Brain Research, 524 (1990) 331-335
331
Elsevier BRES 24215
Prolonged electrophysiological maturation of transplanted hippocampal neurons Lori A. Mudrick, Jasmine Stabel, Roland S.G. Jones and Uwe Heinemann lnstitut far Neurophysiologie, Universitiit zu K6ln, Cologne (E R. G.)
(Accepted 24 April 1990) Key words: Hippocampal CA1 region; Cerebral ischemia; Neuronal transplantation; Intracellular recording; Calcium; Excitatory amino acid;
Development
The CA1 region of the rat hippocampal formation was lesioned by transient forebrain ischemia and subsequently repopulated with dispersed fetal hippocampal neurons. Using the hippocampal slice preparation, 7-12 months post-transplantation, intracellular recordings were made during synaptic activation and extracellular calcium measurements were made during iontophoretic application of excitatory amino acids. The data indicate that the developmental period of the transplanted neurons is prolonged with respect to N-methyl-D-aspartate (NMDA) receptor-mediated responses and may be involved in maintaining calcium dependent developmental processes such as fiber outgrowth. In the rat hippocampal formation almost the entire population of dorsal CA1 hippocampal neurons are lost following transient forebrain ischemia 3'19'25. This region can be repopulated by the injection of dispersed fetal hippocampal neurons 15. Within the transplants CAl-like pyramidal neurons have been identified, both electrophysiologically and anatomically, and they comprise approximately 70% of the transplanted cell population 15' 16. A good number of cells contain parvalbumin which represent a population of ~-aminobutyric acid (GABAergic) interneurons 2'9. This suggests the presence of local inhibitory circuitries. We have been investigating the ability of the transplanted CAl-like pyramidal neurons (TCPN) to reinstate synaptic connections and to attain intrinsic properties that are characteristic of the adult CA1 pyramidal neurons (ACPN) they have replaced. By using morphological criteria the TCPN appear to have attained an adult state by 1-2 months post-transplantation. In contrast, although the TCPN have developed adult like biophysical properties (resting membrane potential, spike amplitude, input resistance, time constant) at 2 months, the evoked activity recorded from the TCPN is different from that recorded from ACPN. This difference is primarily manifested by very prolonged EPSP's and the firing of multiple action potentials 16. The presence of IPSP's arising from local inhibitory circuits are not obvious following subthreshold or suprathreshold activation despite the anatomical localization of parvalbumin- (GABAergic) positive terminal varicosities surrounding the TCPN. These properties are characteristic
of immature pyramidal neurons 1'23'24. The presence of such properties could be explained if the inhibitory synapses were intact but the hyperpolarizing events were masked by enhanced excitatory events. The present investigation was designed to follow the synaptic development of the TCPN. The results show that maturation of synaptic characteristics of TCPN is prolonged for up to 12 months. It appears that NMDA receptors may have an enhanced role in mediating synaptic transmission in TCPN during this prolonged developmental period. Hippocampal slices were obtained from 6 age-matched control animals and from 6 animals which had been subjected to cerebral ischemia, to lesion the CA1 region, and subsequently transplanted unilaterally with fetal hippocampal neurons 15'16. Seven to 14 months posttransplantation (PT) slices nominally 400 ,um thick were prepared from the ischemic/transplanted hemisphere, the ischemic hemisphere and the control brain and were distributed between 3 recording chambers. Methodologies for preparation and maintenance have been described previously 13. Either intracellular measurements were made of synaptic activity or measurements were made of the extracellular C a 2÷ changes following the iontophoretic application of the excitatory amino acids (EAAs) NMDA, glutamate (Glu), and quisqualate (Quis). Intracellular electrodes (o.d. 1.2 mm) were filled with 1-3 M potassium acetate (40-80 MI2) and used to probe the transplanted region or the CA1 stratum pyramidale (SP) of control and ischemic slices. Once a
