The question then arises as to whether neurones lying deeper in the brain such as those belonging to the temporal lobe and to the hippocampus also have growth factors which will allow for their survival. The neurotrophic factor which keeps retinal ganglion cells alive is not the growth factor that Levi-Montalcini discovered in smooth and cardiac muscle that keeps autonomic neurones alive. The question then is to what extent can we reconstitute an injured temporal lobe or hippocampus by adding in a neurotrophic factor. The hippocampus, a relatively old and primitive type of cortex, abuts onto the temporal lobe (see figure 11). Despite its relative simplicity, the hippocampus is crucially involved in the formation of memories. If a transverse section is cut through this region of the brain many different classes of neurones can be identified (see figures 12 and 13). Some of these are the first neurones to degenerate anywhere in your brain if you have Alzheimer's Disease, particularly those neurones which project into the hippocampus from the part of brain called the septum. Surprisingly, these septal neurones are kept alive by Levi-Montalcini's growth factor, first found to keep autonomic neurones alive in the peripheral nervous system. So if a neurotrophic factor is discovered that saves a certain class of neurones in the periphery it may well work on a class of neurones in the brain.

Figure 11 [Click on image to enlarge] The limbic or "border" system of the brain comprises primarily the inferior temporal lobe, hippocampus and amygdala. The hippocampus and amygdala lie on the medial surface of the brain rather than on its lateral aspect. Not only is the system most frequently involved in epileptic fits but it is also one of the first parts of the brain to degenerate in Alzheimer's disease.

Figure 12 [Click on image to enlarge] A section through the hippocampus, isolated from the rest of the brain. The diagram also shows the layout of the principal neurononal types (indicated as spheres with branching trees of processes called dendrites) together with their interconnections (indicated by the long thin processes, called axons, with arrows attached). The specific input, bringing information about all sensations from the cortex (such as sight, sound or smell), come from the axons arising in entorhinal cortex which synapse on the dendritic processes of granule cell neurones in the so called fascia dentata. These neurones themselves relay this transformed information to the pyramidal neurones in the CA3 region via their axons that form synapses on the CA3 neurones. These neurones are known to code for memories; they connect with each other through processes called recurrent collaterals (only two of which are shown). It is the excitability of these neurones, in part conferred upon them by these recurrent collaterals, that makes this region of the brain the most likely to trigger an epileptic seizure. The CA3 neurones in turn project to the CAl pyramidal neurones on which they synapse. These neurones also code for memories and their axons project to the region called the subiculum in the old paleocortex and from there to the neocortex. Thus the trisynaptic pathway is from entorhinal cortex to fascia dentata to CA3 pyramids to CA1 pyramids back to the cortex. There is another, less specific input (on the left) necessary for the normal functioning of the hippocampus and that comes from the subcortical structure called the septum. These axons synapse on all neurones in the hippocampus. They are the first neurones to degenerate in the brain of people suffering from Alzheimer's disease. This leads to the loss of memory formation that characterized this disease.

Figure 13. [Click on image to enlarge] The functional circuit between different types of neurones, shown in figure 12, the synaptic connections within this circuit have the very special property of remembering over very long periods of time if they have been subjected to impulse traffic. 'W' shows a transverse section through the hippocampus, like that shown in figure 12, except that stimulating electrodes have been placed on the nerves from the entorhinal cortex (called the perforant pathway axons) and a recording electrode in the dendritic layer of the granule cells, where the perforant pathway axons synapse. "B" shows an enlargement of the boxed area in "A", with sample electrical recordings of the compound population spike occurring because many granule cells fire in synchrony due to stimulating the perforant path axons; the population excitatory postsynaptic potential (epsp) is recorded from the synaptic regions between the perforant path axons and the granule cells an stimulation and gives a measure of the efficacy of synaptic transmission. "C" shows the results of stimulating the perforant pathway at a frequency of 15Hz for 10 seconds at the four times indicated in the graph of the relative amplitude of the population epsp against time; if the perforant nerves are stimulated every few minutes with a single impulse before, during and after the 15HZ stimulating periods then the amplitude of the population epsp at each 6 minute period is shown to grow over 3 hours until it settles down to a size 300% that of the control (in which no stimulation occurred at 15Hz); two examples of this long-term potentiation of the population epsp are shown one before and one after the 15HZ conditioning period, This enhanced efficacy of transmission through the synapses of over 300% is shown to last for 3 hours after the I5Hz stimulation but may continue for days or months. The mechanisms responsible for this potentiation are required to retain a memory.

