Localization and quantitation of proenkephalin-derived neuropeptides in the rat hippocampus

We have measured the content of met- and leu-enkephalin and dynorphin B in the rat hippocampus, and localized these opioid peptides within the intrinsic hippocampal neuronal circuitry with specific lesions. Several enkephalins, two of which were shown to be met- and leu-enkephalin, were identified in rat hippocampus. The levels of the enkephalin-related peptides were unaffected by intrahippocampal injections of colchicine, which destroyed the great majority of the hippocampal granule cells, while the level of dynorphin B, which serves as a marker for the proenkephalin B-derived peptides, was markedly depleted. Entorhinal cortical lesions ablating the perforant pathway input to the hippocampus did not significantly alter dynorphin B nor enkephalin levels in the hippocampus. Unilateral fimbrial transection caused a small but significant increase in dynorphin B on the side of the lesion relative to the non-lesioned side, although neither side was significantly different from control, while at the same time causing a significant bilateral increase in both met- and leu-enkephalin levels. This may result from loss of a direct or indirect stimulatory input to peptide-containing neurons within the hippocampus. The enkephalins appear to be located in neuronal cell bodies intrinsic to the body of the hippocampus, while the dynorphins are likely to be intrinsic only to the granule cell-mossy fiber system originating in the dentate gyrus.     

Localization and quantitation of dynorphin B in the rat hippocampus

The present study measures the content of dynorphin B in the rat hippocampus, and localizes the dynorphins within the intrinsic hippocampal neuronal circuitry. The level of dynorphin B, which is representative of the prodynorphin-derived peptides, was markedly depleted by intrahippocampal injection of colchice, which destroyed the great majority of the hippocampal granule cells and the associated mossy fiber pathway. The hippocampus contralateral to the injection demonstrated a slight, non-significant rise in dynorphin B levels after colchicine. Entorhinal cortical lesions ablating the perforant pathway input to the hippocampus did not significantly alter dynorphin B levels in the hippocampus. Unilateral fimbrial transection caused a small but significant increase in dynorphin B on the side of the lesion relative to the unlesioned side, but neither side was significantly different from control.     

Expression of the 70 kdalton neurofilament protein in clonal lines of mouse neuroblastoma

The present study measures the content of dynorphin B in the rat hippocampus, and localizes the dynorphins within the intrinsic hippocampal neuronal circuitry. The level of dynorphin B, which is representative of the prodynorphin-derived peptides, was markedly depleted by intrahippocampal injection of colchicine, which destroyed the great majority of the hippocampal granule cells and the associated mossy fiber pathway. The hippocampus contralateral to the injection demonstrated a slight, non-significant rise in dynorphin B levels after colchicine. Entorhinal cortical lesions ablating the perforant pathway input to the hippocampus did not significantly alter dynorphin B levels in the hippocampus. Unilateral fimbrial transection caused a small but significant increase in dynorphin B on the side of the lesion relative to the unlesioned side, but neither side was significantly different from control.     

Nicotine-induced alterations in peripheral tissue concentrations of native and cryptic Met- and Leu-enkephalin

This study examined the peripheral tissue distribution of native and cryptic Met- and Leu-enkephalin, and regulation of tissue enkephalins by nicotine. Met- and Leu-enkephalin concentrations showed widespread variation in tissue concentration and degree of processing. HPLC characterization of homogenate of spleen revealed that both native and cryptic immunoreactive Met-enkephalin are comprised of two peaks, one representing authentic Met-enkephalin pentapeptide and the other its sulfoxide. Subacute repeated administration of nicotine 0.1 mg/kg ip, six times at 30 min intervals, increased native Met- and Leu-enkephalin in adrenal medulla without affecting cryptic Met- and Leu-enkephalin concentrations, consistent with increased processing of larger peptides to Met- and Leu-enkephalin. Subacute nicotine decreased splenic concentrations of native and cryptic Met-enkephalin and native Leu-enkephalin, consistent with increased release of Met- and Leu-enkephalin from spleen and decreased synthesis of proenkephalin A or inadequate processing of larger peptides to enkephalin pentapeptides in spleen to compensate for the increased release during this period. HPLC characterization revealed that nicotine-induced decrease in native Met-enkephalin in spleen resulted from reductions in both pentapeptide and its sulfoxide. Nicotine also increased native Met-enkephalin in jejunum, decreased cryptic Met-enkephalin in heart atrium, increased native Leu-enkephalin in anterior pituitary and decreased cryptic Leu-enkephalin in jejunum. Nicotine may produce some of its effects through alterations in release of enkephalins from peripheral tissues.     