Correspondence: L.A. Mudrick, Institut ffir Neurophysiologie, Universit~it zu K61n, Robert-Koch-Strasse 39, 5000 Cologne 41, F.R.G.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
332 stable recovery had been obtained following impalement, the neurons were activated by shocks to the Schaffer collaterals (bipolar electrodes, square wave pulses 0.1 ms, 0.2 Hz, 5-40 V) or by current injection (0.3-2.0 nA; 125-225 ms) through the recording electrode utilizing conventional current clamp techniques. Ca2+-sensitive/ reference electrodes were pulled from theta glass and the reference barrel was filled with 150 mM NaC1. The Ca2+-sensitive barrel was filled with 100 mM CaCi 2 and the tip with Ca2+-sensitive resin (Fluka cocktail 21048) (tip diameter 1-3 #m, ref. 1-10 MI2, Ca2+-sensitive 5-40 GO). Iontophoretic electrodes were filled with Quis (Cambridge Research Biochemicals), NMDA (CRB) and/or Glu (Sigma) (100 mM in 150 mM NaC1, pH 7.4) and glued to the calcium-sensitive electrodes with a tip separation of 15-30 ~m. The microelectrode complex was placed in the stratum pyramidale of a control slice to record the changes in [Ca2+]o and associated negative field potentials following iontophoretic application (15 s) of the EAAs. Five min intervals were allowed between each application. After 3-5 measurements along the somato-dendritic extent (100/~m increments) the electrode complex was positioned within the shrunken CA1 region of an ischemic slice and measurements were made. Finally, the microelectrode complex was positioned in slices prepared from ischemic/transplanted animals and measurements were made from the opaque transplanted regions. After recording in the transplanted slices the electrode complex was returned to the control slice and measurements were made to confirm that the sensitivity of the electrode had not been altered during the experiment. To quantify the [Ca2+]o decreases the ischemic and transplant values were normalized with respect to the control value of the experimental day. After recording, the slices were immersion fixed in paraformaldehyde (4% in phosphate buffer) and cryostat sections were made. These sections were stained with thionin (0.5%) to confirm the ischemic lesion and the presence of the transplant. Transplanted CAl-like pyramidal neurons (TPCN) were identified by their general membrane properties and response to depolarizing current injection 21'22 which were not obviously different from adult CA1 pyramidal neurons (ACPN) measured in the same chamber. In an earlier study we had shown that in younger TCPN (2-7 months post-transplantation) the excitatory postsynaptic potential (EPSP; measured just subthreshold for spike activation) rose sharply and was followed by a prolonged decay to baseline (EPSP duration 25-200 ms) when compared to control values. The strong hyperpolarization, due to the early inhibitory postsynaptic potential (IPSP), which normally follows the EPSP in ACPN was not observed in these transplanted neurons 16. In some
A
B
~ 2 mV 10 ms
~ 2 mV 10 ms
G
Washout
10 ms
Fig. 1. A and B are examples to illustrate the effects of bath-applied DL-APV (30 /~M) on the EPSPs recorded from the transplanted neurons. The amplitude and duration could be reduced by APV (5 min) in TCPN with a very prolonged EPSP (A). APV did not affect the EPSP when the duration was less prolonged (B) (RMP --76 mV, - - 73 mV, respectively). C illustrates an EPSP followed by a strong IPSP (RMP --57 mV). The IPSP was blocked by bicuculline (2.5 /~M; 15 min) which increased the amplitude and duration of the EPSP. The IPSP recovered after washout (25 min) of bicucuiline ( R M P - - 5 3 mV).
