied by enhanced sensitivity to zolpidem and increased expression of GABAAR alpha1 mRNA, whereas the level of alpha3 mRNA decreased. This result is consistent with the hypothesis that the IPSC kinetics of developing collicular neurons is determined by the level of alpha1/alpha3. As alpha1/alpha3 peaked when N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic currents reached their maximum (P12) it was asked whether NMDAR activity can shape the kinetics of GABAergic IPSCs. Cultured collicular neurons were treated with NMDA or NMDAR block, and it was found that the former resulted in faster and the latter in slower IPSC decay. Group I mGluR blockade had no effect. Experiments with bdnf-/- mice revealed that, with some delay, the increase of alpha1/alpha3 mRNA also occurred in the chronic absence of brain-derived neurotrophic factor (BDNF) and, again, this was accompanied by the shortening of IPSCs. In addition, there was an age-dependent depression of IPSC amplitudes by endogenous BDNF, which might reflect the developmental increase in the expression of GABAAR gamma2L, as opposed to gamma2S. Together, these experiments suggest that the GABAAR alpha subunit switch and the associated change in the IPSC kinetics were specifically controlled by NMDAR activity and independent on the signalling through group I mGluRs or TrkB.

During postnatal development, the retinocollicular pathway undergoes activity-dependent refinement, resulting in the precise retinotopic map seen in adults. Previous studies established that retinal efferents reach the mouse superior colliculus (SC) by embryonic day 16. Morphologically, synapses were found in the rat SC before birth. As part of an extended project aimed at understanding the development of synaptic transmission in the visual layers of the SC, we report here the presence of functionally active synapses immediately after birth. Circuit activity in mouse SC neurons was detected in horizontal slices of the visual layers using cell-attached voltage clamp. The spontaneous discharge of action potentials was abolished by glutamatergic blockers and facilitated by bicuculline, showing that circuit activity is based on synaptic transmission and that the action of gamma-aminobutyric acid is inhibitory. Using whole-cell voltage clamp, spontaneous glutamatergic postsynaptic currents as well as miniature GABAergic postsynaptic currents were recorded on postnatal day 1. Excitatory and inhibitory postsynaptic currents could also be evoked by electrical stimulation. Glutamatergic postsynaptic currents comprised both (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated components. The early function of glutamatergic and GABAergic synaptic transmission in the visual layers of SC suggests that SC neurons are able to process information originating from retinal axons immediately after birth.

In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.

This study was aimed at clarifying the role of metabotropic glutamate receptors (mGluRs) in the regulation of intracellular Ca2+ concentration ([Ca2+]i in postnatal mouse retinal ganglion neurons (RGNs). RGNs were maintained for 1-2 weeks in vitro by adding brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) to the culture medium. In order to select these cells for electrophysiological measurements, RGNs were vitally labelled with an antibody against Thy-1.2. Voltage-activated Ca2+ currents [ICa(V)] were recorded with patch electrodes in the whole-cell configuration. It was found that racemic +/--1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) or its active enantiomer 1S,3R-ACPD rapidly and reversibly either enhanced or depressed ICa(V). Quisqualate (QA), L-2-amino-4-phosphonobutyrate (L-AP4) and the endogenous transmitter glutamate induced similar effects when ionotropic glutamate receptors were blocked with D-2-amino-5-phosphonovalerate (D-APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). omega-Conotoxin GVIA (omega-CgTx GVIA), but not nifedipine prevented modulation of ICa(V) by mGluR agonists. The depression of ICa(V) by t-ACPD was irreversible when cells were dialysed with guanosine-5'-O-(3-thiotriphosphate) (GTP[gamma-S]). Ratio measurements of fura-2 fluorescence in Thy-1+ cells showed that neither t-ACPD, QA nor L-AP4 affected [Ca2+]i by liberation of Ca2+ from intracellular stores. Our results suggest that cultured RGNs express mGluRs. These receptors cannot induce Ca2+ release from intracellular stores but regulate [Ca2+]i by a fast and reversible, G-protein-mediated action on a subpopulation of voltage-activated Ca2+ channels.