Characterization of the prodynorphin and proenkephalin neuropeptide systems in rat hippocampus

Opioid peptides derived from prodynorphin were localized immunocytochemically to dentate granule cells and mossy fibers of the rat hippocampus with antisera against dynorphin A(1-17) and dynorphin B. Extracts of microdissected hippocampal regions were resolved by reverse phase and molecular exclusion chromatography to identify the molecular forms of the dynorphin A immunoreactivity and to quantify regional contents. Results demonstrated that the relative concentration of dynorphin A within each dissected region of hippocampus agreed well with the distribution of dynorphin A detected by immunocytochemical methods. Immunostaining of proenkephalin-derived opioid peptides, [Leu5]enkephalin and bovine adrenal medullary peptide-22P, was concentrated in cell bodies of the entorhinal cortex, nerve fibers in the perforant pathway, and terminals in the outer molecular layer of the dentate gyrus. Light immunostaining of granule cells and mossy fibers with these antisera was also found. The relative concentration of [Leu5]enkephalin immunoreactivity in each microdissected region of the hippocampus also agreed well with the distribution of [Leu5]enkephalin immunostaining. Chromatography of hippocampal regional extracts demonstrated that the immunoreactivity measured was due to the presence of authentic [Leu5]enkephalin. The probable neurotransmitter function of both [Leu5]enkephalin and dynorphin A was shown by their calcium-dependent release after in vitro depolarization of hippocampal tissue. The reported presence of beta-endorphin in hippocampus was not verified. Comparison of the hippocampal distribution and content of prodynorphin and proenkephalin-derived opioids suggests that separate populations of neurons containing these two peptide families form distinct neurotransmitter systems of roughly equal concentration.     

Comparative localization of enkephalin and cholecystokinin immunoreactivities and heavy metals in the hippocampus

The present study is concerned with the cellular origins and identities of the hippocampal enkephalin and CCK-immunoreactive fibers and terminals. In the hippocampus of the rat, the guinea pig and the European hedgehog a system of enkephalin immunoreactive nerves emerges in the hilus of area dentata and can be followed to the apical dendrites of the hippocampal regio inferior pyramidal cells. This pattern of immunoreactive nerves corresponds to the hippocampal mossy fiber system as visualized by the Timm staining. Cholecystokinin immunoreactive nerve fibers and terminals reveal the same distribution in the guinea pig and the European hedgehog whereas in the rat the mossy fiber zone contains little or no cholecystokinin immunoreactivity. In the guinea pig degeneration of the mossy fibers after stereotactic lesions of the mossy fibers causes a complete loss of both enkephalin and cholecystokinin immunoreactivity in the mossy fiber zone. Only a few enkephalin immunoreactive cell bodies were scattered throughout the granular cell layer of area dentata, but inhibition of the axoplasmic transport by colchicine dramatically increased the number of enkephalin immunoreactive granule cell bodies. Enkephalin immunoreactive cell bodies were also detected in the hilus, throughout the pyramidal cell layer as well as in the stratum radiatum and stratum moleculare. Cholecystokinin immunoreactive cell bodies were seen in the hilus of the area dentata and in the stratum oriens, stratum pyramidale and stratum radiatum and the cell-rich layer of subiculum. No cholecystokinin immunoreactive cell bodies were observed in the granular cell layer of area dentata. Even after colchicine treatment the granule cells were devoid of cholecystokinin immunoreactivity. In the rat a system of nerves displaying enkephalin immunoreactivity was also observed in the superficial one-third of the molecular layer of area dentata, a zone which corresponds to the termination of the lateral perforant path. Another observation was that in the rat, but not in the guinea pig and the hedgehog, the terminal zone of both the medial perforant path and the zone of commissural and associational fibers of area dentata contained cholecystokinin immunoreactive molecules. In summary, our data show: (1) that the hippocampal mossy fibers contain enkephalin immunoreactive molecules; (2) that the cholecystokinin immunoreactivity in the mossy fiber zone is most likely also localized in the mossy fibers per se, although the granule cells seem devoid of cholecystokinin immunoreactivity; (3) zinc, here visualized as a Timm-positive substance, is also localized in the mossy fiber terminals; further, (4) other intrinsic cell bodies than the granule cells may contribute to both the enkephalin and cholecystokinin immunoreactive terminals within the hippocampus; (5) in the rat the lateral perforant path may be enkephalinergic; and (6) both the terminal zone of the medial perforant path and the associational and commissural fibers of the rat contain cholecystokinin immunoreactivity.     