Figure 14. [Click on image to enlarge] Procedure for transplanting embryonic septal neurones from the hippocampal region of a foetus to the hippocampus of a mature animal with a degenerating septum. The septal region (black area) is first removed from the foetal brain and placed in a culture dish with enzymes that loosen the tissue into separate neurones. The partly separated neurones are then placed in a test tube and the isolation process taken further by rapidly shaking the neurones up and down in a pipette in the test tube. The completely dissociated neurones are next taken up into a syringe. The syringe needle is then located with great accuracy in the appropriate part of the hippocampus and the dissociated septal neurones injected into this area of the brain.

As already mentioned, rather than introducing growth factors into the brain in the hope of saving a certain class of neurones that are degenerating because of a neurological disease, it is possible to introduce embryonic neurones of the same class that have been destroyed by the disease (figure 14). These can be obtained from a set of neurones that have been genetically manipulated to be of a similar kind to those that have degenerated. If you introduce these neurones into the brain (see figure 14), they reconstitute the normal circuitry, making the correct functional connections. In this way they restore the normal function, or at lease almost the normal function, of that part of the brain which had degenerated.

There are now standard techniques for introducing neurones into the brains of animals. Neurones are taken from an early embryonic rat brain, for example from that part of the brain that degenerates in Alzheimer's Disease. Further purification of these then occurs using various cell sorting techniques. Finally just the specific class of neurones required are placed in a tube from where they are sucked up into a pipette (see figure 14). This pipette is then used to inject the neurones into just that part of the brain which has degenerated or been injured (figure 14).

How do we test out if we have reconstituted normal function after say a transplant into the hippocampus of a set of neurones to replace those that have degenerated? Figure 15 shows one procedure that is used to determine this. It is called the alternating T-maze test. A rat is put at the end of one arm of the T-maze and is allowed to run to the other end where a choice is made of either turning left or right. In the first trial food is placed on the right arm and the rat is forced to turn right because a trapdoor prevents it from turning left. On the second trial there is no trapdoor to force the rat to turn a particular way so tha it may now turn either left or right. However a nasty trick is carried out before this second trial. Before the rat is allowed to run the second trial food is placed on the left arm of the maze, opposite its original position. After this trial the rat is taken out of the maze and allowed to rest for a while before another alternating trial is attempted. Altogether this is repeated about six times a day. It doesn't take very long before the rat appears to figure out what is going on. It turns right on the first trial and gets the food; on the second trial, when you put the food on the opposite arm, the rat immediately turns left and doesn't make the mistake of turning right. The graph in figure 15 shows the rate at which the rat learns to make the correct choice on the second trial, namely to turn left. In a sham trial the neocortex is exposed without any experimentation, and then closed; over a period of about one and a half weeks the rat learns on 100% of occasions to always turn left to get the food on the second trial. If however there has been an injury to the septum, then the rat turns left on the second occasion at random frequency (namely on 50% of the occasions). It hasn't learnt at all; it cannot lay down the memory that it should turn left always on the second trial. But if we do a transplant of healthy septal neurones from an embryonic rat into the hippocampus of this injured adult rat or of a rat whose septal neurones have degenerated because it has a form of senile dementia, then it only takes a matter of about two months before we find that the rats can learn at the 90% level to turn left (figure 15). The rats that received the transplant have learnt to nearly always turn left on the alternate tmaze performance because normal hippocampal functioning has been reconstituted by this septal transplant.

You might well ask how is it that the rat can tell where it is in the T-maze because the maze is symmetrical; how can the rat tell its left from its right despite the fact that it has no visual clues to orientate itself. Well the trick here is that the t-maze is set in a room which has lots of interesting objects in it and these allow the rat to work out exactly where it is. These interesting objects may include curtains, clocks, and pictures of female rats as shown in figure 16. This allows the rat to determine, when it is sitting at the start position n the T-maze, the geometry of the situation around it. The hippocampus calculates the spatial layout of the room from this information, allowing the rat to determine what is its left and its right. These spatial clues are very necessary for it to be always able to distinguish left from right in the alternating T-maze performance.