Peripheral nerve injury or amputation leads to extensive changes within the central representations of the mammalian body surface. The mechanisms responsible for post-traumatic reorganization of these maps in adults may also, at least partly, underlie a more general feature of the somatosensory system—the capacity for stimulus-dependent plasticity. Acetylcholine has been implicated in both of these processes. We studied the binding of the ligands [ 3 H]QNB and [ 3 H]pirenzepine in rat hindlimb somatosensory cortex from 1 to 14 days following sciatic nerve transection. Although the [ 3 H]QNB binding was not different from normal levels in tissue homogenates of the affected somatosensory cortex, differences were demonstrated when binding was measured on a layer-by-layer basis. [ 3 H]QNB binding was changed only in certain layers, at certain times. The predominant effects appeared to be a decrease in binding in the middle layers from 4 to 14 days after the transection. Combining the [ 3 H]QNB data with data obtained from the more M 1 -selective ligand [ 3 H]pirenzepine suggested that complex changes occur among several muscarinic receptors, including receptors with non-M 1 subtype characteristics. Moreover, unilateral nerve transection affects the hindlimb somatosensory regions in both hemispheres.

A method for the rapid dissection of circumscribed areas of rat cortex is described. The technique does not depend on skull-derived landmarks but uses for stereotaxic orientation the cross-point of the interhemispheric gap with the caudal margin of the cortex. An application of this dissection method to the biochemical analysis of cholinergic markers within the hindlimb representation of the primary somatosensory cortex revealed that both the activity of the enzyme choline acetyltransferase as well as the binding of [3H]quinuclidinyl benzilate to muscarinic cholinergic receptors do not seem to be affected drastically three days after unilateral transection of the sciatic nerve. The only significant effect detected was a slight decrease in the activity of the choline acetyltransferase within the hindlimb representation of the primary somatosensory cortex contralateral to the transected sciatic nerve. In the primary visual cortex, the cholinergic markers investigated did not show significant alterations after sciatic nerve injury.

By comparing behavioral performance and cortical theta activity (4–8 Hz) on a trial by trial basis we examined how the different behavioral stages of tone-induced avoidance learning in the shuttle-box may be distinguishable by theta power as a potential correlate of changing strategies of information processing. Electrocorticograms with pronounced theta content were recorded across the cortical surface of gerbils during avoidance learning and analyzed in each trial in conjunction with reaction times and unconditioned and conditioned responses. The focus of theta analysis in this paradigm with a 5-s delay between tone and foot-shock onsets was on the 14-s periods after hurdle crossing where feedback information from a trial is available. The strongest theta activity occurred in stage 1 of initial tone conditioning which was sharply reduced to a minimum during stage 2 of optimization of unconditioned escape responses from the foot shock. A few initial successful avoidance responses gave rise to a reversal of the decline of theta activity that later reached a second maximum. A systematic increase of theta activity during this stage 3 of avoidance conditioning was found for the occasional trials with unconditioned responses and not for the increasing number of conditioned responses suggesting that error processing is a major correlate of this new increase of theta power. After the second maximum the theta power slowly declined together with a further improvement of behavioral performance indicating that stage 4 of retrieval of the consolidated avoidance response was reached. The results suggest that behind a previously reported general trend of decreasing theta power with increasing performance in this paradigm there is a hidden microstructure of theta activity across trials which separates stages of avoidance conditioning and is partially mirrored by known changes of prefrontal dopamine release.