Localization of cytochrome oxidase (COX) activity and COX mRNA in the hippocampus and entorhinal cortex of the monkey brain: correlation with specific neuronal pathways

Cytochrome oxidase (COX) activity and COX II mRNA expression were localized in the hippocampal formation and entorhinal cortex of the rhesus monkey brain by means of enzyme histochemistry and in situ hybridization, respectively. Within the hippocampal formation, the terminal field of the perforant pathway showed the highest levels of COX activity, whereas COX II mRNA was localized mainly in neuronal cell bodies. In the entorhinal cortex. COX II mRNA was detected in neuronal cell bodies of layers II and IV. These results indicate that the pattern of localization of COX and its mRNA in entorhinal cortex correlates with the input and output pathways of the hippocampus.     

Perforant pathway changes and the memory impairment of Alzheimer's disease

The perforant pathway is a large neuronal projection that arises from layers II and III of the entorhinal cortex of the parahippocampal gyrus. It is the principal source of cortical input to the hippocampal formation. In 11 cases of Alzheimer's disease, we have found that neurofibrillary tangles develop in the cells of origin of the perforant pathway. In addition, the termination zone of the perforant pathway, in the outer two thirds of the molecular layer of the dentate gyrus, contains a distinct layer of neuritic plaques. None of the 8 control subjects had such changes. These profound alterations effectively disconnect the hippocampal formation from the association and limbic cortices. Because of the central role of the hippocampus and parahippocampal gyrus in learning, it is likely that pathological changes in the perforant pathway, by precluding normal hippocampal operation, account for some aspects of the memory impairment in Alzheimer's disease.     

Outgrowth-promoting molecules in the adult hippocampus after perforant path lesion

Lesion-induced neuronal plasticity in the adult central nervous system of higher vertebrates appears to be controlled by region- and layer-specific molecules. In this study we demonstrate that membrane-bound hippocampal outgrowth-promoting molecules, as present during the development of the entorhino-hippocampal system and absent or masked in the adult hippocampus, appear 10 days after transection of the perforant pathway. We used an outgrowth preference assay to analyse the outgrowth preference of axons from postnatal entorhinal explants on alternating membrane lanes obtained from hippocampus deafferented from its entorhinal input taken 4, 10, 20, 30 and 80 days post-lesion and from adult control hippocampus. Neurites from the entorhinal cortex preferred to extend axons on hippocampal membranes disconnected from their entorhinal input for 10 days in comparison with membranes obtained from unlesioned adult animals. Membranes obtained from hippocampi disconnected from their entorhinal input for 10 days were equally as attractive for growing entorhinal cortex (EC) axons as membranes from early postnatal hippocampi. Further analysis of membrane properties in an outgrowth length assay showed that entorhinal axons extended significantly longer on stripes of lesioned hippocampal membranes in comparison with unlesioned hippocampal membranes. This effect was most prominent 10 days after lesion, a time point at which axonal sprouting and reactive synaptogenesis are at their peak. Phospholipase treatment of membranes obtained from unlesioned hippocampi of adult animals strongly promoted the outgrowth length of entorhinal axons on these membranes but did not affect their outgrowth preference for deafferented hippocampal membranes. Our results indicate that membrane-bound outgrowth-promoting molecules are reactivated in the adult hippocampus following transection of the perforant pathway, and that neonatal entorhinal axons are able to respond to these molecules. These findings support the hypothesis of a temporal accessibility of membrane-bound factors governing the layer-specific sprouting of remaining axons following perforant path lesion in vivo.     