TCPN (2-3 months post-transplantation) more than one action potential could be evoked and were followed by a long lasting hyperpolarization. The present study showed that between 7 and 14 months post-transplantation two groups of TCPN could be identified. One group demonstrated prolonged EPSPs that were not different from the younger TCPN (n = 15). Bath application of 2-amino-phosphonovaleric acid (APV, 30/ira) reduced the EPSP duration in 3 of 5 of these neurons. The EPSP duration ( > 60 ms) was prolonged in these neurons wheras the EPSP duration was much shorter (< 30 ms) in the neurons not demonstrating an obvious APV sensitive component (Fig. 1A,B). The second group of older TCPN (n = 25) demonstrated synaptic responses with EPSP-IPSP sequences characteristic of ACPN. In these cases the early IPSP could be blocked by bicuculline (2.5-5/~m) in a reversible manner (Fig. 1C). The slices containing the greatest proportion of TCPN demonstrating EPSP-IPSP sequences were made from the oldest animals (12-14 months post-transplantation). In the CA1 of control slices NMDA and Glu applica-
333 concentration-dependent changes in [Ca2+]o and corresponding negative potential shifts, the kinetics of which were comparable to those observed in the control CA1 region. In 3 of 6 experiments NMDA- and Glu-evoked decreases in [Ca2+]o were similar to or greater in amplitude than those recorded in the normal CA1 region in spite of the fact that stimulus induced ionic changes were smaller than in control area CA1 (Mudrick, K6hr and Heinemann, in preparation). In two experiments comparable responses could be evoked with double the ejection current and in one experiment even with high ejection current the reponses were smaller. Changes in [Ca2+]o during Quis application always required very high ejection currents to produce similar responses (Fig. 2). A comparison of the normalized data showed that the decreases in [Ca2+]o evoked by Quis, using equivalent ejection currents, were only 34% _+ 14 (S.D., n = 5) of the control values whereas the decreases in [Ca2+]o evoked similarly by NMDA and Glu were 91% + 42 (S.D., n = 6) of the control values. Early in development neurons usually receive an extensive afferent component which is attenuated greatly over time. This results from 'programmed' cell death and
tion (20-60 nA) produced concentration-dependent decreases in [Ca2+]o with similar kinetics. Control experiments have shown the Glu-evoked decrease to be due to activation of both NMDA receptors and voltage-dependent calcium channels. Decreases in [Ca2+]o could be observed at the onset of iontophoresis and continued until the current was discontinued. The [Ca2+]o then returned rapidly to baseline. Quis (45-100 nA) evoked a decrease in [Ca2+]o at the onset of application if the neurons were depolarized sufficiently to activate voltagedependent Ca 2÷ channels 1°'11. Calcium levels then returned to baseline and usually increased from baseline after termination of Quis application which has been attributed to active extrusion 4"1°'2°. All responses to E A A application were accompanied by a slow negative field potential. When similar recordings were made in area CA1 of ischemic slices changes in [Ca2+]o were very small even at high ejection currents (approx. 15% of control). This was due to the lack of viable neurons in the CA1 region of these slices and Ca z+ uptake by the surviving interneurons. Within the transplants Quis, Glu and NMDA induced
A
Control
Ischemic
B
Transplant
++ - 4 0 nA Glu
- 4 0 nA Glu
Control
Transplant
09
,.,,.-V- 4 0 nA
- 25 nA NMDA
Glu
]++ - 35 nA N M D A
2.0
+
1.6 m M
t+a+,Jol
1.3
~
1.3
+ +50 nA Quis
1.6 m M
[Ca2'
- 85 nA Quis
(],2+ -70 nA Quis 20 s
- 60 nA Quis
- 150 nA Quis 20 s
Fig. 2. A: a comparison of the changes in [Ca:+]o and corresponding cxtracellular field potentials evoked by the iontophoretic application (15 s) of Glu and Quis in control, ischcmic and transplanted slices. The reponse to Glu in the transplant is similar to that evoked in the control. In contrast, the response to Quis application is much smaller in the transplanted slices. The responses are minimal in the ischcmic slice. B : the action of H M D A on the extraccllular potential and [Ca2+]o is similar to that evoked by Glu. Evoked responses are strong during N M D A but minimal during Quis application even at extremely high ejection currents.
334 removal of excess synaptic contacts. Afferent fiber distribution to the transplanted neurons may occur in a similar manner, with extensive sprouting by the adult host, but the programmed cell death is not likely to occur. Therefore, it may take much longer for the density and distribution of afferent fibers to approach normal levels. The ability of the transplanted neurons to obtain a totally mature membrane composition may be dependent upon a normal complement of afferent input 12. In younger TCPN, excitatory events may result in an excessive increase in Ca 2+ influx which could mask the inward CI-- current mediating the IPSP 7. Some evidence for this contention has been obtained from immature neurons in which N M D A evoked currents are less voltage dependent and can be activated at quite negative membrane potentials ~. A n enhancement of N M D A receptor-mediated decreases in [Ca2+]o has been shown in area C A I of young slices suggesting participation of N M D A receptors during low frequency synaptic transmission 5. This compares to our findings in the transplants and may explain the prolongation of the EPSP. Indeed, prolonged EPSPs were reduced by A P V application. The extracellular calcium measurements suggest that receptors for the excitatory amino acids are abundant in the transplanted neurons. Although Quis induced decreases in [Ca2+]o were smaller than in control slices, which is what could be expected from a lesser number of neurons, the decreases in [Ca2+]o in response to N M D A were large when compared to control slices in spite of the fact that the neuronal packing density in the transplants is lower than in intact area CA1 16 (Mudrick & Baimbridge, submitted). Similarly, changes in [Ca2+]o within
This research was supported by the Canadian Heart and Stroke Foundation (L.A.M.) and the SFB 200(C8) (U.H.). We acknowledge gratefully M. Groenenwald for her assistance in preparing the ion-sensitive electrodes and the figures.