Serotonergic neurones are among the first to develop in the central nervous system. Their survival and maturation is promoted by a variety of factors, including serotonin itself, brain-derived neurotrophic factor (BDNF) and S100beta, an astrocyte-specific Ca(2+) binding protein. Here, we used BDNF-deficient mice and cell cultures of embryonic raphe neurones to determine whether or not BDNF effects on developing serotonergic raphe neurones are influenced by its action on glial cells. In BDNF-/- mice, the number of serotonin-immunoreactive neuronal somata, the amount of the serotonin transporter, the serotonin content in the striatum and the hippocampus, and the content of 5-hydroxyindoleacetic acid in all brain regions analysed were increased. By contrast, reduced immunoreactivity was found for myelin basic protein (MBP) in all brain areas including the raphe and its target region, the hippocampus. Exogenously applied BDNF increased the number of MBP-immunopositive cells in the respective culture systems. The raphe area displayed selectively reduced immunoreactivity for S100beta. Accordingly, S100beta was increased in primary cultures of pure astrocytes by exogenous BDNF. In glia-free neuronal cultures prepared from the embryonic mouse raphe, addition of BDNF supported the survival of serotonergic neurones and increased the number of axon collaterals and primary dendrites. The latter effect was inhibited by the simultaneous addition of S100beta. These results suggest that the presence of BDNF is not a requirement for the survival and maturation of serotonergic neurones in vivo. BDNF is, however, required for the local expression of S100beta and production of MBP. Therefore BDNF might indirectly influence the development of the serotonergic system by stimulating the expression of S100beta in astrocytes and the production MBP in oligodendrocytes.

The effects of brain-derived neurotrophic factor (BDNF) on the development of presynaptic terminals and of neuronal subtypes in various brain areas were studied in BDNF-knockout (BDNF-/-) mice at postnatal days 15-17. Western analysis revealed no changes in the overall amount of a variety of synaptic proteins in BDNF-/- mice as compared to wild type mice. In addition, the complex between the vesicular proteins, synaptophysin and synaptobrevin, as well as their respective homodimers were unaltered. Moreover, no changes in the density of neurons were found in, e.g., the CA3 region of the hippocampus and the nucleus nervi facialis of BDNF-/- mice. However, cholinergic cells were reduced by 20% in the medial septum of BDNF-/- mice associated with a decrease in the activity of choline acetyltransferase and protein levels of nerve growth factor in the hippocampus by 16% and 44%, respectively. In the striatum, however, the total number of cholinergic cells were comparable in both groups, although the activity of choline acetyltransferase was decreased by 46%. In GABAergic interneurons, the expression of neuropeptides in various brain areas was differentially affected by BDNF deletion as revealed by immunohistochemistry. In the hippocampus and cortex of BDNF-/- mice, the density of neuropeptide Y-, somatostatin-, and parvalbumin-immunoreactive cells was drastically reduced, whereas the density of calretinin-positive cells was increased. The extent of these changes in neuropeptide-containing cells varied among hippocampal subregions. In the striatum, only the density of parvalbumin-immunoreactive cells was decreased by approximately 45%. In conclusion, BDNF deficiency is accompanied by a differential dysregulation in the expression of neuropeptides and calcium-binding proteins in otherwise intact GABAergic and glutamatergic neurons in a region-specific manner.

Using microdialysis from medial prefrontal cortex of gerbils during aversive auditory conditioning in the shuttle-box we have previously shown a transient increase of dopamine efflux correlated with the establishment of avoidance behavior. We hypothesized that the acquisition of a new behavioral strategy is generally accompanied by this extra prefrontal dopamine release. The present experiment aimed at further testing this hypothesis. In a pre-training period in the shuttle-box the gerbils acquired an active avoidance response by generalizing two different tone signals to a GO-meaning (change of shuttle-box compartment). Thereafter, they were subjected in relearning sessions to differentially associate the known tone stimuli with GO- and NOGO- (no change of shuttle-box compartment) conditions, respectively. The following formation of discrimination behavior led to a similar extra dopamine increase as found during establishment of the avoidance strategy. This significant enhancement was limited to rapidly relearning individuals. Furthermore, the dopamine increase attenuated in these animals with increasing performance during the course of the discrimination training, similar to the retrieval stage of the avoidance strategy. Therefore, the dopamine system seems to be critically involved in the initial formation of associations for new behavioral strategies, i.e. learning. We assume that the prefrontal dopamine increase during initial learning of the complex discrimination behavior indicates an involvement of working memory principles and a goal-directed formation of a behavioral strategy.