Regional and subcellular distribution of Met- and Leu-enkephalin-degrading activity in rat brain

Met- and Leu-enkephalin were degraded rapidly by brain aminopeptidases with the K_m's 9.1 mM and 5.7 mM respectively; the V_max, 100 μmol/mg protein per min for Met-enkephalin and 50 μmol/mg protein per min for Leu-enkephalin. The major product for Met-enkephalin was des-Tyr-Met-enkephalin. The enkephalin-degrading activity (EDA) in the brain was 16-fold higher than in plasma and was 15% of that in the kidney. The hydrolytic activity was heterogeneous in rat brain regions. For Met-enkephalin, the activity in decreasing order was striatum, hypothalamus, hippocampus, cerebellum, cortex, mid-brain, and medulla oblongata; for Leu-enkephalin the order was hippocampus, striatum, mid-brain, cortex, hypothalamus, cerebellum, and medulla oblongata. The subcellular distribution of the EDA in the whole brain, the hippocampus, and the striatum was similar, with the soluble fraction having the highest, the synaptosomal fraction the lowest, activity. The distribution of EDA was different from the arylamidase activity with Tyr-β-naphthylamide and Leu-β-naphthylamide as substrates. Our results indicate that a group of aminopeptidases is responsible for the degradation of both enkephalins.     

Reinnervation of the hippocampal perforant pathway zone in Alzheimer's disease

The perforant pathway originates from the entorhinal cortex of the anterior parahippocampal gyrus and terminates on the outer dendritic branches of the granule cells of the dentate gyrus and pyramidal cells of the subiculum and hippocampus. It carries the principal cortical input to the hippocampal formation. Destruction of the perforant pathway in experimental animals leads to a partial deafferentation of its target neurons, followed by a robust sprouting of acetylcholinesterase (AChE) terminals in the deafferented perforant pathway zone. In Alzheimer's disease, the cells of origin of the perforant pathway are laden with neurofibrillary tangles. AChE staining in the terminal zone of the perforant pathway in Alzheimer's disease shows several distinct patterns that are not found in control brains. These changes are consistent with the results of experimental studies demonstrating reinnervation in laboratory mammals, including nonhuman primates. The results suggest that in Alzheimer's disease sprouting of AChE-containing systems occurs in the hippocampal formation in response to disease-related cellular damage in the entorhinal cortex.     

Long-term potentiation recruits a trisynaptic excitatory associative network within the mouse dentate gyrus

Granule cells of the hippocampal dentate gyrus receive two powerful excitatory inputs: the perforant path, originating from the entorhinal cortex, and the associational pathway, originating from mossy cells, the principal neurons of the dentate gyrus hilus. We examined the electrophysiological properties of the less well-studied associational pathway and its interaction with the perforant path in the intact mouse hippocampus and then tested homosynaptic, trans-synaptic and associative long-term potentiation of these pathways. The associational pathway was either monosynaptically activated by stimulation within the inner molecular layer or trisynaptically activated after stimulation of the perforant path. Laminar profiles of extracellularly recorded associational pathway field potentials demonstrated a bell-shaped curve with a peak in the inner molecular layer. Tetanization of the perforant path induced not only homosynaptic potentiation of the perforant path (162.4 +/- 6.7% at 0.5-1.5 h after tetanus) but also heterosynaptic potentiation of the associational pathway (115.7 +/- 4.9%). Direct tetanization of the associational pathway within the inner molecular layer was ineffective in either the septo-temporal (97.2 +/- 4.5%) or temporal-septal (104.4 +/- 4.6%) direction. In contrast, conjoint tetanization of the associational pathway with the perforant path potentiated the associational pathway responses in both the septo-temporal (123.4 +/- 5.8%) and the temporal-septal (124.8 +/- 7.3%) directions. Paired-pulse facilitation was attenuated by long-term potentiation in the perforant path and the associational pathway, suggesting pre-synaptic involvement. These results demonstrate that long-term potentiation of the associational pathway and the perforant path is a product of the network properties of the dentate gyrus rather than of each monosynaptic input alone. The architecture of this neural network may be designed for flexible dynamic associations of the afferent perforant path inputs to configure encoded information within hippocampal neuronal ensembles.     