Figure 15. [Click image to enlarge] Use of the forced alternation T-maze to show that following degeneration of septal neurones transplanted embryonic septal neurones can reconstitute the memory system of the hippocampus. The rat is put in the starting position in the T-maze. In the first trial the door on the right, door 1, is open; the door on the left, door 2, is closed and food is placed at 'a '; the rat runs and is forced to turn to the right where the door is open (it can neither seenor smell the food at the starting position). In the second trial the door 1 is open and now the doow 2 is also open and the food placed at 'b'. A correct response is regarded as one in which the rat turns left on trial 2. The trial 1- trial 2 sessions are repeated 6 times per day. The room in which the T-maze is placed contains many items, such as curtains, clocks and computers which despite the rats poor visual acuity appear to allow the rat to orientate itself on the T-maze. The graph shows the percentage of correct responses performed by the rat on the T-maze alternation task over time. Following lesion or degeneration of the septal region of the hippocampus there is a 50% chance that the rat will turn left on the second trial, so nothing has been remembered and the choice is random. Following a sham operation, in which only a harmless pladebo substance is injected into the hippocampus, the rat learns to make 100% left-hand turns on the second trial within 3 weeks of testing so that at this time its' memory for the T-maze performance is perfect. Following transplantation of embryonic septal neurones into a rat with a lesioned septum. the rat learns to perform at the 90% correct level of Performance within about 10 weeks after the operation.

In the above experiment an attempt was made to mimic the effects of senile dementia or stroke that lead to degeneration of the septal neurones innervating the hippocampus by introducing a lesion into the hippocampus. However it is now possible to distinguish aged rats that suffer from a form of senile dementia from those that do not; the former have a natural loss of septal neurones as a consequence of the dementia. The method used to distinguish rats with senile dementia from those that do not suffer from this complaint involves placing rats in a water tank of the kind shown in figure 16. Rats don't like being forced to swim around in a tank any more than we do so a little stand is placed about an inch under the water, which is opaque so that the rat cannot see the stand; as the rat swims around its feet sometimes bump into this little stand and not being stupid it sits on the stand, rests, and looks around the room. We can trace the movements of the rat in the water before it finds the stand using a TV camera elevated above the trough which is shown in figure 16. In this way the actual locus of movement of the rat in the water before it sits on the stand can be followed, as shown in figures 16 and 17. The rat knows it's position in the water because it can see the interesting objects in the room, such as the clock etc, so that it can form a spatial map of the room in its hippocampus as was described above in relation to the T-maze. When the rat forms this map it has the coordinates of the stand with respect to the spatial layout of the objects in the room.

Figure 16. The Morris water tank used to determine the spatial memory of rats. The water tank is placed in about the middle of the room. It contains opaque water and a stand (shown in the cut-away of the tank wall) which is about one inch beneath the surface of the water; this is sufficiently deep for the rat not to see it when swimming in the tank so that it only becomes aware of the stand if its, feet come in contact with it. Surrounding the tank are objects on the wall, such as curtains, clocks and potplants (and a large picture of other rats) which allow the swimming rat to determine its' orientation; the spatial location of the rat in the water tank is laid down as a spatial memory in the hippocampus and this allows a healthy rat to determine the position of the unseen stand with respect to the objects in the room, once its' feet have- come in contact with the stand.. A television camera is placed above the water tank which allows 'the operator at the television computer terminal shown to monitor the locus of the swimming pathway of the rat on::e it has been placed in the tank at an arbitrary position.

Figure 17. The locus of the swimming pathway of rats (determined by the methods given in the legend to figure 16) after they have been placed in the Morris water tank. The view is looking down on the tank, and shows the position of the stand beneath the opaque water in the tank. Each row shows the results for series of trials which determined if a rat found the stand (and then sat on it) during a 5 minute period; there were five trials on each of five successive days and the results are shown for the first trial on the first day (1.1), the fifth trial on the first day (5.1), the fifth trial on the fourth day (5.4) and the fifth trial on the fifth day (5.5). In the first row a young control rat (about 6 months old) was placed at a random site in the tank at 1.1 and left to swim; it will be noted that at 1.1 the rats feet did not accidently make contact with the stand; by 5.1 this had happened and the spatial memory system, of the hippocampus had located the position of the unseen stand with respect to the objects in the room enabling the rat to swim directly to the stand and sit on it, as shown; this also occurred at 5.4; at 5.5 the stand was removed and the rat swam repeatedly over the site where the site had been, seeking a rest. In the second row an Aged Impaired rat (about 3 years old) is shown to be unable to lay down a spatial memory of the position of the stand, even though its' feet accidently come in contact with it several times over the 25 trials. In the third row an Aged Unimpaired rat (again about 3 years old) is shown to be able to lay down a spatial memory as well as the Young Control rat, ancl performs in a like manner. The final row shows an Aged Impaired rat that had a transplant of embryonic neurones from the septum in its' hippocampus; the locus of the swimming pathway of the rat in each case shows that it has formed a spatial memory of the stand as quickly as the Young Control.