The neurotrophin brain-derived neurotrophic factor (BDNF) is involved in numerous aspects of synapse development and plasticity. The present study was aimed at clarifying the significance of endogenous BDNF for the synaptically driven spontaneous network activity and GABAergic inhibition in the superficial layers of the mouse superior colliculus. In this structure neuron survival is unaffected by the absence of BDNF. Two experimental approaches were used: comparison of BDNF-deficient (–/–) and wild-type (+/+) mice and blockade of BDNF receptor signaling by the tyrosine kinase inhibitor K-252a. Patch-clamp recordings were performed on horizontal slices during postnatal days 15 and 16. The lack of BDNF in –/– mice caused a significant reduction of the spontaneous action potential frequency and an increase in the pharmacologically induced disinhibition of spike discharge. This change was accompanied by an increase in the amplitudes of GABAergic evoked, spontaneous, and miniature inhibitory postsynaptic currents (IPSCs). BDNF gene inactivation had no effect on the degree of paired-pulse facilitation or the frequency of miniature IPSCs. The increase of IPSC amplitudes by chronic BDNF deprivation was completely mimicked by acute exposure to K-252a in +/+ animals. The enhancement of GABAergic IPSCs in –/– animals was reversed by acute application of 100 ng/ml BDNF, but this rescue was completely prevented by blocking postsynaptic protein kinase C (PKC) activation with the PKC inhibitor peptide 19–31. From these results we conclude that BDNF increases spontaneous network activity by suppressing GABAergic inhibition, the site of action of BDNF is predominantly postsynaptic, BDNF-induced suppression of GABAergic synaptic transmission is caused by acute downregulation of GABAAreceptors, and BDNF effects are mediated by its TrkB receptor and require PKC activation in the postsynaptic cell.

Recently, we cloned and began to characterize a new N-methyl-d-aspartate receptor (NMDAR) subunit, NR3A. Here we extend our earlier findings by showing that recombinantly expressed NR3A in COS cells is biochemically associated with both NR1 and NR2 subunits. In the oocyte or HEK 293 cell expression systems, co-injection of NR3A with NR1/NR2 subunits acts in a dominant-interfering manner, resulting in a decrease in NMDAR unitary conductance, decrease in Ca2+ permeability, decrease in Mg2+ sensitivity, and slight increase in mean open time compared with NR1/NR2 channels. The smaller unitary conductance channel has also been observed in primary cortical neurons cultured from wild-type rodent on postnatal day 8( P8) and similarly found to be relatively insensitive to Mg2+ block. Consistent with these findings, whole cell NMDA-evoked currents are larger in NR3A-deficient mice compared with wild-type mice, and this effect follows a developmental pattern similar to that of NR3A protein expression on Western blots, with peak expression at P8. Finally, a new longer splice variant of NR3A has been cloned and found to be expressed in rodent cortical neurons by single-cell RT-PCR and in situ hybridization.