Ben-Ari, Y., Cherubini, E. and Krnjevic, K., Changes in voltage dependence of NMDA currents during development, Neurosci. Len., 94 (1988) 88-92. Celio, M.R., Parvalbumin in most gamma-aminobutyric acidcontaining neurons of the rat cerebral cortex, Science, 231 (1986) 995-997. Diemer, N.H. and Siemkowicz, E., Regional neurone damage after cerebral ischemia in normo- and hypoglycemic rats, Neuropathol. Appl. Neurobiol., 7 (1981) 217-227. Hamon, B. and Heinemann, U., Effects of GABA and bicuculline on N-methyl-D-aspartate- and quisqualate-induced reductions in extracellular free calcium in area CA1 of the hippocampal slice, Exp. Brain Res., 64 (1986) 27-36. Hamon, B. and Heinemann, U., Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus, Dev. Brain Res., 38 (1988) 286-290. Heinemann, U., Jones, R.S.G., K6hr, G., Mudrick, L. and Stabel, J., Functional properties of neuronal grafts in area CA1 studied in hippocampal slices from ischaemic rats, J. Physiol., 413 (1989) 32P. Inoue, M., Oomura, Y., Yakushiji, T. and Akaike, N., Intracellular calcium ions decrease the affinity of the GABA receptor, Nature, 324 (1986) 156-158. Jones, R.S.G. and Heinemann, U., Spontaneous activity mediated by NMDA receptors in immature rat entorhinal cortex in
vitro, Neurosci. Lett., 104 (1989) 93-98. 9 Kosaka, T., Katsumaru, H., Hama, K., Wu, J.-Y. and Heizmann, C.W., GABA-ergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus, Brain Research, 419 (1987) 119-130. 10 Lambert, J.D.C. and Heinemann, U., Aspects of the action of excitatory amino acids on hippocampal CA1 neurons. In U. Heinemann, M. Klee, E. Neher and W. Singer (Eds.), Calcium Electrogenesis and Neuronal Functioning, Springer, Heidelberg, 1986, pp. 279-290. 11 Lambert, J.D.C. and Heinemann, U., Ionic movements associated with the action of excitatory amino acids on pyramidal neurons in area CA1 of rat hippocampal slices, 1989, submitted. 12 Lipton, S.A. and Kater, S.B., Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, Trends Neurosci., 12 (1989) 265-270. 13 Mody, I., Stanton, P.K. and Heinemann, U., Activation of N-methyl-D-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling, J. Neurophysiol., 59 (1988) 1033-1054. 14 Mody, I., Lambert, J.D.C. and Heinemann, U., Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices, J. Neurophysiol., 57 (1987) 869-888. 15 Mudrick, L.A., Leung, P.P.-H., Baimbridge, K.G. and Miller,
1
2 3 4
5 6
7 8
the transplants during repetitive stimulation were enhanced 4-fold in low Mg 2+ medium, compared to normal medium 6 (Mudrick, K6hr, Heinemann, in preparation), which is much greater than the 1- to 2-fold enhancement observed in the intact CA1 region 14. These data indicate that in the TCPN the efficacy and/or density of N M D A receptors is enhanced which would suggest that the T C P N are maintaining a state which they reach about 2 weeks postnatal 5. Whether there is also a difference in voltage dependency remains to be determined. It has been suggested that, in immature neurons, an enhanced N M D A - e v o k e d Ca 2+ influx may have an important role in promoting neuronal growth and differentiation 1'17'18. In addition, neurons in the entorhinal cortex have been shown to possess an enhanced N M D A component during the period of fiber outgrowth to and synapse formation with the dentate granule cells which seems to correspond with the prolonged ontogenesis and development of these cells 8. The same may be true for the transplanted neurons but it may be necessary for this growth enhancing property to remain 'turned on' for a longer time. Since development in an adult environment is quite different from that which occurs in a perinatal environment, this potential growth enhancing property observed in immature neurons may play an important role in the fiber outgrowth and integration of the TCPN. Although this hypothesis has not been tested in vivo it would be interesting to investigate the effects of N M D A receptor blockade on the development of transplanted neurons.