Characterization of perforant path lesions in rodent models of memory and attention

Early stage Alzheimer's disease (AD) pathology is associated with neurodegeneration of systems within the temporal cortex, e.g. the entorhinal cortex, perforant pathway and hippocampus. The perforant pathway provides the major neuronal input to the hippocampus from the entorhinal cortex and thus relays multimodal sensory information derived from cortical zones into the hippocampus. The earliest symptoms of AD include cognitive impairments, e.g. deficits in short-term memory and attention. Consequently, we have investigated the effect of bilateral knife cut lesions to the perforant path on cognition in rats using models measuring primarily short-term memory (operant delayed match to position task), attention (serial five-choice reaction time task) and spatial learning (Morris water maze). Rats receiving bilateral perforant path lesions showed normal neurological function and a mild hyperactivity. The lesion produced little effect on attention assessed using the five-choice task. In contrast, animals with equivalent lesions showed a robust delay-dependent deficit in the delayed match to position task. Spatial learning in the water maze task was also severely impaired. The delay-dependent deficit in the match to position task was not reversed by tacrine (3 mg/kg) pretreatment. The present data support a selective impairment of cognitive function following perforant path lesions that was confined to mnemonic rather than attentional processing. These findings complement primate and human studies identifying a critical role of the perforant pathway and associated temporal lobe structures in declarative memory. Degeneration of the perforant pathway is likely to contribute to the mnemonic deficits characteristic of early AD. The failure of tacrine to ameliorate these deficits may be relevant to an emerging clinical literature suggesting that cholinomimetic therapies improve attentional rather than mnemonic function in AD.     

Evidence for amelioration of ischaemic neuronal damage in the hippocampal formation by lesions of the perforant path

The effect of lesions of two excitatory afferent pathways on the cellular damage in the hippocampus following complete cerebral ischaemia was investigated in the rat. Lesions transecting the perforant path led to a significant decrease in cellular damage in the hippocampal CA1 region ipsilateral to the lesion as compared to the contalateral side and to control. Lesions of the fimbria-fornix, on the other hand, had no significant effects. We propose that the protective effect of the perforant path lesions is due to removal of glutamatergic/aspartergic pathways and that release of these excitatory amino acids might be a critical factor for neuronal necrosis following cerebral ischaemia.     

Evidence for amelioration of ischaemic neuronal damage in the hippocampal formation by lesions of the perforant path

The effect of lesions of two excitatory afferent pathways on the cellular damage in the hippocampus following complete cerebral ischaemia was investigated in the rat Lesions transecting the perforant path led to a significant decrease in cellular damage in the hippocampal CA1 region ipsilateral to the lesion as compared to the contatterai side and to control. Lesions of the fimbria-fornix, on the other hand, had no significant effects. We propose that the protective effect of the perforant path lesions is due to removal of glutamatergic/aspartergic pathways and that release of these excitatory amino acids might be a critical factor for neuronal necrosis following cerebral ischaemia.     