Previous work by Cellerino et al. has shown that chronic absence of brain-derived neurotrophic factor (BDNF) resulted in hypomyelination of the optic nerve. Since myelination is influenced by neuronal activity, it is possible that a deficiency in BDNF during early development could alter the firing properties of retinal neurons. To test this hypothesis, patch-clamp recordings were performed in retinal whole mounts from BDNF-deficient (bdnf-/-), heterozygote (bdnf+/-) or wild-type control mice (bdnf+/+). Ganglion cell layer neurons (RGNs) were tested at different age [postnatal day (P)1-11] for their ability to encode graded depolarization with variable action potential frequency. At all developmental ages examined, RGNs exhibiting frequency coding were less frequently encountered in bdnf-/- than in bdnf+/+ mice. At P1, none of the RGNs from bdnf-/- mice displayed repetitive firing compared to 50% in bdnf+/+ mice, and by P7-11, only 50% of bdnf-/- RGNs exhibited repetitive firing compared to 100% in bdnf+/+ mice. Moreover, in bdnf-/- RGNs repetitive discharge was characterized by a reduced frequency increment per current change. Acquisition of repetitive firing was paralleled by a decrease in input resistance and a steep increase of sodium current density. In bdnf-/- mice, the onset of this increase occurred at later stages of development than in wild-type controls (bdnf-/-: P6-11; bdnf+/+: P2-6). The discharge pattern of P7-11 bdnf-/- RGNs resembled that of RGNs in neonatal wild-type mice and was mimicked by acute application of a Ca(2+) channel blocker. We conclude that BDNF plays an important role in the ontogeny of repetitive firing of RGNs.

The NMDA (N -methyl-D-aspartate) subclass of glutamate receptor1 is essential for the synaptic plasticity thought to underlie learning and memory2,3,4 and for synaptic refinement during development5,6. It is currently believed that the NMDA receptor (NMDAR) is a heteromultimeric channel comprising the ubiquitous NR1 subunit and at least one regionally localized NR2 subunit7,8,9,10,11. Here we report the characterization of a regulatory NMDAR subunit, NR3A (formerly termed NMDAR-L or χ-1), which is expressed primarily during brain development12,13. NR3Aco-immunoprecipitates with receptor subunits NR1 and NR2 in cerebrocortical extracts. In single-channel recordings from Xenopus oocytes, addition of NR3A to NR1 and NR2 leads to the appearance of a smaller unitary conductance. Genetic knockout of NR3A in mice results in enhanced NMDA responses and increased dendritic spines in early postnatal cerebrocortical neurons. These data suggest that NR3A is involved in the development of synaptic elements by modulating NMDAR activity.

A culture system of the postnatal rat retina was established to investigate Ca2+ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca2+ currents of RGNs and to clarify the potential use of the lanthanide Gd3+ as a selective Ca2+ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca2+ currents (ICa(LVA)) and high voltage activated Ca2+ currents (ICa(HVA)). The remaining 2/3 of RGNs in short term culture and most RGNs in long-term culture displayed only ICa(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of ICa(HVA) was irreversible blocked by the N channel antagonist ω-CgTx (5 μM). The L channel antagonist nifedipine (10 μM) eliminated about 25% of ICa(HVA). Thus, about 1/3 of the HVA Ca2+ or Ba2+ current remained unaffected by either ω-CgTx or nifedipine. ω-AgaTx (200 nM) completely failed to block HVA Ca2+ or Ba2+ currents in RGNs. Gd3+ exerted contrasting actions on LVA and HVA Ca2+ currents. While ICa(LVA) consistently increased in the presence of Gd3+ (0.32–3.2 μM), ICa(HVA) always decreased, especially when using higher concentrations of Gd3+ (10–32 μM). The blocking action of Gd3+ was not restricted to the ω-CgTx-sensitive HVA current component, but also concerned ω-CgTx- and nifedipine-resistant components. The decay of Ca2+ currents was accelerated in the presence of Gd3+. Even in RGNs lacking ICa(LVA), application of 3.2 μM Gd3+ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from −90 to 0 mV; 10 mM [Ca2+]0; 26°C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd3+. Gd3+ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd3+ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca2+ currents, that resemble in some aspects T, N and L channel currents. A significant component of the HVA Ca2+ current was resistant to the available HVA channel blockers suggesting the presence of a pharmacologically distinct type of HVA Ca2+ channel type in RGNs. Our experiments also show that Gd3+ is not suitable for isolation of HVA subcomponents in RGNs, but it can be used to distinguish between LVA and HVA Ca2+ currents, as these currents reacted to Gd3+ in an opposite way. The purely depressive effect of this lanthanide on spontaneous synaptic activity is consistent with the assumption that in retinal neurons LVA Ca2+ channels are not involved in the regulation of glutamate release.