335
16
17 18
19 20
J.J., Neuronal transplants used in the repair of acute ischemic injury in the CNS, Prog. Brain Res., 78 (1988) 87-93. Mudrick, L.A., Baimbridge, K.G. and Peet, M.J., Hippocampal neurons transplanted into ischemically lesioned hippocampus: electroresponsiveness and reestablishment of circuitries, Exp. Brain Res., 76 (1989) 333-342. Patterson, P.H., On the importance of being inhibited, or saying no to growth cones, Neuron, 1 (1988) 263-267. Pearce, I.A., Cambray-Deakin, M.A. and Burgoyne, R.D., Glutamate acting on NMDA receptors stimulates neurite outgrowth from cerebellar granule cells, FEBS Lea., 223 (1987) 143-147. Pulsinelli, W.A. and Brierly, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. Pumain, R., Kurcewicz, I. and Louvel, J., Ionic changes induced
21 22 23 24 25
by excitatory amino acids in the rat cerebral cortex, Can. J. Physiol. Pharmacol., 65 (1987) 1067-1077. Schwartzkroin, P.A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Research, 85 (1975) 423-436. Schwartzkroin, P.A., Further characteristics of hippocampal CA1 cells in vitro, Brain Research, 128 (1977) 53-68. Schwartzkroin, P.A., Development of rabbit hippocampus: physiology, Dev. Brain Res., 2 (1982) 469-486. Schwartzkroin, P.A., Regulation of excitability in hippocampal neurons. In The Hippocampus, Vol. 3, Plenum, New York, 1986, pp. 113-136. Smith, M.-L., Auer, R.N. and Siesjo, B.K., The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia, Acta Neuropathol., 64 (1984) 319-332.
331
Elsevier BRES 24215
Prolonged electrophysiological maturation of transplanted hippocampal neurons Lori A. Mudrick, Jasmine Stabel, Roland S.G. Jones and Uwe Heinemann lnstitut far Neurophysiologie, Universitiit zu K6ln, Cologne (E R. G.)
(Accepted 24 April 1990) Key words: Hippocampal CA1 region; Cerebral ischemia; Neuronal transplantation; Intracellular recording; Calcium; Excitatory amino acid;
Development
The CA1 region of the rat hippocampal formation was lesioned by transient forebrain ischemia and subsequently repopulated with dispersed fetal hippocampal neurons. Using the hippocampal slice preparation, 7-12 months post-transplantation, intracellular recordings were made during synaptic activation and extracellular calcium measurements were made during iontophoretic application of excitatory amino acids. The data indicate that the developmental period of the transplanted neurons is prolonged with respect to N-methyl-D-aspartate (NMDA) receptor-mediated responses and may be involved in maintaining calcium dependent developmental processes such as fiber outgrowth. In the rat hippocampal formation almost the entire population of dorsal CA1 hippocampal neurons are lost following transient forebrain ischemia 3'19'25. This region can be repopulated by the injection of dispersed fetal hippocampal neurons 15. Within the transplants CAl-like pyramidal neurons have been identified, both electrophysiologically and anatomically, and they comprise approximately 70% of the transplanted cell population 15' 16. A good number of cells contain parvalbumin which represent a population of ~-aminobutyric acid (GABAergic) interneurons 2'9. This suggests the presence of local inhibitory circuitries. We have been investigating the ability of the transplanted CAl-like pyramidal neurons (TCPN) to reinstate synaptic connections and to attain intrinsic properties that are characteristic of the adult CA1 pyramidal neurons (ACPN) they have replaced. By using morphological criteria the TCPN appear to have attained an adult state by 1-2 months post-transplantation. In contrast, although the TCPN have developed adult like biophysical properties (resting membrane potential, spike amplitude, input resistance, time constant) at 2 months, the evoked activity recorded from the TCPN is different from that recorded from ACPN. This difference is primarily manifested by very prolonged EPSP's and the firing of multiple action potentials 16. The presence of IPSP's arising from local inhibitory circuits are not obvious following subthreshold or suprathreshold activation despite the anatomical localization of parvalbumin- (GABAergic) positive terminal varicosities surrounding the TCPN. These properties are characteristic