Enkephalin convertase: localization to specific neuronal pathways

3H-Guanidinoethylmercaptosuccinic acid (GEMSA) selectively labels the carboxypeptidase B-like enzyme enkephalin convertase (EC) in rat brain tissue sections. We have used autoradiography with 3H-GEMSA to map membrane-bound EC in the rat forebrain and, in conjunction with lesioning techniques, to localize EC to specific neuronal pathways. The highest levels of EC are in the median eminence. High levels of EC also occur in the hypothalamic magnocellular nuclei, in several nuclei of the amygdala, the lateral septum, and the bed nuclei of the stria terminalis. Knife-cut lesions of the stria terminalis increase EC posterior to the lesion in the stria and deplete EC from the stria adjacent to the bed nucleus, suggesting that EC, like enkephalins, is axonally transported within the stria terminalis. Ibotenic acid lesions of the caudate nucleus destroy binding in the substantia nigra pars reticulata ipsilateral to the lesion, suggesting that nigral EC is associated with axons originating in the caudate nucleus. We have also mapped EC in detail in the hippocampus. EC levels are highest near pyramidal cells of CA 3-4 and the dentate gyrus granule cells. Quinolinic acid lesions destroy both the granule and pyramidal cells and destroy all of the 3H-GEMSA labeling except for a small amount in the molecular layer of the dentate gyrus. Selective destruction of CA 3-4 pyramidal cells with kainic acid eliminates EC in the pyramidal cell region. Destruction of granule cells of the dentate gyrus with colchicine depletes binding in the dentate gyrus without any change in the area surrounding field CA 3-4. High levels of 3H-GEMSA binding are present in the hippocampus at least 3 d before birth. These observations suggest that in the hippocampus the majority of EC is associated with pyramidal cells, which have not been shown to contain enkephalins. 3H-GEMSA autoradiography of the trigeminal ganglion localizes EC to the sensory neurons and not to white matter tracts there. These studies demonstrate that while EC is contained in enkephalinergic pathways, it is also present in some neurons that do not contain enkephalins.     

Enkephalin inhibition of inhibitory input to CA1 and CA3 pyramidal neurons in the hippocampus

Enkephalin-induced excitation in the hippocampus has been attributed to the attenuation of inhibitory input as well as to augmentation of excitatory input to pyramidal neurons. We have further examined these possible mechanisms of enkephalin action, as well as the possibility that enkephalins may be affecting intrinsic membrane properties, by recording intracellularly from CA1 and CA3 pyramidal cells in the guinea pig hippocampal brain slice preparation. It was observed that the inhibitory synaptic potential was significantly decreased in the presence of leucine enkephalin and D-alanine, D-leucine-enkephalin (DADL), whereas the excitatory synaptic potential, revealed by block of the inhibitory postsynaptic potential (IPSP) by bicuculline, was unaltered. In addition, the response of pyramidal cells to pressure-applied GABA was unaffected by enkephalin, as were the voltage-dependent membrane conductances. The increase in excitability which was observed in both field potential and intracellular recordings to drop application of DADL must, then, be due to a purely presynaptic block of inhibitory interneurons in both the CA1 and CA3 areas of the hippocampus.     

Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata

The hippocampus and fascia dentata receive their major extrinsic input from the entorhinal area through the so-called perforant path. This pathway is now shown to be composed of at least two distinct fiber systems: (1) A medial perforant path coming from the medial part of the entorhinal area and terminating in the middle of the dentate molecular layer and in the deep half of the stratum lacunosum-moleculare of the hippocampal subfield CA3. (2) A lateral perforant path from the lateral part of the entorhinal area to a superficial zone in the dentate molecular layer and to the superfcial part of the stratum lacunosum-moleculare of CA3. This paper deals specifically with the lateral perforant path. A third group of perforant fibers, bing intermediate to the others with regard to both origin and termination has been noticed in one animal. The fiber-course of the lateral perforant path is found to be identical to that previously described for the medial path. The terminal field is present along the whole axial extent of the hippocampus and fascia dentata, i.e., from the temporal tip to the subsplenial portion. No sings of degeneration corresponding to the so-called alvear path were observed following lesions of either the medial or the lateral part of the entorhinal cortex. Terminal degeneration appeared in the molecular layer of the subiculum and CA1 and in the anterior continuation of the hippocampal formation subsequent to lesions including the prepyriform cortex.