We have studied the changes in the GABAergic system in the rat somatosensory cortex 1-14 d after sensory deprivation of the hind-limb representation area. Glutamate decarboxylase (GAD) activity was measured in the individual cortical layers using serial sections cut on a freezing microtome parallel to the cortical surface. Gamma-aminobutyric acid (GABA) high-affinity uptake was studied in cortical homogenates of the hind-limb representation area. There was a less than or equal to 13% decrease in GAD activity in layer II-IV in both cortical hemispheres 3 d after sciatic nerve injury. In contrast, we found that high-affinity uptake of GABA is not affected. The data mirror only small changes in GABAergic transmission, probably as a result of the methods employed. These changes correspond to electrophysiological studies suggesting that peripheral manipulation of the somatosensory system, e.g., nerve transection, is accompanied by changes in GABAergic transmission.

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.

Three days after systemic administration of kainic acid (15 mg/kg, s.c.), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high-affinity choline uptake) and GABAergic parameters [benzodiazepine and gamma-aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle-injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium-dependent high-affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid-induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and cholinoceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.

The postnatal development of [3H]dihydroalprenolol binding to beta-adrenergic receptors has been studied in frontal cortex, cerebellum, striatum, and hypothalamus of the rat after prenatal and perinatal exposure to diazepam. Dams were injected subcutaneously with single daily doses of 1 mg of diazepam/kg from day 7 to 20 of gestation or from day 15 of gestation to day 6 after birth. Prenatal exposure had no effect on litter size or length of gestation or on the postnatal development of body and brain weights of the progeny. However, a reduced mortality of the pups was observed in relation to vehicle-treated controls until postnatal day 10. Prenatal diazepam administration decreased [3H]dihydroalprenolol binding in frontal cortex, striatum, and hypothalamus but not in cerebellum. This decrease in beta-adrenergic receptor binding was due to a decrease in receptor density rather than in receptor affinity. In contrast, perinatal diazepam exposure led to a transient decrease in [3H]dihydroalprenolol binding limited to the frontal cortex. The permanent reduction in number of beta-adrenergic receptors, which depends on the scaling and duration of the drug application period, points to the necessity of a prolonged evaluation of effects of exposure to psychotropic drugs in early stages of brain development.

In 25-day-old rats with one eyelid sutured at the age of 10 days, the binding of [3H]flunitrazepam in the visual structures (retina, lateral geniculate nucleus, superior colliculus, visual cortex) and frontal cortex was determined. Monocular visual deprivation (MD) resulted in a significant decrease of the [3H]flunitrazepam binding in the retina of the open eye to about 76% of the control value. No changes in [3H]flunitrazepam binding were detectable under these conditions in the central visual structures examined and the non-visual cortical region. Scatchard analysis indicated that the changes found in the retina of the open eye of MD rats are due to a decreased binding affinity only, the maximum receptor number being unaffected. Eight hours after re-opening the sutured eyelid of 25-day-old MD rats, benzodiazepine binding in the open eye was increased to the control level, whereas the binding in the retina of the re-opened eye remained unchanged in comparison to control animals. Dark adaptation of 25-day-old control rats resulted in an increased [3H]flunitrazepam binding in the retina by 28% compared to that detectable in the retina of light-adapted animals. In contrast, dark-adaptation of MD rats did not affect [3H]flunitrazepam binding in the retina of both eyes in comparison to that found in the corresponding retina of light-adapted MD animals. The data obtained suggest a physiological coupling between both retinas, possibly mediated through centres inside of the central nervous system.