of immature pyramidal neurons 1'23'24. The presence of such properties could be explained if the inhibitory synapses were intact but the hyperpolarizing events were masked by enhanced excitatory events. The present investigation was designed to follow the synaptic development of the TCPN. The results show that maturation of synaptic characteristics of TCPN is prolonged for up to 12 months. It appears that NMDA receptors may have an enhanced role in mediating synaptic transmission in TCPN during this prolonged developmental period. Hippocampal slices were obtained from 6 age-matched control animals and from 6 animals which had been subjected to cerebral ischemia, to lesion the CA1 region, and subsequently transplanted unilaterally with fetal hippocampal neurons 15'16. Seven to 14 months posttransplantation (PT) slices nominally 400 ,um thick were prepared from the ischemic/transplanted hemisphere, the ischemic hemisphere and the control brain and were distributed between 3 recording chambers. Methodologies for preparation and maintenance have been described previously 13. Either intracellular measurements were made of synaptic activity or measurements were made of the extracellular C a 2÷ changes following the iontophoretic application of the excitatory amino acids (EAAs) NMDA, glutamate (Glu), and quisqualate (Quis). Intracellular electrodes (o.d. 1.2 mm) were filled with 1-3 M potassium acetate (40-80 MI2) and used to probe the transplanted region or the CA1 stratum pyramidale (SP) of control and ischemic slices. Once a
Correspondence: L.A. Mudrick, Institut ffir Neurophysiologie, Universit~it zu K61n, Robert-Koch-Strasse 39, 5000 Cologne 41, F.R.G.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
332 stable recovery had been obtained following impalement, the neurons were activated by shocks to the Schaffer collaterals (bipolar electrodes, square wave pulses 0.1 ms, 0.2 Hz, 5-40 V) or by current injection (0.3-2.0 nA; 125-225 ms) through the recording electrode utilizing conventional current clamp techniques. Ca2+-sensitive/ reference electrodes were pulled from theta glass and the reference barrel was filled with 150 mM NaC1. The Ca2+-sensitive barrel was filled with 100 mM CaCi 2 and the tip with Ca2+-sensitive resin (Fluka cocktail 21048) (tip diameter 1-3 #m, ref. 1-10 MI2, Ca2+-sensitive 5-40 GO). Iontophoretic electrodes were filled with Quis (Cambridge Research Biochemicals), NMDA (CRB) and/or Glu (Sigma) (100 mM in 150 mM NaC1, pH 7.4) and glued to the calcium-sensitive electrodes with a tip separation of 15-30 ~m. The microelectrode complex was placed in the stratum pyramidale of a control slice to record the changes in [Ca2+]o and associated negative field potentials following iontophoretic application (15 s) of the EAAs. Five min intervals were allowed between each application. After 3-5 measurements along the somato-dendritic extent (100/~m increments) the electrode complex was positioned within the shrunken CA1 region of an ischemic slice and measurements were made. Finally, the microelectrode complex was positioned in slices prepared from ischemic/transplanted animals and measurements were made from the opaque transplanted regions. After recording in the transplanted slices the electrode complex was returned to the control slice and measurements were made to confirm that the sensitivity of the electrode had not been altered during the experiment. To quantify the [Ca2+]o decreases the ischemic and transplant values were normalized with respect to the control value of the experimental day. After recording, the slices were immersion fixed in paraformaldehyde (4% in phosphate buffer) and cryostat sections were made. These sections were stained with thionin (0.5%) to confirm the ischemic lesion and the presence of the transplant. Transplanted CAl-like pyramidal neurons (TPCN) were identified by their general membrane properties and response to depolarizing current injection 21'22 which were not obviously different from adult CA1 pyramidal neurons (ACPN) measured in the same chamber. In an earlier study we had shown that in younger TCPN (2-7 months post-transplantation) the excitatory postsynaptic potential (EPSP; measured just subthreshold for spike activation) rose sharply and was followed by a prolonged decay to baseline (EPSP duration 25-200 ms) when compared to control values. The strong hyperpolarization, due to the early inhibitory postsynaptic potential (IPSP), which normally follows the EPSP in ACPN was not observed in these transplanted neurons 16. In some
A
B
~ 2 mV 10 ms
~ 2 mV 10 ms
G
Washout
10 ms
Fig. 1. A and B are examples to illustrate the effects of bath-applied DL-APV (30 /~M) on the EPSPs recorded from the transplanted neurons. The amplitude and duration could be reduced by APV (5 min) in TCPN with a very prolonged EPSP (A). APV did not affect the EPSP when the duration was less prolonged (B) (RMP --76 mV, - - 73 mV, respectively). C illustrates an EPSP followed by a strong IPSP (RMP --57 mV). The IPSP was blocked by bicuculline (2.5 /~M; 15 min) which increased the amplitude and duration of the EPSP. The IPSP recovered after washout (25 min) of bicucuiline ( R M P - - 5 3 mV).