If we take a young rat about 6 months old and put it into the water for a few minutes, then the locus of its movements on this first trial on the first day are given in the first row and column of figure 17: in this particular trial the rats legs didn't hit the stand beneath the water so the rat just swam around for a couple of minutes. But a few trials later, namely the fifth trial on this first day (see figure 17), the rat has learnt in the intermediate trials the position of the stand and so it swims immediately to it and sits down. Presumably this rat has formed a spatial memory of the position of the stand as a consequence of the normal functioning of its septo-hippocampal circuits. Of course by the fourth trial on the fifth day the rat has no trouble at all, no matter where it enters the water, it immediately locates where the stand is (see figure 17). on the fifth trial of the fifth day a nasty trick is carried out: the stand is removed and the rat is entering the water tank at a random position swims around frantically trying to find the stand; the locus of its movements are then centred on the position where the stand was, as shown in figure 17. We can use this technique to pick out those rats which are suffering senile dementia from those that are not. In the second row of figure 16 the results are shown for a three and a half year old rat, about the equivalent of a human at eighty: it is apparent that even by the fifth trial on the fourth day, after the rat had bumped into- the stand many times during the preceding days, it could still not locate the stand using spatial clues. This was an aged impaired rat, that is it had senile dementia. All old rats do not get senile dementia any more than all old humans do: in the fourth row of figure 17 the results are shown for another three and a half year old rat: by the fifth trial on the first day it had evidently formed a good spatial map of the whereabouts of the stand. On succeeding days it did as well as the young rat whose performance is shown in the first row.

Using this water tank technique, invented by Morris, we are able to sort out rats suffering from senile dementia from those that are not suffering from senile dementia. This is we can separate out those whose septo-hippocampal circuit is functioning from those in which these circuits are in bad shape. If we take an aged rat suffering from senile dementia now and operate on it in the way I previously described, that is introducing embryonic septal neurones into its hippocampus, then after a month r so has elapsed to allow the implanted neurones to make connections, the water tank test shows that the rat has recovered its ability to lay down spatial memories (see the last row in figure 17). This approach clearly demonstrates that transplants of neurones from embryonic material which allows the reconstitution of damaged or degenerating neuronal circuits involved in the formation of spatial memory.

In conclusion then I have been proposing that we are confronted in the 21st Century with what I regard as the third great area of mystery concerning natural knowledge. The first involved the discovery of quantum mechanics in 1926 by Schrodinger and Heisenberg. The second was to some extent begun by Schrodinger himself with his little book "What is Life", which attracted physicists of high quality into biology. One of these was Francis Crick who after codiscovering the structure of DNA in 1952 went on to delineate the concept of the genetic code in specific ways which Schrodinger had only dreamed of in his little book. This helped lay the foundations of molecular biology that has come to dominate a good deal of science in the latter part of the 20th century, illuminating our understanding of embryology in particular. The third challenge for us now is the human brain.

Figure 18. A human embryo at 5 weeks (15 mm. long) The hands are clearly visible with the just clearly delineated fingers. The heart, with liver below, can be seen between the hands; the diaphragm separates the heart and liver. Most striking, are the two halves of the cerebrum which can be seen through the transparent skin of the forehead above the developing eyes.

There are two questions that I have laid stress on which will dominate science in the 21st century. one of these is what are the actual workings of the brain that give rise to consciousness and how does this develop (figure 18) . We have seen that consciousness involved in visual phenomena requires the correct funptioning of parietal cortex and inferior temporal lobe. The second question that we have considered is whether our increased knowledge of the workings of the brain, particularly in relation to the search for the neuronal concomitants of consciousness, will provide us with insights into means of ameliorating various neuronal diseases. In particular those diseases which arise as a consequence of vascular stroke and as a consequence of schizophrenia, diseases which produce hallucinations and which completely distort our consciousness of reality. This essay has stressed one possible approach to the problem. This is through the introduction into the appropriate parts of the degenerating brain of neurotrophic factors that allow for the survival of specific classes of neurones. Alternatively a transplant of appropriate viable neurones may be made to replace degenerating ones. The next century offers us the possibility of understanding our own brains.

Acknowledgments

I am extremely grateful to my colleague, Dr. Bogdan Dreher, for his critical reading of the manuscript and his many helpful suggestions.

Further Reading

Crick, F. (1988) "What Mad Pursuit." Weidenfeld and Nicolson. London.

Judson, H.F. (1979) "The Eighth Day of Creation". Simon and Schuster. New York.

Moore, W. (1989) "Schrodinger". Cambridge University Press. Cambridge.

Schrodinger, E. (1944) "What is Life?" Cambridge University Press. Cambridge.

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