TCPN (2-3 months post-transplantation) more than one action potential could be evoked and were followed by a long lasting hyperpolarization. The present study showed that between 7 and 14 months post-transplantation two groups of TCPN could be identified. One group demonstrated prolonged EPSPs that were not different from the younger TCPN (n = 15). Bath application of 2-amino-phosphonovaleric acid (APV, 30/ira) reduced the EPSP duration in 3 of 5 of these neurons. The EPSP duration ( > 60 ms) was prolonged in these neurons wheras the EPSP duration was much shorter (< 30 ms) in the neurons not demonstrating an obvious APV sensitive component (Fig. 1A,B). The second group of older TCPN (n = 25) demonstrated synaptic responses with EPSP-IPSP sequences characteristic of ACPN. In these cases the early IPSP could be blocked by bicuculline (2.5-5/~m) in a reversible manner (Fig. 1C). The slices containing the greatest proportion of TCPN demonstrating EPSP-IPSP sequences were made from the oldest animals (12-14 months post-transplantation). In the CA1 of control slices NMDA and Glu applica-
333 concentration-dependent changes in [Ca2+]o and corresponding negative potential shifts, the kinetics of which were comparable to those observed in the control CA1 region. In 3 of 6 experiments NMDA- and Glu-evoked decreases in [Ca2+]o were similar to or greater in amplitude than those recorded in the normal CA1 region in spite of the fact that stimulus induced ionic changes were smaller than in control area CA1 (Mudrick, K6hr and Heinemann, in preparation). In two experiments comparable responses could be evoked with double the ejection current and in one experiment even with high ejection current the reponses were smaller. Changes in [Ca2+]o during Quis application always required very high ejection currents to produce similar responses (Fig. 2). A comparison of the normalized data showed that the decreases in [Ca2+]o evoked by Quis, using equivalent ejection currents, were only 34% _+ 14 (S.D., n = 5) of the control values whereas the decreases in [Ca2+]o evoked similarly by NMDA and Glu were 91% + 42 (S.D., n = 6) of the control values. Early in development neurons usually receive an extensive afferent component which is attenuated greatly over time. This results from 'programmed' cell death and
tion (20-60 nA) produced concentration-dependent decreases in [Ca2+]o with similar kinetics. Control experiments have shown the Glu-evoked decrease to be due to activation of both NMDA receptors and voltage-dependent calcium channels. Decreases in [Ca2+]o could be observed at the onset of iontophoresis and continued until the current was discontinued. The [Ca2+]o then returned rapidly to baseline. Quis (45-100 nA) evoked a decrease in [Ca2+]o at the onset of application if the neurons were depolarized sufficiently to activate voltagedependent Ca 2÷ channels 1°'11. Calcium levels then returned to baseline and usually increased from baseline after termination of Quis application which has been attributed to active extrusion 4"1°'2°. All responses to E A A application were accompanied by a slow negative field potential. When similar recordings were made in area CA1 of ischemic slices changes in [Ca2+]o were very small even at high ejection currents (approx. 15% of control). This was due to the lack of viable neurons in the CA1 region of these slices and Ca z+ uptake by the surviving interneurons. Within the transplants Quis, Glu and NMDA induced
A
Control
Ischemic
B
Transplant
++ - 4 0 nA Glu
- 4 0 nA Glu
Control
Transplant
09
,.,,.-V- 4 0 nA
- 25 nA NMDA
Glu
]++ - 35 nA N M D A
2.0
+
1.6 m M
t+a+,Jol
1.3
~
1.3
+ +50 nA Quis
1.6 m M
[Ca2'
- 85 nA Quis
(],2+ -70 nA Quis 20 s
- 60 nA Quis
- 150 nA Quis 20 s
Fig. 2. A: a comparison of the changes in [Ca:+]o and corresponding cxtracellular field potentials evoked by the iontophoretic application (15 s) of Glu and Quis in control, ischcmic and transplanted slices. The reponse to Glu in the transplant is similar to that evoked in the control. In contrast, the response to Quis application is much smaller in the transplanted slices. The responses are minimal in the ischcmic slice. B : the action of H M D A on the extraccllular potential and [Ca2+]o is similar to that evoked by Glu. Evoked responses are strong during N M D A but minimal during Quis application even at extremely high ejection currents.
334 removal of excess synaptic contacts. Afferent fiber distribution to the transplanted neurons may occur in a similar manner, with extensive sprouting by the adult host, but the programmed cell death is not likely to occur. Therefore, it may take much longer for the density and distribution of afferent fibers to approach normal levels. The ability of the transplanted neurons to obtain a totally mature membrane composition may be dependent upon a normal complement of afferent input 12. In younger TCPN, excitatory events may result in an excessive increase in Ca 2+ influx which could mask the inward CI-- current mediating the IPSP 7. Some evidence for this contention has been obtained from immature neurons in which N M D A evoked currents are less voltage dependent and can be activated at quite negative membrane potentials ~. A n enhancement of N M D A receptor-mediated decreases in [Ca2+]o has been shown in area C A I of young slices suggesting participation of N M D A receptors during low frequency synaptic transmission 5. This compares to our findings in the transplants and may explain the prolongation of the EPSP. Indeed, prolonged EPSPs were reduced by A P V application. The extracellular calcium measurements suggest that receptors for the excitatory amino acids are abundant in the transplanted neurons. Although Quis induced decreases in [Ca2+]o were smaller than in control slices, which is what could be expected from a lesser number of neurons, the decreases in [Ca2+]o in response to N M D A were large when compared to control slices in spite of the fact that the neuronal packing density in the transplants is lower than in intact area CA1 16 (Mudrick & Baimbridge, submitted). Similarly, changes in [Ca2+]o within
This research was supported by the Canadian Heart and Stroke Foundation (L.A.M.) and the SFB 200(C8) (U.H.). We acknowledge gratefully M. Groenenwald for her assistance in preparing the ion-sensitive electrodes and the figures.
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the transplants during repetitive stimulation were enhanced 4-fold in low Mg 2+ medium, compared to normal medium 6 (Mudrick, K6hr, Heinemann, in preparation), which is much greater than the 1- to 2-fold enhancement observed in the intact CA1 region 14. These data indicate that in the TCPN the efficacy and/or density of N M D A receptors is enhanced which would suggest that the T C P N are maintaining a state which they reach about 2 weeks postnatal 5. Whether there is also a difference in voltage dependency remains to be determined. It has been suggested that, in immature neurons, an enhanced N M D A - e v o k e d Ca 2+ influx may have an important role in promoting neuronal growth and differentiation 1'17'18. In addition, neurons in the entorhinal cortex have been shown to possess an enhanced N M D A component during the period of fiber outgrowth to and synapse formation with the dentate granule cells which seems to correspond with the prolonged ontogenesis and development of these cells 8. The same may be true for the transplanted neurons but it may be necessary for this growth enhancing property to remain 'turned on' for a longer time. Since development in an adult environment is quite different from that which occurs in a perinatal environment, this potential growth enhancing property observed in immature neurons may play an important role in the fiber outgrowth and integration of the TCPN. Although this hypothesis has not been tested in vivo it would be interesting to investigate the effects of N M D A receptor blockade on the development of transplanted neurons.
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